Notch Signaling Is Antagonized by SAO-1, A Novel GYF - Genetics (PDFDrive)

!
"#$%&!'()*+,(*)!('!+*$+)#*(-./!01!234567!+!*#8.,!9:;5/#<+(*!=>#$.(*!$&+$!
(*$.>+%$'!?($&!$&.!@A!B0(CB($(*!,()+'.!2@D56E!(*!!"#$%$&'()!
!"#$%&$'()'*"#$+',-".'/)'01&.$2+'/&.34$2'5)'06#37$%8+''964$:;&.$'*)'*"316.8+'''
<"##&$'=)'>?"%@#$%+''>"@;$%&.$'A)'=B".864+''<"%6#&.$'061@@$'
'
'
C$:"%@D$.@'6E'F&6#682+'(D;$%4@'<6##$8$+'(D;$%4@+'G('HIHHJ'
'
'
Hale et al.
SAO-1 antagonizes notch signaling
2
'
FB**(*)!G($,.H!=(KLI'36?.'%$81#"@$4'M6@B;'4&8."#&.8'
'
I.1!J#>/'H'M6@B;+'$D7%26+'8"DD"L4$B%$@"4$+'05N+'=<N'
'
K#>>.'=#*/(*)!3B$&#>H''
<"%6#&.$'061@@$'
'
JJOP'GB01&%$'/&E$'=B&$.B$4'
C$:"%@D$.@'6E'F&6#682'
(D;$%4@'<6##$8$'
(D;$%4@+'G('HIHHJ'
'
Q$#R'SIOLTSJLUOJI'
E"VR'SIOLTSJLPWTT'
$D"&#R'B$861@@$X"D;$%4@)$31'
Hale et al.
3
'
(F=QY(<Q'
'
M6@B;'4&8."#&.8':"@;?"24'B".'7$'%$81#"@$3'@;%618;'"'-"%&$@2'6E'B$##1#"%'D$B;".&4D4+'
".3'8$.$@&B"##2'B6D:%6D&4$3'424@$D4':%6-&3$'14$E1#':#"@E6%D4'E%6D'?;&B;'@6'4$"%B;'
E6%'@;$'%$4:6.4&7#$'D631#"@6%4)''Q;$'!"#$%&'"()*+*,-#.#/"$,-8$.$'"0'12'$.B63$4'"'
B6D:6.$.@'6E'8"DD"L4$B%$@"4$+'?;&B;'&4'$44$.@&"#'E6%'M6@B;'4&8."#&.8'$-$.@4'
@;%618;61@'3$-$#6:D$.@)''F2'#66Z&.8'E6%'41::%$446%4'6E'@;$'&.B6D:#$@$#2':$.$@%".@''
"0'123452678'D1@"@&6.+'?$'&3$.@&E2'"'.$?'8$.$+',"%12'[,1::%$446%'6E'":;L%.$\+'@;"@'
.$8"@&-$#2'%$81#"@$4'"0'123452678'"B@&-&@2'&.'@;$'$"%#2'$D7%26)''Q;$',"%12'8$.$'
$.B63$4'"'.6-$#':%6@$&.'@;"@'B6.@"&.4'"'05N':%6@$&.L:%6@$&.'&.@$%"B@&6.'36D"&.'".3'
&.@$%"B@4'4:$B&E&B"##2'?&@;'=,/LIH+'".'N7?P'B6D:6.$.@'6E'=<N',O'17&]1&@&.'#&8"4$4)''
A$'3$D6.4@%"@$'@;"@'@;$'$D7%26.&B'#$@;"#&@2'6E'"0'123452678'D1@".@4'B".'7$'
41::%$44$3'72'%$D6-&.8',"%12'"B@&-&@2'6%'72'D1@"@&6.4'@;"@'3&4%1:@'@;$'=(KLI'L'=,/L
IH':%6@$&.'&.@$%"B@&6.)''C$B%$"4$3',"%12'"B@&-&@2'"#46'&.E#1$.B$4'M6@B;'4&8."#&.8'
$-$.@4'?;$.'@;$2'"%$'B6D:%6D&4$3'"@'3&EE$%$.@'D6#$B1#"%'4@$:4'6E'@;$':"@;?"2+'41B;'
"4'"@'@;$'#$-$#'6E'@;$'M6@B;'%$B$:@6%'0/^LI'6%'@;$'36?.4@%$"D'@%".4B%&:@&6.'E"B@6%'
/(0LI)''<6D7&.$3'"."#24&4'6E'@;$'=(KLI'L'=,/LIH':%6@$&.'&.@$%"B@&6.'".3'B6D:"%&46.4'
6E',"%12'".3',#.129'8$.$@&B'&.@$%"B@&6.4'4188$4@'"':644&7#$'%6#$'E6%'=(KLI'"4'".'
"BB$446%2':%6@$&.'@;"@':"%@&B&:"@$4'?&@;'=,/LIH'&.'36?.'%$81#"@&6.'6E'M6@B;'4&8."#&.8)''
Q;&4'?6%Z':%6-&3$4'@;$'E&%4@'D1@".@'"."#24&4'6E'"'05NL36D"&.':%6@$&.'&.'$&@;$%'!:-
#.#/"$,'6%';&%,%0'*."+'".3'&.@%631B$4'"'.$?'@2:$'6E'N7?PL&.@$%"B@&.8':%6@$&.'@;"@'"B@4'
&.'"'4174$@'6E'N7?P'E1.B@&6.4)'
Hale et al.
4
_MQYKC`<Q_KM'
'
Q;$'M6@B;'4&8."#&.8':"@;?"2':#"24'"'B%&@&B"#'%6#$'&.'D".2'B$##LE"@$'B;6&B$4'
31%&.8'".&D"#'3$-$#6:D$.@)''^"@;?"2'"B@&-"@&6.'7$8&.4'?&@;'@;$'&.@$%"B@&6.'6E'"'C=/'
[C$#@"a=$%%"@$a/"8LJ\'#&8".3'".3'"'B$##L41%E"B$'M6@B;'%$B$:@6%)''`:6.'#&8".3'7&.3&.8+'
@;$'M6@B;'%$B$:@6%'1.3$%86$4'@?6'4$]1$.@&"#':%6@$6#2@&B'B#$"-"8$4R'".'(C(GL
:%6@$"4$'%$#$"4$4'@;$'$V@%"B$##1#"%'36D"&.'".3'@;$.'8"DD"L4$B%$@"4$'%$#$"4$4'@;$'
&.@%"B$##1#"%'36D"&.'?;&B;'@%".4#6B"@$4'@6'@;$'.1B#$14)''0"DD"'4$B%$@"4$'&4'"'
B6D:#$V'6E'E61%'&.@$8%"#'D$D7%".$':%6@$&.4'[:%$4$.&#&.+'(^*LI+'(^*LJaM&B"4@%&.+'".3'
^,MLJ\+'@;"@'"#46'B#$"-$4'"'-"%&$@2'6E'6@;$%'@%".4D$D7%".$':%6@$&.'4174@%"@$4+'
&.B#13&.8',YFFS'%$B$:@6%'@2%64&.$'Z&."4$+'MLB"3;$%&.+'".3'@;$'"D2#6&3L!':%$B1%46%'
:%6@$&.'[(^^\'"446B&"@$3'?&@;'(#b;$&D$%c4'3&4$"4$'[4$$'^(Y>='".3'<`YQ_='JHHP\)''K.B$'
&.'@;$'.1B#$14+'@;$'M6@B;'&.@%"B$##1#"%'36D"&.'&.@$%"B@4'?&@;'@;$'B6.4$%-$3'
@%".4B%&:@&6.'E"B@6%'<=/'[<FNIa=1::%$446%'6E'*"&%#$44a/(0LI\'@6'%$81#"@$'
@%".4B%&:@&6.'6E'@"%8$@'8$.$4'[E6%'%$-&$?'4$$'>K^(M'".3'_/(0(M'JHHW\)''
'
Q;$%$'"%$'@?6'%$#"@$3'M6@B;'%$B$:@6%4'&.'!:-#.#/"$,<-0/^LI-".3-/_MLIJ+'?;&B;'
D$3&"@$'"'-"%&$@2'6E'B$##'&.@$%"B@&6.4'@;%618;61@'3$-$#6:D$.@)''_.'@;$'$"%#2'$D7%26+'"@'
#$"4@'4&V'3&4@&.B@'B$##'&.@$%"B@&6.4'"%$'D$3&"@$3'72'0/^LI'6%'/_MLIJ'".3'4$%-$'@6'
:"@@$%.'@;$'3$-$#6:D$.@"#'E"@$'6E'$D7%26.&B'B$##4'[%$-&$?$3'&.'^Y_,=='JHHT\)''Q;$'E&%4@'
@?6'41B;'4&8."#&.8'$-$.@4'6BB1%'"@'@;$'SLB$##'".3'IJLB$##'4@"8$4'6E'$D7%268$.$4&4'".3'
"%$'D$3&"@$3'72'D"@$%."##2L41::#&$3'4&8."#&.8'B6D:6.$.@4'&.B#13&.8'0/^LI)''
Y$D6-"#'6E'D"@$%."#'0/^LI'6%'/(0LI+'6%'".2'6E'@;$'E61%'8"DD"L4$B%$@"4$'
Hale et al.
5
B6D:6.$.@4+':%$-$.@4'M6@B;'4&8."#&.8'&.'@;$4$'$"%#2'4@"8$4'6E'$D7%268$.$4&4+'
%$41#@&.8'&.'D&44:$B&E&B"@&6.'6E'4$-$%"#'7#"4@6D$%$'E"@$4'".3'$-$.@1"#'"%%$4@'6E'@;$'
$D7%26'[4$$'^Y_,=='JHHT\)''(@'#$"4@'46D$'6E'@;$'#"@$%'$D7%26.&B'4&8."#&.8'$-$.@4'B".'
7$'D$3&"@$3'72'$&@;$%'0/^LI'6%'/_MLIJ+'41B;'@;"@'"'D1@".@':;$.6@2:$'&4'6.#2'
"::"%$.@'&.'@;$4$'B$##LE"@$'B;6&B$4'&E'76@;'%$B$:@6%4'"%$'"74$.@'[/(GF_,'".3'>_GF/,'
IWWI\)'''
'
C1%&.8':64@L$D7%26.&B'3$-$#6:D$.@'0/^LI'".3'/_MLIJ';"-$'3&4@&.B@'%6#$4)''
0/^LI'"B@&-"@&6.'&4'%$]1&%$3'B6.@&.1614#2'@6'&.31B$'8$%D'B$##':%6#&E$%"@&6.'&.'@;$'
3&4@"#'86."3+'@;14':#"2&.8'"'B%&@&B"#'%6#$'&.'%$81#"@&.8'@;$'7"#".B$'7$@?$$.'
:%6#&E$%"@&6.'".3'D$&64&4'[>_GF/,'".3'<Y_QQ,MC,M'JHHT\)''/644'6E'0/^LI'"B@&-&@2'
%$41#@4'&.'4@$%&#&@2'31$'@6'8$%D#&.$'1.3$%':%6#&E$%"@&6.+'?;$%$"4';2:$%"B@&-$'0/^LI'
:%6@$&.'B"14$4'6-$%':%6#&E$%"@&6.'6E'@;$'8$%D'#&.$'"@'@;$'$V:$.4$'6E'8$%D'B$##'
3&EE$%$.@&"@&6.'[F,YY5-#+-".:'IWWPd'>_GF/,'".3'<Y_QQ,MC,M'JHHTd'^,^^,Y-#+-".:'JHHO\)''
/_MLIJ'&4'#"%8$#2'%$4:6.4&7#$'E6%'D$3&"@&.8'B$##'&.@$%"B@&6.4'@;"@'3&B@"@$'46D"@&B'B$##'
E"@$'B;6&B$4+'41B;'"4'@;64$'@;"@'"%$'B%&@&B"#'@6'-1#-"#'D6%:;68$.$4&4)''/644'6E'/_MLIJ'
E1.B@&6.'%$41#@4'&.'".'$88L#"2&.8'3$E$B@&-$':;$.6@2:$'[,8#\'7$B"14$'6E'D&44:$B&E&B"@&6.'
6E'4$-$%"#'-1#-"#'".3'1@$%&.$'B$##'E"@$4'[E6%'%$-&$?'4$$'0Y,,MA(/C'JHHT\)'
'
0$.$@&B'&.@$%"B@&6.4'7$@?$$.'M6@B;'4&8."#&.8'B6D:6.$.@4'".3'6@;$%'B$##1#"%'
:%6B$44$4'"%$'1.B6-$%&.8'"'-"%&$@2'6E'B$##1#"%'D$B;".&4D4'@;"@'%$81#"@$'M6@B;'
:"@;?"2'"B@&-&@2)''F6@;':64&@&-$'".3'.$8"@&-$'D631#"@6%4';"-$'7$$.'&3$.@&E&$3'
@;%618;'D1@"@&6.4'@;"@'"#@$%'@;$'"D61.@'6E'M6@B;'4&8."#&.8'"B@&-"@&6.'&.'".&D"#4'?&@;'
Hale et al.
6
D1@".@'M6@B;'%$B$:@6%4'[E6%'$V"D:#$'GK`Y_>_=-#+-".:'JHIHd'=`MC(Y(G'".3'0Y,,MA(/C'
IWWO7d'!,Y*,5,M-#+-".:'IWWed'E6%'%$-&$?'4$$'NKYQ_M_'JHHW\)''C6?.'%$81#"@6%4'&3$.@&E&$3'
@;%618;'@;&4'"::%6"B;'&.'!:-#.#/"$,'&.B#13$'B6D:6.$.@4'6E'$.36:#"4D&B'%$@&B1#1D'
"446B&"@$3':%6@$&.'3$8%"3"@&6.'[,Y(C\'[0Y(MQ'".3'0Y,,MA(/C'IWWe\+'B"%86'
4$#$B@&-&@2'E6%',Y'@6'06#8&'@%".4:6%@'[A,M'".3'0Y,,MA(/C'IWWW\+'$.36B2@&B'@%"EE&BZ&.8'
[C,'=K`f(-#+-".:'JHHP\+'".3'17&]1&@&.LD$3&"@$3':%6@$646D$'3$8%"3"@&6.'[*`FF(YC-#+-
".:'IWWP\)''Q;$'D$B;".&4D4'6E'M6@B;':"@;?"2'%$81#"@&6.'"%$':%6-&.8'@6'7$'
E1.B@&6."##2'B6.4$%-$3+'71@'@;$'%$#"@&-$'%6#$'6E'$"B;'6E'@;$4$'D631#"@&.8'$EE$B@4'&4'
#&Z$#2'@6'3&EE$%'E6%'3&4@&.B@'B$##1#"%'B6.@$V@4)''M6@B;':"@;?"2'D631#"@&6.'&.'!:-#.#/"$,'
;"4'7$$.'?$##'4@13&$3'&.'#"%-"#'".3'"31#@'4&8."#&.8'$-$.@4+'71@'#&@@#$'&4'Z.6?.'"761@'@;$'
%$81#"@&6.'6E'M6@B;'"B@&-"@&6.'&.'@;$'$D7%26)'
'
=,/LIH'?"4'&3$.@&E&$3'"4'"'36?.'%$81#"@6%'6E'/_MLIJ'&.'!:-#.#/"$,+'".3'?"4'
4;6?.'@6'"B@'@;%618;'17&]1&@&.LD$3&"@$3':%6@$646D"#'3$8%"3"@&6.'[*`FF(YC-#+-".:'
IWWPd'/_-#+-".:'JHHJd'A`-#+-".:'JHHI\)''=,/LIH'&4'"'D$D7$%'6E'@;$'E"D&#2'6E'N7?P'
:%6@$&.4'[NL76V'".3'AC'%$:$"@'36D"&.LB6.@"&.&.8'P\'@;"@'&.B#13$4'@;$'2$"4@'".3'
;1D".'<3BS':%6@$&.4'[4$$'A,/<>,Y'".3'</`YG(M'JHHU\E6%'%$-&$?\)''Q;$'D6#$B1#"%'
%6#$'6E'N7?P':%6@$&.4+'#&Z$'6@;$%'NL76V':%6@$&.4+'&4'@6':%6-&3$'"'4174@%"@$'%$B68.&@&6.'
36D"&.'E6%'@;$'D1#@&4171.&@'=<N'[=Z:IL<1##&.ILNL76V\'@2:$',O'17&]1&@&.'#&8"4$4'[4$$'
FK=`'".3'>_^Y,K='JHHU\)''Q;$'4174@%"@$4'@;"@'"%$'@"%8$@$3'E6%'17&]1&@&."@&6.'72'N7?P'
:%6@$&.4'&.B#13$':%6@$&.4'?;64$'#$-$#4'D14@'7$'@&8;@#2'B6.@%6##$3'31%&.8'B$##'3&-&4&6.'
".3'3&EE$%$.@&"@&6.'[$)8)+'B2B#&.',+'91.+'G2B+'".3'M6@B;\)''/644L6ELE1.B@&6.'D1@"@&6.4'&.'
N7?P'8$.$4'"%$'"446B&"@$3'?&@;'@1D6%&8$.$4&4+'"4'&@4'4174@%"@$4'7$B6D$'
Hale et al.
7
&."::%6:%&"@$#2'4@"7&#&b$3'[A,/<>,Y'".3'</`YG(M'JHHU\)'=@13&$4'&.'76@;'!:-#.#/"$,'
".3'D"DD"#4';"-$'$4@"7#&4;$3':%$4$.&#&.'".3'@;$'M6@B;'&.@%"B$##1#"%'36D"&.'"4'
3&%$B@'@"%8$@4'6E'@;$'=,/LIH'N7?P':%6@$&.+'?;&B;':%6D6@$4'@;$&%'17&]1&@&."@&6.'".3'
:%6@$646D"#'3$8%"3"@&6.'[0`^Q(LYK==_-#+-".:'JHHId'*`FF(YC-#+-".:'IWWPd'/_-#+-".:'JHHJd'
A`-#+-".:'IWWUd'A`-#+-".:'JHHI\d';6?$-$%+'41B;'"'%6#$'E6%'=,/LIH'&.'@;$'$D7%26';"4'.6@'
2$@'7$$.'$V:#6%$3)''0$.$@&B'&.@$%"B@&6.4'7$@?$$.'!:-#.#/"$,',#.129'".3'8$.$4'6E'@;$'
4$VL3$@$%D&."@&6.':"@;?"2':6&.@'@6'"33&@&6."#'@"%8$@4'6E'=,/LIHLD$3&"@$3'36?.'
%$81#"@&6.'[9(0,Y-#+-".:'JHHS\+'D"Z&.8'@;$'4@132'6E',#.129'@;%618;61@'!:-#.#/"$,'
3$-$#6:D$.@'"'14$E1#'D63$#'424@$D'&.'?;&B;'@6'"."#2b$'@;$'32."D&B'E1.B@&6.'6E'N7?P'
:%6@$&.4)''
'
_.'@;&4'4@132'?$'4618;@'@6'&3$.@&E2'B$##1#"%'B6D:6.$.@4'@;"@'%$81#"@$'M6@B;'
4&8."#&.8'&.'@;$'$"%#2'$D7%26)''A$'7$8".'?&@;'"'8$.$@&B"##2'4$.4&@&b$3'424@$D'@;"@'
B6.4&4@$3'6E'"'D1@".@'E6%D'6E'(^*LI+'@;$'B6.4$%-$3'4$-$.L:"44'@%".4D$D7%".$'
:%6@$&.'@;"@'&4':"%@'6E'@;$'8"DD"L4$B%$@"4$'B6D:#$V)''Q;$'.6.4$.4$'"##$#$'"0'1
23452678'&4':%$3&B@$3'@6'$.B63$'"'@%1.B"@$3'(^*LI':%6@$&.'@;"@'#"BZ4'@;$'<L@$%D&."#'
OO'%$4&31$4'[0K`QQ,-#+-".:'JHHJ\)''Q;$4$'@$%D&."#'%$4&31$4'"%$'.6@'$44$.@&"#'E6%'(^*LI'
E1.B@&6.+'"4';6D6b28614'"0'123452678'".&D"#4'"%$'E$%@&#$'".3'$88L#"2&.8':%6E&B&$.@+'
?&@;'6.#2'"':"%@&"##2':$.$@%".@'D"@$%."#L$EE$B@'$D7%26.&B'#$@;"#&@2)''_.'B6.@%"4@+'
4@%6.8$%'"0'12'D1@".@'"##$#$4'B"14$'"'E1##2':$.$@%".@',8#':;$.6@2:$'".3'D6%$'4$-$%$'
D"@$%."#L$EE$B@'#$@;"#&@2'[NY(M<_=-#+-".:'JHHJd'0K`QQ,-#+-".:'JHHJ\)''G64@'$D7%264'E%6D'
"0'123452678';$%D":;%63&@$4';"-$'41EE&B&$.@'(^*LI'E1.B@&6.'@6'41::6%@'M6@B;'
4&8."#&.8'"@'@;$'SLB$##'4@"8$+'71@'.6@'$.618;'E6%'@;$'IJLB$##'4@"8$'B$##'&.@$%"B@&6.'
Hale et al.
8
[0K`QQ,-#+-".:'JHHJ\)''Q;14'"0'123452678'".&D"#4':%6-&3$'"';&8;#2'4$.4&@&b$3':#"@E6%D'
E%6D'?;&B;'@6'4$"%B;'E6%'D631#"@6%4'6E'@;$4$'$"%#2'$D7%26.&B'4&8."#&.8'$-$.@4)''A$'
%$"46.$3'@;"@'#644L6ELE1.B@&6.'D1@"@&6.4'&.'8$.$4'@;"@'.$8"@&-$#2'%$81#"@$'M6@B;'
4&8."#&.8'&.'@;$'$"%#2'$D7%26'D&8;@'B6D:$.4"@$'E6%'&.41EE&B&$.@'"0'123452678'"B@&-&@2+'
@;14'"##6?&.8'"';&8;$%':%6:6%@&6.'6E'@;$'$D7%264'E%6D'"0'123452678'D6@;$%4'@6'
41%-&-$)''A$'3$4B%&7$';$%$'"'.$?'8$.$+',"%12'[41::%$446%'6E'":;L6.$\+'@;"@'?$'&46#"@$3'
@;%618;'41B;'"'41::%$446%'4B%$$.)''Y$D6-"#'6E',"%12-E1.B@&6.'41::%$44$4'@;$'
$D7%26.&B'#$@;"#&@2'B"14$3'72'"0'123452678'".3'"#46'4;6?4'8$.$@&B'&.@$%"B@&6.4'?&@;'
D1@".@'E6%D4'6E'/.012'".3'."/12)'''`4&.8'2$"4@'@?6L;27%&3'"."#24&4+'?$'3$D6.4@%"@$'
@;"@'@;$'=(KLI':%6@$&.'&.@$%"B@4'?&@;'@;$',O'17&]1&@&.'#&8"4$'B6D:6.$.@'=,/LIH+'
4188$4@&.8'"':644&7#$'D$B;".&4D'E6%',"%12LD$3&"@$3'41::%$44&6.)''=(KLI'&4'"'.6-$#'
:%6@$&.'@;"@'B6.@"&.4'"'05N'36D"&.+'".'$-6#1@&6."%&#2'B6.4$%-$3':%6@$&.L:%6@$&.'
&.@$%"B@&6.'36D"&.)''F$B"14$'#&@@#$'E1.B@&6."#'"."#24&4'B1%%$.@#2'$V&4@4'E6%'05N'36D"&.'
:%6@$&.4'&.';&8;$%'$1Z"%26@$4+'"."#24&4'6E'@;$'!:-#.#/"$,',"%12'8$.$'"@'@;$'#$-$#'6E'
D1@".@':;$.6@2:$'".3':%6@$&.L:%6@$&.'&.@$%"B@&6.':%6-&3$4'"'B6.@$V@'&.'?;&B;'@6'
$V:#6%$'@;$'%6#$'6E'"'4:$B&E&B'05N'36D"&.':%6@$&.'&.'D$@"b6".4)!
!
Hale et al.
9
G(Q,Y_(/='(MC'G,Q*KC='
2$>+(*'!+*/!9.*.$(%!L+*(=B,+$(#*'H!
(##'4@%"&.4'?$%$'3$%&-$3'E%6D'@;$'F%&4@6#'MJ'4@%"&.'".3'D"&.@"&.$3'"BB6%3&.8'
@6'4@".3"%3':%6B$31%$4'"@'JH6<'1.#$44'6@;$%?&4$'.6@$3'[FY,MM,Y'IWPS\)''G1@"@&6.4'
?$%$'41::#&$3'72'@;$'<"$.6%;"73&@&4'0$.$@&B4'<$.@$%'[`)'6E'G&..$46@"\'1.#$44'.6@$3+'
".3'"%$'#&4@$3'72'#&.Z"8$'8%61:'[/0\R''/0_R'5$=1>?3#297>8<'"0'12345267<-%&>@<-".3-
452>A8'[<)'061@@$\+'3#02698+'.*$1223$BCC8)'/0___R'5$=1A>3#2@?8+'/.0123($2@8<-3DAB8'[9)'
>&D7#$\+'3#>266-".3-#>26>8'[9)'^%&$44+'4$$'.6@$'7$#6?\+'3"&>9>8+''.*$12>3$A9>-".3-
$C7C$?A98:''/0_!R'."/123%E2A8)''/0!R')0F1223#>>68<-,D+1A3,=@8<-.%$1A3#>27B8<-,#.1
293$2977-".3-(=>6A8'[F)'<6.%"3@\+',#.1293"&628<-,"%123*G28'[@;&4'4@132\+',"%123%GAAAB8'
[!:-#.#/"$,'>.6BZ61@'<6.46%@&1D\+'5$=17C3#?228<-*+;H>-".3-$;H6>:''gR',#.12>3"&2A2<-
"&272<-+F228'".3'@;$'7"#".B$%''I>J(.*163#?A78-.#+1K3'CC28h'[_d___\)'N6%'=M^'D"::&.8+'
@;$'?&#3L@2:$'*"?"&&".'4@%"&.'<FSUTe'?"4'14$3)''Q;$'E6##6?&.8'%$:6%@$%'B6.4@%1B@4'
?$%$'14$3'E6%'B$##'&3$.@&E&B"@&6.R'4E012LL/H0'%$:6%@$%'E6%'".B;6%'B$##4[A(M0'".3'
=Q,YMF,Y0'JHHH\+'=%/1>LL/H0'%$:6%@$%'E6%'1@$%&.$':&'B$##4[*(MM(LYK=,'".3'*(M'IWWW\+'
".3'+0'12LL/H03MNO-#+-".:->9998'E6%'*=M4)''Q;$'/.0123#>2668'"##$#$'14$3';$%$'?"4'
67@"&.$3'E%6D'@;$'^%&$44'#"7'?;$%$'&@'?"4'D&44'%$E$%%$3'@6'"4'/.0123#>2628'[C(/NK-#+-
".:'JHIH\d'?$'4$]1$.B$3'61%'"##$#$'@6'B6.E&%D'&@4'&3$.@&@2'"4'8#:LI[#>2668'[BJPUT@'
D1@"@&6.\+'".3'14$'@;$'B6%%$B@'.6D$.B#"@1%$';$%$)'
'
M500&#,,%&-,=&##$L'5$=1>?3#297>8-"0'123452678-.*$122'3$BCC8P-'I>-;$@$%6b28614'
;$%D":;%63&@$4'?$%$'D1@"8$.&b$3'?&@;',G='".3'"##6?$3'@6'4$#ELE$%@&#&b$)''A;$.'@;$'
Hale et al.
10
NJ':6:1#"@&6.';"3'%$"B;$3'@;$'/S'4@"8$+'I'D#'6E'IHDG'#$-"D&46#$'[=&8D"'/WPTe\'?"4'
"33$3'@6'$"B;':#"@$'@6'$.%&B;'E6%';6D6b28614'`.BLJW-NJ'&.3&-&31"#4d':#"@$4'?$%$'
4B%$$.$3'6.$'@6'@?6'3"24'#"@$%'E6%'7"84'6E'?6%D4+'&.3&B"@&-$'6E';&8;':%68$.2'41%-&-"#)''
N%6D'IH+SHH'D1@"8$.&b$3';":#6&3'8$.6D$4+'4&V'$V@%"8$.&B'D1@"@&6.4+'B6%%$4:6.3&.8'
@6'E61%'6%'E&-$'8$.$4+'?$%$'E61.3'@6'3%"D"@&B"##2'&.B%$"4$'@;$':$%B$.@'6E'-&"7#$'
:%68$.2)''Q;$'*G2'D1@"@&6.'?"4'61@B%644$3'"@'#$"4@'E&-$'@&D$4'7$E6%$'"."#24&4)''Q;$'
3$#$@&6.'"##$#$'%GAAAB'?"4'8$.$%"@$3'72'@;$'!:-#.#/"$,'>.6BZ61@'<6.46%@&1D+'
KZ#";6D"'G$3&B"#'Y$4$"%B;'N61.3"@&6.)'A$'4$]1$.B$3'@;$',"%12'8$.$'&.'
;6D6b28614'%GAAAB'".&D"#4'".3'E61.3'@;"@'eWI'7:'"%$'3$#$@$3'?&@;&.'@;$',"%12'KYN+'
&.B#13&.8'D64@'6E'$V6.'6.$'".3'"##'6E'$V6.4'@?6+'@;%$$+'".3'E61%)''''
'
I#,+*$/-+'#-E"+#&$".-#HH#=+-%H-,"%12L''Q6'3$@$%D&.$'&E'$D7%26.&B',"%12'8$.6@2:$':#"24'"'
%6#$'&.'@;$'41%-&-"#'6E'"0'123452678'$D7%264+'#&-$':%68$.2'?$%$'B6##$B@$3'E%6D'
:"%@&"##2'41::%$44$3'"0'123452678d',"%12ai'D6@;$%4'".3'@;$&%'8$.6@2:$'?"4'
3$@$%D&.$3'7"4$3'6.'@;$':%$4$.B$'6E'@;$')0F122'D"%Z$%'".3'6.'@;$'8$.$%"@&6.'6E'#&-$'
:%68$.2'&.'@;$'.$V@'8$.$%"@&6.)''_.'"##'$V:$%&D$.@4+';6D6b28614',"%12'D1@".@'
$D7%264'%$:%$4$.@$3'%618;#2'JTj'6E'@;$'#&-$':%68$.2'"4'3&3';6D6b28614',"%12[i\'
:%68$.2+'&.3&B"@&.8'@;"@'@;$'3$8%$$'6E'41::%$44&6.'?"4'3$@$%D&.$3'72'@;$'D"@$%."#'
8$.6@2:$'%"@;$%'@;".'@;$'$D7%26.&B'8$.6@2:$)''N6%'$V"D:#$+'@;$'#&-$':%68$.2'E%6D'
5$=1>?3#297>8-"0'123452678d')0F1223#>>68',"%123*G28ai'D6@;$%4'?$%$'Jej'C:2'="6+'
Jej'.6.LC:2'.6.L="6+'".3'SUj';$@$%6b28614'[.'k'PP\d'&.'"'4$:"%"@$'$V:$%&D$.@+'
Jej'6E'@;$'#&-$':%68$.2'E%6D'5$=1>?-3#297>8-"0'123452678d',"%123%GAAAB8P)0F1
223#>>68-D6@;$%4'?$%$';6D6b28614'C:2'[.kJHTT\)''^"@$%."#'?&#3L@2:$',"%123Q8'3&3'
Hale et al.
11
.6@'3$B%$"4$'@;$'41::%$44&6.'@;"@'D"@$%."#-,"%123*G28'&D:64$3'6.'"0'123452678'
$D7%264R';$%D":;%63&@$4'6E'8$.6@2:$'5$=1>?3#297>8-"0'123452678R-)0F1223#>>68-
,"%123*G28'2&$#3$3'UJj'-&"7#$'4$#EL:%68$.2'[.kSWe\'B6D:"%$3'@6'UHj'-&"7#$'B%644L
:%68$.2'[.kOWT+'"##'.6.LC:2\'"E@$%'D"@&.8'@6'D"#$4'6E'8$.6@2:$'5$=1>?3#297>8-"0'1
23452678:'
'
S'#$%+F0*=-T$".F,*,L'"0'12'D1@".@'4@%"&.4'?$%$'D"&.@"&.$3'"4';$@$%6b286@$4'?&@;'@;$'
E6##6?&.8'8$.6@2:$R'5$=1>?3#297>8-"0'12P'I>J(.*163#?A78-.#+1K3'CC28h+'".3'
;6D6b28614'"0'12'".&D"#4'?$%$'4$#$B@$3'E6%':;$.6@2:&B'"."#24&4'72'4$#$B@&.8'?6%D4'
?&@;'@;$'`.BLJW':;$.6@2:$'[;6D6b28614''I>'".&D"#4'"%$'$D7%26.&B'#$@;"#'31$'@6'@;$'
'CC2'D1@"@&6.'&.'".'1.&3$.@&E&$3'8$.$\)''_.'"##'B"4$4'@$4@'4@%"&.4'?$%$':%$:"%$3'".3'
4B6%$3'4&D1#@".$614#2'?&@;'@;$'%$#$-".@'B6.@%6#'4@%"&.)''!&"7#$':%68$.2'?$%$'
D$"41%$3'72'B61.@&.8'$884'#"&3'6-$%'"'TLU';61%'&.@$%-"#+'B61.@&.8'@;$'.1D7$%'6E'
1.;"@B;$3'$884'JHLJS';%4'#"@$%+'".3'B61.@&.8'@;$'.1D7$%'6E'-&"7#$'".&D"#4'@;"@'
%$"B;$3'"@'#$"4@'@;$'/S'4@"8$'"E@$%'".'"33&@&6."#'SULeH';61%4'[@&D$4'?$%$'&.B%$"4$3'
E6%'IT6<'$V:$%&D$.@4'".3'3$B%$"4$3'E6%'JT6<'$V:$%&D$.@4'"BB6%3&.8#2\d'$884'?$%$'
"."#2b$3'E%6D'"'D&.&D1D'6E'IH'3&EE$%$.@'D6@;$%4'E6%'$"B;'8$.6@2:$)''^%68$.2'
-&"7&#&@2'?"4'B"#B1#"@$3'"4'@;$':%6:6%@&6.'6E'@6@"#'$884'#"&3'@;"@'8%$?'@6'"@'#$"4@'@;$'/S'
4@"8$)''*"@B;&.8'%"@$'?"4'B"#B1#"@$3'"4'@;$':%6:6%@&6.'6E'$D7%264'@;"@'41BB$44E1##2'
;"@B;'[3$31B$3'E%6D'@;$'B61.@$3'.1D7$%'6E'1.;"@B;$3'$D7%264\)''/"%-"#'-&"7&#&@2'
?"4'B"#B1#"@$3'"4'@;$':%6:6%@&6.'6E';"@B;$3'$D7%264'@;"@'8%$?'&.@6'-&"7#$'/Sa"31#@4)''
,D7%264'?$%$'674$%-$3'72'M6D"%4Z&'G&B%64B6:2'[f$&44'(V&6:#".\'E6%'D$"41%$D$.@4'
6E'$D7%26.&B'D6%:;68$.$4&4'".3':;"%2.V'E6%D"@&6.d'@;$'B%&@$%&"'14$3'E6%'4B6%&.8'".'
Hale et al.
12
$D7%26'"4';"-&.8'41BB$44E1#'D6%:;68$.$4&4'?"4'@;$'E1##'$.B#641%$'72';2:63$%D"#'
B$##4+'%$8"%3#$44'6E'4#&8;@'"7.6%D"#'4;":$4)''N6%'@$D:$%"@1%$'4$.4&@&-$'/.012'".3'."/12'
"##$#$4+'4@%"&.4'?$%$'D"&.@"&.$3'"@'@;$':$%D&44&-$'@$D:$%"@1%$'1.@&#';"@B;&.8+'"@'
?;&B;':6&.@'@;$2'?$%$'8%6?.'"@'@;$'&.3&B"@$3'$V:$%&D$.@"#'@$D:$%"@1%$)''N6%'
$D7%26.&B'"44$44D$.@'6E'4@%"&.4'@;"@'"%$'4@$%&#$'"@'@;$'%$4@%&B@&-$'@$D:$%"@1%$+'41B;'
"4'/.0123($2@8<-/.0123#>2668<-"$)-/.0123"&>9>8+'$D7%264'?$%$'67@"&.$3'72'"##6?&.8'
D6@;$%4'@6'3$-$#6:'"@'@;$':$%D&44&-$'@$D:$%"@1%$'1.@&#'D&3'/S'4@"8$'@6'"##6?'46D$'
8$%D#&.$':%6#&E$%"@&6.d'"E@$%'IJLJS';61%4'6E'"BB#&D"@&6.'"@'@;$'%$4@%&B@&-$'
@$D:$%"@1%$+'$D7%264'?$%$'@;$.'B6##$B@$3'".3'"44"2$3'"4'"76-$)''=@$%&#&@2'?"4'
D$"41%$3'72'8%6?&.8'4@%"&.4'"@'@;$'&.3&B"@$3'@$D:$%"@1%$'E%6D'@;$'@&D$'6E';"@B;&.8d'
&.3&-&31"##2':#"@$3'".&D"#4'?$%$'4B6%$3'"4'E$%@&#$'&E'".2'$D7%264'?$%$'D"3$+'".3'@;$'
8$.$%"#'"::$"%".B$'6E'@;$&%'86."34'1.3$%'7%&8;@E&$#3'D&B%64B6:2'?"4'.6@$3)'
'
L#,.%B,+>!M/.*$(N(%+$(#*!#N!)'*+,-!
Q;$'*G2'D1@"@&6.'?"4'D"::$3'@6'B;%6D646D$'!+'"@'T)T'D1'E%6D')0F1223#>>68:''
[TaSe'*G2';6D6b286@$4'E%6D'*G2P)0F122':&BZ$3'1:'@;$')0F122\)''F%&4@6#a*"?"&&".'
%$B6D7&.".@4'?$%$'B6##$B@$3'E6%'=M^'"."#24&4'E%6D'@;$';$@$%6b28614'4@%"&.',D+1A3,=@8'
,"%123*G28'5$=17C3#?228-P-Q'[*"?"&&".'E%6D'<FSUTe\)'''Ie'Y6#L.6.L`.B'%$B6D7&.".@4'
?$%$'&3$.@&E&$3+'".3';"#E'6E'@;$4$'%$B6D7&.$3'@6'@;$'%&8;@'6E'*G2'".3';"#E'@6'@;$'#$E@)''
Q;$4$'Ie'%$B6D7&.".@4'?$%$'"."#2b$3'?&@;'=M^4'&.'@;$',D+1A-U-5$=17C'%$8&6.d'*G2'?"4'
E61.3'@6'#&$'7$@?$$.'=M^'NTTFIJlIh'".3'=M^'NTU,IHlOh+'".'&.@$%-"#'6E'IPT'>F+'?&@;'
*G2'D"::&.8'@6'@;$'D&33#$'6E'@;$'&.@$%-"#'[Oae'%$B6D7&.".@4'7$@?$$.'=M^'NTTFIJlIh'
".3'*G2+'".3'Oae'7$@?$$.'*G2'".3'=M^'NTU,IHlOh\)''Q?6'B64D&34'D":'@6'@;$'B$.@$%'6E'
Hale et al.
13
@;&4'IPTZ7'%$8&6.R''YIHCIJ'".3'<HeFO'[41::#&$3'72'@;$'A$##B6D$'Q%14@'=".8$%'
_.4@&@1@$+'<"D7%&38$+'`>\)''Q;$4$'?$%$'&.m$B@$3'&.@6'5$=1>?3#297>8'"0'123452678',"%1
23*G28'?6%D4'["4':%$-&614#2'3$4B%&7$3'&.'[0K`QQ,-#+-".:'JHHJ\'@6'#66Z'E6%'".@&L
41::%$44&6.)''(#@;618;'4D"##'"D61.@4'6E'3$"3'$D7%264'?$%$'6BB"4&6."##2'674$%-$3'&.'
4@%"&.4'B6.@"&.&.8'@%".48$.$4'B"%%2&.8'$&@;$%'B64D&3+'4$-$%"#'YIHCIJ'@%".48$.&B'
?6%D4'?$%$'E61.3'@6'8&-$'OH'n'THj'3$"3'$D7%264)''<".3&3"@$'KYN4'?&@;&.'@;$'
YIHCIJ'B64D&3'%$8&6.'?$%$'@;$.'"."#2b$3'72'YM(&'E6%'@;$'"7&#&@2'@6'41::%$44'@;$'
D"@$%."#L$EE$B@'#$@;"#&@2'6E'"0'123452678';6D6b28614'".&D"#4)''IHLIT';6D6b28614'
5$=1>?3#297>8'"0'123452678'/S'?6%D4'?$%$'&.m$B@$3'?&@;'3617#$'4@%".3$3'YM('
B6%%$4:6.3&.8'@6'6.$'6E'E61%'KYN4R''YIHCIJ)J+'YIHCIJ)U'YIHCIJ)IO'".3'YIHCIJ)IS)''
C617#$'4@%".3$3'YM('?"4'8$.$%"@$3'72'%1.'6EE'@%".4B%&:@&6.'E%6D'QPL7&.3&.8L4&@$'
@"88$3'^<Y'@$D:#"@$4'14&.8'(D:#&4B%&7$'QP'*&8;'5&$#3'Q%".4B%&:@&6.'>&@'E%6D'
,:&B$.@%$'[:%&D$%'4$]1$.B$4'"-"&#"7#$'1:6.'%$]1$4@\+'".3'&.m$B@$3'"@'"::%6V&D"@$#2'
Io8ao#'34YM()''_.'@;$'E&%4@'@;%$$'B"4$4+'"0'123452678'?6%D4'B6.@&.1$3'@6'8$.$%"@$'
D64@#2'3$"3'$D7%264d'&.'B6.@%"4@+'?6%D4'&.m$B@$3'?&@;'YIHCIJ)IS'YM('2&$#3$3'"'
4@%&Z&.8'.1D7$%'6E'#&-$':%68$.2+'?;&B;'@;$.'8%$?'1:'@6'2&$#3'#&-$':%68$.2'@;$D4$#-$4)'''
Q?6'&.3$:$.3$.@'BCM(4'B6%%$4:6.3&.8'@6'@;$'YIHCIJ)IS'KYN'?$%$'B#6.$3'E%6D'$"B;'
6E'?&#3L@2:$'".3',"%123*G28'D1@".@'?6%D4'[D&V$3'4@"8$\R''Q6@"#'BCM('?"4'8$.$%"@$3'
72'YQL^<Y'14&.8'_.-&@%68$.c4'=1:$%4B%&:@'___'>&@'".3'@;$.'14$3'"4'@$D:#"@$'E6%'^<Y'
?&@;'<#6.$@$B;'Q&@".&1D'Q"]'^6#2D$%"4$'14&.8''=/I'".3'=/J':%&D$%4[=^_,Q*-#+-".:'
IWWO\'&.'B6D7&."@&6.'?&@;'"':%&D$%'@;"@'6-$%#":4'@;$':%$3&B@$3'4@6:'B636.'6E'
YIHCIJ)IS'[:%&D$%'4$]1$.B$4'"-"&#"7#$'1:6.'%$]1$4@\)'_.'76@;'B"4$4+':%631B@4'?$%$'
&3$.@&E&$3'?&@;'@;$'4:#&B$3'#$"3$%'4$]1$.B$'=/J+'71@'.6@'?&@;'=/I)''=$]1$.B$'"."#24&4'
Hale et al.
14
6E'@;$4$'E61%'&.3$:$.3$.@'BCM('B#6.$4'B6.E&%D$3'@;$'&.@%6.a$V6.'m1.B@&6.4':%$3&B@$3'
72'0,M,N_MC,Y'".3'B6.E&%D$3'72',=Q4'&3$.@&E&$3'E6%'@;$'YIHCIJ)IS'KYN'
[;@@:Raa?6%D7"4$)6%8\)''0,M,N_MC,Y':%$3&B@4'E61%'-$%4&6.4'6E'4:#&B$3'YIHCIJ)IS'
:%631B@4+'@$%D$3'"'@;%618;'3d'61%'4$]1$.B$4'B6%%$4:6.3'@6'YIHCIJ)IS"+'B6.@"&.&.8'
4&V'$V6.4)''Q;$'6.#2'3&EE$%$.B$'7$@?$$.'@;$'?&#3L@2:$'".3'@;$'*G2'B63&.8'%$8&6.'6E'
YIHCIJ)IS'?"4'"'4&.8#$'.1B#$6@&3$'B;".8$'@;"@'%$41#@4'&.'"'/$1'@6'^;$'B;".8$'"@'
:%$3&B@$3'"D&.6'"B&3'TW''[B636.'<QQ'@6'QQQ\)'
'
:.+'$!G?#!O10>(/!3*+,1'('H!
Q;$'/$V(L7"4$3'2$"4@'@?6';27%&3'424@$D'?&@;'31"#'%$:6%@$%4'[."=N'".3'VWMA\'
?"4'14$3[*K//,MF,Y0-#+-".:'IWWT\)''5$"4@'4@%"&.4+'(GYPH'".3'/SH+'".3':#"4D&34'E6%'
71&#3&.8'E14&6.'B6.4@%1B@4'?$%$'41::#&$3'72'^$@$%'^%2B&"ZR':0(CLg^'E6%'(B@&-"@&6.'
C6D"&.'E14&6.4'".3':FQGIIe'E6%'CM('F&.3&.8'C6D"&.'E14&6.4'[^Y5<_(>'".3'
*(YQA,//'IWWe\)''BCM('E6%',#.129'?"4':%6-&3$3'72'9".$'*177"%3'".3'_-"'0%$$.?"#3)''
.*$1>A'BCM('?"4':%6-&3$3'72'K#&-$%'*67$%@)''BCM('4$]1$.B$4'?$%$'^<Y'"D:#&E&$3'
14&.8':%&D$%4'@;"@'&.@%631B$3'".'XE"_'%$4@%&B@&6.'4&@$'"@'@;$'Tc'$.3'6E'"':%6@$&.'B63&.8'
%$8&6.'6%'"'M"._'%$4%&B@&6.'4&@$'"@'@;$'Oc'$.3'6E'@;$'%$8&6.'[:%&D$%'4$]1$.B$4'"-"&#"7#$'
1:6.'%$]1$4@\)''Y$4@%&B@$3'E%"8D$.@4'?$%$'#&8"@$3'&.@6'XE"_LM"._LB1@':0(CLg^'6%'
:FQGIIe)''(##'(C'B6.4@%1B@4'?$%$'@%".4E6%D$3'&.@6'/SH+'".3'"##'CFC'B6.4@%1B@4'?$%$'
@%".4E6%D$3'&.@6'(GYPHd'3$4&%$3':"&%?&4$'B6D7&."@&6.4'?$%$'B%$"@$3'72'D"@&.8'
@%".4E6%D".@4'".3'4$#$B@&.8'E6%'3617#2'@%".4E6%D$3'3&:#6&34'E6%'"."#24&4)''VWMA'
$V:%$44&6.'?"4'"."#2b$3'72'8%6?&.8'6.'n*_='46#&3'D$3&1D'41::#$D$.@$3'?&@;'H)JTG'
6%'I)JTG'OL"D&.6@%&"b6#$)''/&]1&3'!L8"#"B"@64&3"4$'"44"24'?$%$'B"%%&$3'61@'"BB6%3&.8'
Hale et al.
15
@6'4@".3"%3':%6B$31%$4[^Y5<_(>'".3'*(YQA,//'IWWe\)''5$"4@'$V@%"B@4'?$%$'D"3$'E%6D'
$V:6.$.@&"##2'8%6?&.8'B1#@1%$4'6E'@;$'3&:#6&3'4@%"&.4'14$3'&.'."=N'"44"24+'14&.8'@;$'
:%6B$31%$'6E'>%14;.&%6-'$@'"#)'[>`=*M_YK!'JHHH\)''<$##'#24"@$'B6.B$.@%"@&6.4'?$%$'
$]1"#&b$3'"BB6%3&.8'@6'KCeHH'B1#@1%$'D$"41%$D$.@4'".3'$]1"#'-6#1D$4'?$%$'
E%"B@&6."@$3'6.'IHLIJj':6#2"B%2#"D&3$'Q%&4a0#2B&.$'8$#4)''A$4@$%.'7#6@'"."#24&4'?"4'
:$%E6%D$3'14&.8'".@&763&$4'"8"&.4@'@;$'0"#S'(B@&-"@&6.'C6D"&.'[G&##&:6%$'pHeLJUO\+'
@;$'/$V('CM('F&.3&.8'C6D"&.'[G&##&:6%$pHeLPIW\+'".3'@;$'2$"4@'<3BJU'^%6@$&.'
[=".@"'<%1b'F&6@$B;'p4BLePHW\'"4'"'B6.@%6#'E6%':%6@$&.'#$-$#4'&.'3&EE$%$.@'2$"4@'
$V@%"B@4)''<;$D&B%6D$'[QG\'D6#$B1#"%'?$&8;@'D"%Z$%4'[=&8D"\'?$%$'14$3'E6%'4&b$'
4@".3"%34)'',V@%"B@4'6E'2$"4@'4@%"&.4'$V:%$44&.8'=(KLI[i\'".3'=(KLI[&ZI\'E14&6.4'?$%$'
B6D:"%$3'&.'D6%$'@;".'IH'&.3$:$.3$.@#2':%$:"%$3'$V@%"B@4+'46D$'E%6D';":#6&3'B$##4'
$V:%$44&.8'6.#2'@;$'=(KLI'(C'E14&6.'B6.4@%1B@+'".3'46D$'E%6D'3&:#6&3'B$##4'
$V:%$44&.8'@;$'=(KLI'(C'E14&6.'"4'?$##'"4'"'=,/LIH'CFC'E14&6.'["4'4;6?.'&.'N&81%$'
O<\d'"#@;618;'@;$%$'?"4'46D$'4@%"&.L&.3$:$.3$.@'-"%&"7&#&@2'E%6D'$V@%"B@'@6'$V@%"B@+'
@;$%$'?"4'.6'B6.4&4@$.@'3&EE$%$.B$'7$@?$$.'=(KLI[i\'".3'=(KLI[&ZI\'E14&6.':%6@$&.4)'
Hale et al.
16
Y,=`/Q='
'
A$'&46#"@$3'4&V'$V@%"8$.&B'D1@"@&6.4'&.'"'4B%$$.'E6%'41::%$44&6.'6E'"0'1
23452678'D"@$%."#L$EE$B@'#$@;"#&@2'[4$$'G$@;634\+'".3':%$4$.@';$%$'61%'"."#24&4'6E'6.$'
6E'@;$4$'D1@"@&6.4+'*G2+'".3'@;$'.6-$#'8$.$'&@'3$E&.$4+',"%12'[,1::%$446%'6E'":;L%.$\)''
A$'"#46'67@"&.$3'E%6D'@;$'!:-#.#/"$,'0$.$'>.6BZ61@'<6.46%@&1D'@;$'3$#$@&6.'"##$#$'
,"%123%GAAAB8+'".3'&.B#13$'@;&4'D1@"@&6.'&.'61%'"."#24&4'6E',"%12'E1.B@&6.)'
'
F./B%./!)'*+,!+%$(8($1!'B==>.''.'!'./+,012,345#.<0>1#*(%!,.$&+,($1H!!!
Q;$':"%@&"#'#644L6ELE1.B@&6.'"##$#$'"0'123452678'B"14$4'".'&.B6D:#$@$#2'
:$.$@%".@'D"@$%."#L$EE$B@'$D7%26.&B'#$@;"#&@2R''"#D64@'"##':%68$.2':%631B$3'72'".'
"0'123452678';$%D":;%63&@$'"%%$4@'"4'#"@$L4@"8$'$D7%264'6%';"@B;#&.84'?&@;'@;$'
;"##D"%Z'M6@B;':;$.6@2:$'6E'"'D&44&.8'".@$%&6%':;"%2.V+'&.3&B"@&.8'E"&#$3'M6@B;'
4&8."#&.8'"@'@;$'IJLB$##'4@"8$'[N&81%$'I('".3'0K`QQ,-#+-".:'JHHJd'^Y_,=='JHHT\)''_.'4;"%:'
B6.@%"4@+'"0'123452678R-,"%123*G28';6D6b28614'?6%D4':%631B$'-&%@1"##2'"##'-&"7#$'
:%68$.2'[Q"7#$'I\)''=1::%$44&6.'6E'@;$'"0'123452678'$D7%26.&B'3$E$B@'&4'$]1"##2'
3%"D"@&B'?&@;'@;$'3$#$@&6.'"##$#$',"%123%GAAAB8'[3$4B%&7$3'7$#6?\'".3'72',"%12'YM(&'
[Q"7#$'I'".3'N&81%$'IF\+'&.3&B"@&.8'@;"@'@;$'M6@B;'3$E$B@'6E'"0'123452678'$D7%264'&4'
6-$%B6D$'72'%$D6-&.8',"%12-?&#3L@2:$'E1.B@&6.)''^"%@&"#'41::%$44&6.'&4'674$%-$3'&.'
".&D"#4'@;"@'"%$';$@$%6b28614'E6%'$&@;$%',"%123*G28+',"%123%GAAAB8+'6%'@;$'
B;%6D646D"#'3$#$@&6.'*+;H>'[Q"7#$'I'".3'4$$'N&81%$'J(\)''Q;$4$'%$41#@4'4188$4@'@;"@'
@;$',"%12'#6B14'&4';":#6&.41EE&B&$.@+'41B;'@;"@'$-$.'"'4D"##'%$31B@&6.'&.',"%12'E1.B@&6.'
&4'$EE$B@&-$'"@'"0'123452678'41::%$44&6.)''Q;$',"%123*G28'"##$#$'D14@'%$@"&.'46D$'#6?'
Hale et al.
17
#$-$#'6E',"%12-"B@&-&@2'7$B"14$'41::%$44&6.'&.',"%123*G28';$@$%6b286@$4'&4'.6@'"4'4@%6.8'
"4'&.'3$#$@&6.';$@$%6b286@$4'[Q"7#$'I\)''(."#24&4'6E'$D7%264'E%6D',"%12-;$@$%6b28614'
".&D"#4'$4@"7#&4;$4'@;"@'&@'&4'D"@$%."#+'%"@;$%'@;".'b286@&B+',"%12'"B@&-&@2'@;"@'
&.E#1$.B$4'$D7%26.&B'41%-&-"#'[4$$'G$@;634\)''Q"Z$.'@68$@;$%'@;$4$'8$.$@&B'"."#24$4'
4188$4@'@;"@'@;$'=(KLI':%6@$&.';"4'"'.$8"@&-$'$EE$B@'6.'"0'123452678'"B@&-&@2'&.'@;$'
$"%#2'$D7%26)''
G6%$'4$-$%$'"##$#$4'6E'"0'12'[452>A<-%&>@<-".3-#0269\'B"14$'E1##2':$.$@%".@'
$D7%26.&B'#$@;"#&@2'&.'?;&B;'@;$'$D7%264'.6@'6.#2'#"BZ'".'".@$%&6%':;"%2.V'[31$'@6'
E"&#$3'M6@B;'4&8."#&.8'"@'@;$'IJLB$##'4@"8$\+'71@'"#46'E"&#'@6'B6D:#$@$'7632'
D6%:;68$.$4&4'7$B"14$'6E'E"&#$3'M6@B;'4&8."#&.8'"@'@;$'SLB$##'4@"8$'[N&81%$'I<'".3'4$$'
[0K`QQ,-#+-".:'JHHJd'^Y_,=='JHHT\)''M$&@;$%',"%123*G28'.6%',"%123%GAAAB8'B".'%$4@6%$'
$D7%26.&B'-&"7&#&@2'@6'@;$4$'"0'12'D1@".@4'[3"@"'.6@'4;6?.\)''*6?$-$%+'@;$',"%1
23%GAAAB8'D1@".@'7"BZ8%61.3'"##6?4'&D:%6-$3'7632'D6%:;68$.$4&4'E6%'@;$'?$"Z$4@'
6E'@;$4$'"##$#$4+'"0'123452>A8-[N&81%$'IC'".3'Q"7#$'J\'?;&B;'&4'.6@'B6D:#$@$#2'3$-6&3'
6E'"0'12'"B@&-&@2'[0K`QQ,-#+-".:'JHHJ\)''(#@;618;'46D$'"0'123452>A8-,"%123%GAAAB8'
$D7%264'$V;&7&@'41BB$44E1#'7632'D6%:;68$.$4&4+'.6.$'6E'@;$4$'$D7%264'&.31B$'".'
".@$%&6%':;"%2.V+'&.3&B"@&-$'6E':$%4&4@&.8'M6@B;'4&8."#&.8'3$E$B@4'"@'@;$'IJLB$##'4@"8$'
[.kWS\)''Q;14+'%$D6-&.8',"%12-"B@&-&@2'"::$"%4'@6'&.B%$"4$'M6@B;'4&8."#&.8'"B@&-&@2'&.'
$"%#2'"0'123452>A8'$D7%264'@6'"##6?'46D$'41BB$44E1#'M6@B;'4&8."#&.8'"@'@;$'SLB$##'
4@"8$+'71@'.6@'@6'#$-$#4'@;"@'"%$'41EE&B&$.@'E6%'"##'M6@B;'4&8."#&.8'$-$.@4'%$]1&%$3'E6%'
$D7%26.&B'-&"7&#&@2)'<6##$B@&-$#2+'61%'%$41#@4'4188$4@'@;"@'%$31B&.8',"%12'E1.B@&6.'B".'
Hale et al.
18
$#$-"@$'M6@B;'4&8."#&.8'&.'$D7%264'?&@;'3$B%$"4$3'71@'.6@'$.@&%$#2'"74$.@'"0'12'
"B@&-&@2)'
'
_.'".'6@;$%?&4$'?&#3L@2:$'7"BZ8%61.3+',"%123*G28-".3-,"%123%GAAAB8-36'.6@'
B"14$'-&4&7#$'3$#$@$%&614'$EE$B@4'1.3$%'4@".3"%3'8%6?@;'B6.3&@&6.4)''*6?$-$%+'?$'
674$%-$'4$-$%"#'&.B6D:#$@$#2':$.$@%".@':;$.6@2:$4'"D6.8'".&D"#4'@;"@'"%$'
;6D6b28614'E6%'@;$'3$#$@&6.'"##$#$',"%123%GAAAB8'".3'%"&4$3'"@'@;$'$#$-"@$3'
@$D:$%"@1%$'6E'JT6<)''Q;$4$'&.B#13$'$D7%26.&B'#$@;"#&@2'[76@@6D'%6?'6E'Q"7#$'O\+'
%$31B$3'7%663'4&b$+'$88'%$@$.@&6.+'".3'&.B%$"4$3'&.B&3$.B$'6E'D"#$4'[3"@"'.6@'4;6?.\)''
Q;$4$':;$.6@2:$4'"%$'#&Z$#2'@6'7$'"446B&"@$3'?&@;'%$D6-"#'6E',"%12'E1.B@&6.'7$B"14$'
?$'?$%$'1."7#$'@6'4$8%$8"@$'@;$D'"?"2'E%6D'@;$'"0'12'41::%$44&6.':;$.6@2:$'6E'
,"%123%GAAAB8'"E@$%'IO'%61.34'6E'61@B%644&.8+'&.B#13&.8'@?6'%$B6D7&."@&6.'$-$.@4'
?&@;&.'"'I)T'D1'3&4@".B$'6.'$"B;'4&3$'6E',"%123%GAAAB8)''N1%@;$%D6%$+'?$'.6@$'@;"@'
@;$'&.3$:$.3$.@#2L&46#"@$3'*G2'"##$#$'"#46'$V;&7&@4'"'-$%2'#6?+'@;618;'4&8.&E&B".@'
$D7%26.&B'#$@;"#&@2'"@'JTq<'".3'$-$.'D6%$'"@'JPq<'[76@@6D'%6?'6E'Q"7#$'O'".3'3"@"'
.6@'4;6?.\)''Q;$'$V&4@$.B$'6E'@;$4$':;$.6@2:$4'"@';&8;'@$D:$%"@1%$4'&.3&B"@$4'@;"@'
,"%12'E1.B@&6.'&4'.6@'$44$.@&"#'1.3$%'.6%D"#'8%6?@;'B6.3&@&6.4+'71@'&@4'"B@&-&@2'D"2'
7$B6D$'B%&@&B"#'1:6.'4@%$44d'@;$'4:$B&E&B'."@1%$'6E'@;$4$':;$.6@2:$4'&4'7$&.8'
&.-$4@&8"@$3'4$:"%"@$#2)'
'
)'*+,067,5!'B==>.''.'!+!<B$+$(#*!(*!$&.!"#$%&!/#?*'$>.+<!.NN.%$#>!%'&+,H''
_.'6%3$%'@6':%67$'@;$'4:$B&E&B&@2'6E',"%12'E1.B@&6.+'?$'"4Z$3'?;$@;$%',"%12'
D1@"@&6.4'B61#3'&.E#1$.B$'M6@B;'4&8."#&.8'?;$.'B6D:6.$.@4'6@;$%'@;".'"0'12'?$%$'
Hale et al.
19
B6D:%6D&4$3)''Q;$'36?.4@%$"D'M6@B;'@%".4B%&:@&6."#'$EE$B@6%'/(0LI'&4':"%@&"##2'
&."B@&-"@$3'72'@;$'@$D:$%"@1%$'4$.4&@&-$'D1@"@&6.'."/123%E2A8<-?;&B;'&.@%631B$4'"'
4&.8#$'"D&.6'"B&3'B;".8$'[(TTIQ+'C"-&3'0%$$.4@$&.+':$%46."#'B6DD1.&B"@&6.\'".3'
B"14$4'&.B6D:#$@$#2':$.$@%".@'M6@B;'4&8."#&.8'3$E$B@4'&.'76@;'$"%#2'".3'#"@$'
$D7%268$.$4&4'[r_(K-#+-".:'IWWT\)''(@'@;$'%$4@%&B@&-$'@$D:$%"@1%$4'6E'JHq<'6%'JTq<+'
%618;#2';"#E'6E'@;$'$D7%264'E%6D'."/123%E2A8'D6@;$%4'"%$'3$E$B@&-$'&.'$"%#2'M6@B;'
4&8."#&.8'$-$.@4'".3'@;14'E"&#'@6';"@B;)''Q;$'%$D"&.3$%'6E'@;$'$D7%264'"%$'41BB$44E1#'
&.'@;$4$'$"%#2'$-$.@4+'71@'E"&#'&.'#"@$%'$D7%26.&B'M6@B;'4&8."#&.8'$-$.@4'@;"@'3%&-$'
E6%D"@&6.'6E'@;$';$"3+'@;$'$VB%$@6%2'B$##+'@;$'".14+'".3'@;$'%$B@1Dd';$.B$'@;$4$'
$D7%264';"@B;'71@'"%%$4@'"4'#"%-"$'[/(GF_,'".3'>_GF/,'IWWId'r_(K-#+-".:'IWWT\)''A$'
E61.3'@;"@'@;$'41%-&-"#'6E':%68$.2'E%6D'."/123%E2A8';$%D":;%63&@$4'&4'4&8.&E&B".@#2'
&D:%6-$3'&.'"',"%123*G28'D1@".@'7"BZ8%61.3'[Q"7#$'O\)''Q;$'6-$%"##'&.B%$"4$'&.'
-&"7&#&@2'&4'@;$'B6D7&.$3'%$41#@'6E'"'4D"##'&.B%$"4$'&.';"@B;&.8'%"@$'".3'"'#"%8$'
&.B%$"4$'&.'#"%-"#'-&"7&#&@2)''N6%'$V"D:#$+'"@'JT6<+'TSj'6E'."/123%E2A8'$D7%264';"@B;'
-$%414'eOj'6E'."/123%E2A8d',"%123*G28'$D7%264'[@;&4'3&EE$%$.B$'&4'4&8.&E&B".@'
"BB6%3&.8'@6'"'@?6L@"&#$3'N&4;$%',V"B@'Q$4@R'S'-"#1$'s'H)HHHI+'4$$'Q"7#$'O'E6%'.'-"#1$4\'
".3'P)Oj'6E'."/123%E2A8';"@B;#&.84'"%$'-&"7#$'B6D:"%$3'@6'OOj'6E'."/123%E2A8d',"%1
23*G28';"@B;#&.84'[4&8.&E&B".@'3&EE$%$.B$'"BB6%3&.8'@6'"'@?6L@"&#$3'N&4;$%',V"B@'Q$4@R'S'
-"#1$'s'H)HHHI+'.keWP'".3'.kTSO+'%$4:$B@&-$#2\)''Q;$'41::%$44&6.'6E'76@;'$D7%26.&B'
".3'#"%-"#'#$@;"#&@2'4188$4@4'@;"@',"%12'&.E#1$.B$4'76@;'$"%#2'".3'#"@$'$D7%26.&B'M6@B;'
4&8."#&.8'$-$.@4)''A$'36'.6@'674$%-$'"'4&D&#"%'&D:%6-$D$.@'6E'."/123%E2A8'-&"7&#&@2'
&.'@;$',"%123%GAAAB8'D1@".@'7"BZ8%61.3+'@;618;'"44$44D$.@'"@'JT6<'B61#3'.6@'7$'
$"4&#2'&.@$%:%$@$3'31$'@6'@$D:$%"@1%$'4$.4&@&-$'#$@;"#&@2'&.@%631B$3'72'@;$',"%1
Hale et al.
20
23%GAAAB8'"##$#$'"@'@;&4'@$D:$%"@1%$'[Q"7#$'O'".3'4$$'.$V@'4$B@&6.\)''Q;$'8$.$@&B'
&.@$%"B@&6.'7$@?$$.',"%123*G28-".3-."/123%E2A8'4188$4@4'@;"@',"%12'"B@&-&@2'36?.'
%$81#"@$4'M6@B;'4&8."#&.8+'$-$.'&.'".'"0'123Q8'7"BZ8%61.3)'
)'*+,!(*N,B.*%.'!'()*+,(*)!%+=+%($1!#N!+,$.>./!9DP56!>.%.=$#>'!/B>(*)!
.<0>1#).*.'('H!!!
G1@".@'0/^LI'%$B$:@6%4'?$%$'@$4@$3'E6%'4$.4&@&-&@2'@6',"%12'"B@&-&@2'&.'@;$'$"%#2'
$D7%26)'''A$'"."#2b$3'@;$'$EE$B@'6E'@;%$$'#644L6ELE1.B@&6.[#E\'"##$#$4'[#>26><'#>266-".3-
($2@R-^Y_,==-#+-".:'IWUPd'>KCK5_(MM_-#+-".:'IWWJ\'".3'6.$';2:$%"B@&-$'"##$#$-3"&>9>R'
^,^^,Y-#+-".:'JHHO\:--(#6.$+'$"B;'6E'@;$4$'@$D:$%"@1%$L4$.4&@&-$'D1@"@&6.4'B"14$4'
$D7%26.&B'#$@;"#&@2'"@'&@4'%$4@%&B@&-$'@$D:$%"@1%$+'&.3&B"@&.8'@;"@'&.41EE&B&$.@'6%'
$VB$44&-$'0/^LI'"B@&-&@2'3&4%1:@4'$D7%268$.$4&4'[F,YY5-#+-".:'IWWPd'*`QQ,Y'".3'
=<*M(F,/'IWWSd'>KCK5_(MM_-#+-".:'IWWJd'^,^^,Y-#+-".:'JHHOd'^Y_,==-#+-".:'IWUP\)''A$'
E61.3'@;"@',"%12'D1@"@&6.4'4&8.&E&B".@#2'&.B%$"4$3'@;$'3$8%$$'6E'$D7%26.&B'#$@;"#&@2'
674$%-$3'&.'"##'E61%'/.012'D1@".@4'"@':$%D&44&-$'6%'4$D&L:$%D&44&-$'@$D:$%"@1%$4'
[Q"7#$'O\)''Q;$4$'8$.$@&B'&.@$%"B@&6.4'B#$"%#2':6&.@'@6'".'$EE$B@'6E',"%12'E1.B@&6.'6.'
0/^LI'4&8."#&.8'"7&#&@2)''*6?$-$%+'@;$'3&%$B@&6.'6E'@;&4'$EE$B@'[&)$)+':64&@&-$'6%'.$8"@&-$\'
&4'1.B#$"%'E6%'4$-$%"#'%$"46.4R''N&%4@+'$D7%26.&B'#$@;"#&@2'B".'%$41#@'E%6D'&.41EE&B&$.@'
M6@B;'4&8."#&.8'"4'?$##'"4'E%6D'$B@6:&B'M6@B;'4&8."#&.8+'6%'$-$.'E%6D'"'B6D7&."@&6.'6E'
@;$4$'$EE$B@4'"@'3&EE$%$.@'$D7%26.&B'4@"8$4'4&.B$'@;$%$'"%$'"@'#$"4@'4&V'3&4@&.B@'B$##LE"@$'
B;6&B$4'&.'@;$'$D7%26'@;"@'3$:$.3'1:6.'@;$':%6:$%'7"#".B$'6E'M6@B;L&.31B$3'".3'
1.&.31B$3'B$##4'[4$$'^Y_,=='JHHT\)''=$B6.3+'@;$'E"B@'@;"@'76@;'#644'6E'E1.B@&6.'[#E\'".3'
Hale et al.
21
;2:$%"B@&-$'/.012'"##$#$4'B"14$'D6%$'$D7%26.&B'#$@;"#&@2'&.',"%12L3$E&B&$.@'$D7%264'
:%$-$.@4'"'4&D:#$'&.@$%:%$@"@&6.'6E'$D7%26.&B'#$@;"#&@2+'".3'&.4@$"3':6&.@4'@6'@;$'
B6D:#$V&@2'6E':%6:$%#2'%$81#"@&.8'0/^LI'"B@&-&@2'&.'@;$'$D7%26)'''Q;&%3+'&.@$%:%$@"@&6.'
6E'/.0123.H8R-,"%12'3617#$'D1@".@4'&4'B6D:#&B"@$3'72'@;$'$V&4@$.B$'6E'/_MLIJ'%$B$:@6%'
@;"@'B".'D$3&"@$'46D$'$D7%26.&B'M6@B;'4&8."#&.8'&.'@;$'"74$.B$'6E'0/^LI+'".3'?;64$'
$V:%$44&6.'&4'.6%D"##2'%$:%$44$3'&.'M6@B;L&.31B$3'$D7%26.&B'B$##4'[M,!,='".3'^Y_,=='
JHHT\)''C$B%$"4$3'0/^LI'"B@&-&@2'&.'$"%#2'$D7%26.&B'&.@$%"B@&6.4';"4'7$$.'4;6?.'@6'
3$L%$:%$44'.*$12>'".3'6@;$%'M6@B;'4&8."#&.8'B6D:6.$.@'8$.$4d'@;$'$EE$B@'6E'41B;'
&."::%6:%&"@$#2'$V:%$44$3'/_MLIJ'B61#3'7$'$V"B$%7"@$3'72'@;$'%$D6-"#'6E'"'36?.'
%$81#"@6%'6E'M6@B;'4&8."#&.8+'@68$@;$%'B"14&.8'$B@6:&B'M6@B;'"B@&-"@&6.'&.'4174$]1$.@'
B$##4'[4$$'C&4B144&6.'E6%'".6@;$%':644&7&#&@2\)''N61%@;+'@;$':;$.6@2:$'6E'@;$'"%%$4@$3'
$D7%264'E%6D'/.0123.H8d',"%12'3617#$'D1@".@4'&4'-"%&"7#$+'?&@;'46D$'$D7%264'
%$4$D7#&.8'/.012L3$E&B&$.@'$D7%264+'46D$'$D7%264'%$4$D7#&.8'/.012-.*$12>'L3$E&B&$.@'
$D7%264+'".3'6@;$%4'$V;&7&@&.8'#$44'3&4@&.B@':;$.6@2:$4'[1.:17#&4;$3'674$%-"@&6.4\+'
4188$4@&.8'@;"@'"'-"%&$@2'6E'4:$B&E&B'B"14$4'E6%'$D7%26.&B'#$@;"#&@2'$V&4@4'&.'@;$4$'
3617#$'D1@".@4)''F$B"14$'6E'@;$4$'B6.4&3$%"@&6.4+'@;$'&.B%$"4$3'$D7%26.&B'#$@;"#&@2'&.'
/.012R-,"%12'3617#$'D1@".@4'B"..6@'7$'14$3'@6'&.E$%'?;$@;$%'@;$'$EE$B@'6E'=(KLI'6.'
&.3&-&31"#'M6@B;'4&8."#&.8'$-$.@4'&.'@;$'$D7%26'&4':64&@&-$'6%'.$8"@&-$)''Q6'D6%$'
3$B&4&-$#2'B;"%"B@$%&b$'@;$'$EE$B@'6E',"%12'D1@"@&6.4'6.'D1@".@'0/^LI'%$B$:@6%'
"B@&-&@2+'?$'"."#2b$3'8$%D#&.$'3$-$#6:D$.@)''
!
Hale et al.
22
F./B%./!)'*+,!+%$(8($1!(*%>.+'.'!9DP56!>.%.=$#>!'()*+,(*)!/B>(*)!).><,(*.!
/.8.,#=<.*$H!!!
0/^LI'"B@&-"@&6.'&.31B$4'8$%D'B$##':%6#&E$%"@&6.'@;%618;61@'#"%-"#'".3'"31#@'
#&E$+'".3'@;$'$V@$.@'6E'8$%D#&.$':%6#&E$%"@&6.'4$%-$4'"4'"'3&%$B@'%$"361@'6E'0/^LI'
"B@&-&@2d'$-$.':"%@&"#'&.B%$"4$4'&.'0/^LI'4&8."#&.8'B".'B"14$'D$"41%"7#$'E$%@&#&@2'&.'".'
6@;$%?&4$'4@$%&#$'0/^LIL3$E&B&$.@'".&D"#[>_GF/,'".3'<Y_QQ,MC,M'JHHT\)''Q;$':"%@&"#'
#644L6ELE1.B@&6.'/.012'"##$#$4'($2@<-DAB<-".3-#>266'E"&#'@6':%6D6@$'8$%D#&.$'
:%6#&E$%"@&6.'"@'%$4@%&B@&-$'@$D:$%"@1%$4+'%$41#@&.8'&.'IHHj'4@$%&#$'".&D"#4+'?;&#$'"@'
&.@$%D$3&"@$'@$D:$%"@1%$4'@;$2'%$@"&.'46D$'E1.B@&6.'".3':%631B$'"'4D"##'.1D7$%'6E'
E$%@&#$'".&D"#4'[>KCK5_(MM_-#+-".:'IWWJd'G(M0K-#+-".:'IWWId'^Y_,==-#+-".:'IWUP\)''A$'
E61.3'@;"@'?;$.',"%12'"B@&-&@2'&4'$#&D&."@$3+'"'8%$"@$%':$%B$.@"8$'6E'@;$4$'/.012'
D1@".@'".&D"#4'"B;&$-$'E$%@&#&@2'[Q"7#$'S\)'''N1%@;$%D6%$+'E$%@&#$'/.012d',"%12'3617#$'
D1@".@4';"-$'#"%8$%'7%663'4&b$4'@;".'E$%@&#$'/.012'4&.8#$'D1@".@4)''N6%'$V"D:#$+'@;$'
:$%B$.@"8$'6E'E$%@&#$'".&D"#4'@;"@'8$.$%"@$'8%$"@$%'@;".'IHH'$D7%264'"@'JO6<'&4'6.#2'
I)Wj'E6%'/.0123($2@8'[.kIHP\'71@'&4'&.B%$"4$3'@6'W)Ij'E6%'/.0123($2@8d',"%123*G28'[.'k'
TTd'@;&4'3&EE$%$.B$'&4'4&8.&E&B".@'"BB6%3&.8'@6'"'@?6L@"&#$3'N&4;$%',V"B@'Q$4@+'?&@;'"'S'
-"#1$'s'H)HT\)''Q;$4$'%$41#@4'4188$4@'@;"@'=(KLI'"B@&-&@2'".@"86.&b$4'0/^LI'4&8."#&.8'
&.'@;$'8$%D'#&.$)''M6'41::%$44&6.'6E'/.012'4@$%&#&@2'?"4'4$$.'"@'@;$'E1##2'%$4@%&B@&-$'
@$D:$%"@1%$+'&.3&B"@&.8'@;"@'%$D6-"#'6E'=(KLI'E1.B@&6.'$#$-"@$4'71@'36$4'.6@'72:"44'
0/^LI'"B@&-&@2)'
' Q;$'@$D:$%"@1%$L4$.4&@&-$';2:$%"B@&-$'"##$#$'/.0123"&>9>8'"#46'3$B%$"4$4'
E$%@&#&@2+'71@'&@'36$4'46'7$B"14$'M6@B;'4&8."#&.8'&.'@;$'8$%D'#&.$'&4'$B@6:&B"##2'"B@&-"@$3'
Hale et al.
23
%"@;$%'@;".'3$B%$"4$3)''(@'$#$-"@$3'@$D:$%"@1%$4+'/.0123"&>9>8'".&D"#4';"-$'$VB$44'
8$%D'B$##':%6#&E$%"@&6.'"@'@;$'$V:$.4$'6E'8$%D'B$##'3&EE$%$.@&"@&6.[^,^^,Y-#+-".:'JHHO\)''
_E'=(KLI'36?.'%$81#"@$4'0/^LI'4&8."#&.8+'@;$.'%$D6-&.8'=(KLI'"B@&-&@2'?61#3'7$'
:%$3&B@$3'@6'$V"B$%7"@$'@;$'/.0123"&>9>8'8$%D#&.$'3$E$B@4)''_.3$$3+'"@'@;$':$%D&44&-$'
@$D:$%"@1%$'6E'JH6<+'?;$%$'-&%@1"##2'"##'/.0123"&>9>8'".&D"#4'"%$'E$%@&#$+'ITj'6E'/.01
23"&>9>8d',"%123%GAAAB8'3617#$'D1@".@4'"%$'B6D:#$@$#2'4@$%&#$+'".3'$-$.'@;$'E$%@&#$'
".&D"#4'"::$"%'@6';"-$'"7$%%".@'8$%D'#&.$4'[Q"7#$'S\)''Q;$'E$%@&#$'/.0123"&>9>8d',"%1
23%GAAAB8'3617#$'D1@".@4';"-$'4D"##'7%663'4&b$4'"-$%"8&.8'6.#2'TH)e':%68$.2'[i'
JT)P'=Cd'.kOI\'B6D:"%$3'@6'".'"-$%"8$'6E'ITI':%68$.2'[i'eO)e'=Cd'.kJH\'E6%'4&.8#$'
/.0123"&>9>8'".&D"#4)''Q6'E1%@;$%'B6D:"%$'@;$'$EE$B@'6E'@;$'/.0123"&>9>8'D1@"@&6.'&.'
@;$',"%123Q8'-$%414'@;$',"%123%GAAAB8'7"BZ8%61.3+'?$'$V"D&.$3'@;$'3&4@%&71@&6.'6E'
D&@6@&B'8$%D'B$##'.1B#$&'@;%618;61@'@;$'86."34'72'C(^_'4@"&.&.8+'".3'4B6%$3'@;$'
.1D7$%'6E'86."3'"%D4'@;"@'3&4:#"2$3'$&@;$%'6E'@;$'@?6'D64@'4$-$%$':;$.6@2:$4'
"446B&"@$3'?&@;'$B@6:&B'0/^LI'4&8."#&.8R'"'@1D6%614'8$%D'#&.$'[Q1D\+'&.'?;&B;'@;$'
$.@&%$'86."3'"%D'&4'E&##$3'?&@;'D&@6@&B'.1B#$&+'6%':%6V&D"#'D&@64&4'[^%6\'&.'?;&B;'@;$'
:%6V&D"#'$.3'6E'@;$'86."3'&4'E&##$3'?&@;'D&@6@&B'.1B#$&'%"@;$%'@;".'3&EE$%$.@&"@$3'8$%D'
B$##4[F,YY5-#+-".:'IWWPd'^,^^,Y-#+-".:'JHHO\)''Q;$':$%B$.@"8$'6E'86."3'"%D4'@;"@'3&4:#"2'
$&@;$%':;$.6@2:$'"@'JH6<'&4'4&8.&E&B".@#2'8%$"@$%'E6%'@;$'/.0123"&>9>8d',"%123%GAAAB8'
3617#$'D1@".@4'[OPj+'.'k'STJ'86."3'"%D4\'@;".'E6%'@;$'/.0123"&>9>8'4&.8#$'D1@".@4'
[JHj+'.'k'SHU'86."3'"%D4d'4@"@&4@&B"##2'4&8.&E&B".@'3&EE$%$.B$'72'@?6L@"&#$3'N&4;$%'
,V"B@'Q$4@R'S'-"#1$'s'H)HHHI\)''Q;14'72'4$-$%"#'"BB61.@4'@;$'$EE$B@'6E'@;$'/.0123"&>9>8'
D1@"@&6.+'?;&B;'&.B%$"4$4'0/^LI'"B@&-&@2'&.'@;$'8$%D'#&.$+'"::$"%4'@6'7$'D6%$'4$-$%$'
Hale et al.
24
?;$.'=(KLI'"B@&-&@2'&4'$#&D&."@$3+'41::6%@&.8'@;$'D63$#'@;"@'=(KLI'"B@&-&@2'36?.'
%$81#"@$4'0/^LIL3$:$.3$.@'4&8."#&.8'&.'@;$'8$%D'#&.$)''
'
Q;$'4$B6.3'!:-#.#/"$,'M6@B;'%$B$:@6%+'/_MLIJ+'&4':%$36D&.".@#2'&.-6#-$3'&.'
46D"@&B'B$##'E"@$'3$B&4&6.4'&.'@;$'#"@$'$D7%26'".3'#"%-"#'4@"8$4)''`4&.8'?$##'3$4B%&7$3'
%6#$4'6E'/_MLIJ'&.'-1#-"#'3$-$#6:D$.@+'?$'4$"%B;$3'E6%'$-&3$.B$'6E'8$.$@&B'
&.@$%"B@&6.4'7$@?$$.',"%12'D1@"@&6.4'".3'?$"Z'.*$12>'#644L6ELE1.B@&6.'D1@"@&6.4'6%'
8"&.L6ELE1.B@&6.'D1@"@&6.4+'71@'E61.3'.6'4&8.&E&B".@'$EE$B@4R'&)$)+'#6?$%&.8',"%12'
"B@&-&@2'3&3'.6@'&.E#1$.B$'@;$'41BB$44'6E'".B;6%'B$##'[(<\'4:$B&E&B"@&6.+'6%'"#@$%'@;$'
6-$%"##'$88'#"2&.8'"7&#&@2'6E'?6%D4'?&@;'$&@;$%'#6?$%$3'6%'$#$-"@$3'.*$12>'"B@&-&@2'
[Q"7#$'=I\)''Q;$'#"BZ'6E'8$.$@&B'&.@$%"B@&6.4'7$@?$$.',"%12'".3'.*$12>'&4'"'.6@"7#$'
B6.@%"4@'@6'@;$'%$"3&#2'674$%-$3'&.@$%"B@&6.4'7$@?$$.',"%12'".3'/.012)''Q;&4'
3&4@&.B@&6.'D"2'%$E#$B@'"'3&EE$%$.B$'&.'36?.'%$81#"@&6.'D$B;".&4D4'E6%'0/^LI'-$%414'
/_MLIJ'4&8."#&.8+'6%'3&EE$%$.B$4'7$@?$$.'8$%D#&.$'".3'46D"@&B'@&441$4'[$)8)+'&.'@$%D4'
6E'$&@;$%',"%12'$V:%$44&6.'6%'@;$'%$81#"@&6.'6E'M6@B;'4&8."#&.8\)'''
'
'
)'*+,#.*%#/.'!+!*#8.,!9:;!/#<+(*!=>#$.(*H###
A$'3$@$%D&.$3'@;$'D6#$B1#"%'&3$.@&@2'6E'@;$',"%12'8$.$'@;%618;'"'B6D7&."@&6.'
6E'=M^'D"::&.8+'B64D&3'%$4B1$+'".3'YM(&'"."#24&4)''0$.$@&B'D"::&.8'?&@;'-&4&7#$'
D"%Z$%4'".3'=M^'D"%Z$%4'#6B"#&b$3'@;$',"%12'8$.$'@6'"'%$8&6.'6E'IPT'Z7'6.'
B;%6D646D$'!+'E#".Z$3'72'4.:'NTTFIJlIh'".3'4.:'NTU,IHlOh'[N&81%$'J(\)''<64D&34'
E%6D'@;&4'%$8&6.'?$%$'@;$.'@$4@$3'E6%'@;$&%'"7&#&@2'@6'41::#2'E1.B@&6."#',"%12'"B@&-&@2'
Hale et al.
25
72'&.m$B@&.8'@;$D'&.@6'"0'123452678R-,"%123*G28';$%D":;%63&@$4'".3'4B6%&.8'E6%'
@%".48$.&B'%$4B1$'6E'@;$',"%123*G28':;$.6@2:$'[t".@&41::%$44&6.u\+'"4'$-&3$.B$3'72'
3$"3'$D7%264)''('4D"##'.1D7$%'6E'@%".48$.&B'?6%D4'B"%%2&.8'B64D&3'YIHCIJ'8"-$'OH'
n'THj'3$"3'$D7%264)''<".3&3"@$'6:$.'%$"3&.8'E%"D$4'[KYN\'?&@;&.'@;$'YIHCIJ'
B64D&3'%$8&6.'?$%$'@;$.'"."#2b$3'72'YM(&'E6%'@;$'"7&#&@2'@6'41::%$44'@;$'D"@$%."#L
$EE$B@'#$@;"#&@2'6E'"0'123452678';6D6b28614'".&D"#4)''A6%D4'&.m$B@$3'?&@;'34YM('
B6%%$4:6.3&.8'@6'KYN'YIHCIJ)IS'2&$#3$3'"'4@%&Z&.8'.1D7$%'6E'#&-$':%68$.2'[4$$'
Q"7#$'I\'&.'B6.@%"4@'@6'1.&.m$B@$3'?6%D4'6%'?6%D4'&.m$B@$3'?&@;'34YM('E%6D'6@;$%'
KYN4'&.'@;$'%$8&6.'[3"@"'.6@'4;6?.\)'
Q;$'&3$.@&@2'6E'YIHCIJ)IS'"4'@;$',"%12'KYN'?"4'B6.E&%D$3'72'@;$'
&3$.@&E&B"@&6.'6E'"'4&.8#$'.1B#$6@&3$'B;".8$'&.'@?6'YIHCIJ)IS'BCM(4'&.3$:$.3$.@#2'
&46#"@$3'72'YQL^<Y'E%6D',"%123*G28'?6%D4'[4$$'G$@;634\)''K1%'BCM('"."#24&4'&4'
B6.4&4@$.@'?&@;'@;$':%$3&B@&6.4'6E'0$.$E&.3$%'E6%'YIHCIJ)IS"+'".3':%$3&B@4'@;"@'@;$'
,"%12'8$.$':%631B@'&4'"'.6-$#'JJS'"D&.6'"B&3':%6@$&.+'?;64$'4$]1$.B$'&4'4;6?.'&.'
N&81%$'JF)''Q;$',"%123%GAAAB8'"##$#$';"4'".'&.@$%."#'3$#$@&6.'@;"@'%$D6-$4'D64@'6E'@;$'
B63&.8'%$8&6.'[4$$'G$@;634\)''_.'@;$'!:-#.#/"$,'@6:68%":;&B"#'$V:%$44&6.'D":+'@;$'
$V:%$44&6.'6E'YIHCIJ)IS'&4'B#"44&E&$3'"4'8$%D'#&.$L$.%&B;$3'".3'&4'E61.3'&.'@;$'4"D$'
$V:%$44&6.':%6E&#$'B#14@$%'"4'"0'12'".3'"0'1>d'."D$#2+'?&@;&.'G61.@"&.'J[>_G-#+-".:'
JHHId'Y,_M>,-#+-".:'JHHH\)'
'
Q;$':%$3&B@$3'=(KLI':%6@$&.'B6.@"&.4'"'05N'36D"&.+'"'D6@&E'@;"@'D$3&"@$4'
:%6@$&.L:%6@$&.'&.@$%"B@&6.4'".3'&4'"'D$D7$%'6E'@;$'8$.$%"#'B#"44'6E':%6@$&.'D6@&E4'
Hale et al.
26
@;"@'7&.3':%6#&.$L%&B;'4$]1$.B$4[>KN/,Y'".3'NY,`MC'JHHe\)''Q;$'05N'36D"&.'&4'
B;"%"B@$%&b$3'72'"'4&8."@1%$'4$@'6E'B6.4$%-$3'"%6D"@&B'"D&.6'"B&3'%$4&31$4':%$B&4$#2'
:#"B$3'@6'4@"BZ'7$@?$$.'"'4&.8#$'"';$#&V'".3'"'4D"##'!'4;$$@)''Q;$'05N'36D"&.'6E'=(KL
I'B6.@"&.4'.&.$'6E'@;$'@$.'4&8."@1%$'%$4&31$4'6E'05N'36D"&.4'[N&81%$'J<\)''(33&@&6."#'
E$"@1%$4';"-$'7$$.'14$3'@6'417LB"@$86%&b$'05N'36D"&.4'&.@6'@?6'B#"44$4R'@;$'=G5J'
417E"D&#2'".3'@;$'<C<JF^'417E"D&#2[>KN/,Y'".3'NY,`MC'JHHe\)''Q;$'=(KLI'05N'
36D"&.'D6%$'B#64$#2'%$4$D7#$4'@;$'=G5J'B#"44'7$B"14$'6E'@;$'@&8;@'4:"B&.8'7$@?$$.'
@;$'E&%4@'".3'4$B6.3':%$3&B@$3'!'4@%".34+'"';"##D"%Z'6E'=G5JL@2:$'05N'36D"&.4)''
A&@;&.'@;$'=G5J'417E"D&#2+'=(KLI'".3'"'4174$@'6E':%6@$&.4+'&.B#13&.8';1D".'0_05NI'
".3'0_05N'J+'4;6?'E1%@;$%'B6.4$%-"@&6.'6E'05N'36D"&.'4$]1$.B$4'#2&.8'36?.4@%$"D'
6E'@;$'@$.'4&8."@1%$'%$4&31$4'[N&81%$'J<\)''M6@"7#2+'6.$'6E'@;$4$'B6.4$%-$3'%$4&31$4+'
#$1B&.$'TW+'&4'@;$'%$4&31$'@;"@'&4'"#@$%$3'&.'@;$',"%123*G28'D1@"@&6.'[/TWN\)'''
Q;$'<L@$%D&."#':6%@&6.'6E'=(KLI';"4'.6'3&4B$%."7#$':%6@$&.'D6@&E4'6%'%$8&6.4'
6E'B6.4$%-"@&6.'"B%644'@;$'".&D"#'Z&.836D+'71@'"D6.8'.$D"@63$'4:$B&$4+'4$]1$.B$'
B6.4$%-"@&6.'7$26.3'@;$'05N'36D"&.'%$-$"#4':1@"@&-$',"%12'6%@;6#684''[N&81%$'JC\)'
0&-$.'@;$'"7&#&@2'6E'05N'36D"&.4'@6'7&.3':%6#&.$L%&B;'4$]1$.B$4+'&@'&4'.6@"7#$'@;"@'@;$'
=(KLI'<L@$%D&.14'&4'$.%&B;$3'&.':%6#&.$4+'".3'&.'$"B;'6E'@;$':%$3&B@$3'%$#"@$3'
.$D"@63$':%6@$&.4+'@;$'<L@$%D&.14'B6.@"&.4'"'^^0'4$]1$.B$'[N&81%$'JC\'?;&B;'&4'@;$'
B6%$'%$B68.&@&6.'4$]1$.B$'E6%'05N'36D"&.4[>KN/,Y-#+-".:'JHHT\)''N1%@;$%D6%$+'@;$'
^^0'4$]1$.B$'6E'=(KLI'&4'E6##6?$3'72'"'#$1B&.$+'B6.4&4@$.@'?&@;'@;$':%$E$%%$3'7&.3&.8'
4&@$'$4@"7#&4;$3'E6%'=G5JL@2:$'05N'36D"&.4+'?;&#$'@;64$'6E'@;$'<CJF^'B#"44':%$E$%'
^^0Ya>[>KN/,Y-#+-".:'JHHT\)''Q;$':%$4$.B$'6E'"':1@"@&-$'05N'%$B68.&@&6.'D6@&E'&.'@;$'
<'@$%D&."#';"#E'6E'=(KLI'&4'4188$4@&-$'6E';6D6@2:&B'6%'&.@%"D6#$B1#"%'&.@$%"B@&6.4)'
Hale et al.
27
23456!=>#$.(*!(*$.>+%$'!/(>.%$,1!?($&!$&.!;0?Q!=>#$.(*!2@D56EH!!!
_.'8$.6D$L?&3$'2$"4@'@?6L;27%&3'"."#24$4+'@;$'YIHCIJ)IS'KYN'[,"%12\'?"4'
E61.3'"4'6.$'6E'4$-$.':%6@$&.4'@;"@'&.@$%"B@'?&@;'=,/LIH+'".',O'17&]1&@&.'#&8"4$'
B6D:6.$.@+'".3'6.$'6E'.&.$@$$.':%6@$&.4'@;"@'&.@$%"B@4'?&@;'32.$&.'#&8;@'B;"&.'I'
[C/<LI\'[/_-#+-".:'JHHSd'A(/*K`Q-#+-".:'JHHH\)''Q;$4$'&.@$%"B@&6.4'D"2'7$':$%@&.$.@'@6'
=(KLIc4'&.E#1$.B$'6.'M6@B;'4&8."#&.8)''A$'&.-$4@&8"@$3'@;$'&.@$%"B@&6.'?&@;'=,/LIH'
7$B"14$'6E'&@4'$4@"7#&4;$3'%6#$'"4'"'36?.'%$81#"@6%'6E'@;$'M6@B;'4&8."#&.8':"@;?"2'&.'
!:-#.#/"$,'".3'D"DD"#4'[*`FF(YC-#+-".:'IWWPd'/_-#+-".:'JHHJd'A`-#+-".:'IWWUd'A`-#+-".:'
JHHI\)''Q;$'ML@$%D&."#'36D"&.'6E'=,/LIH'B6.@"&.4'".'NL76V'D6@&E'@;"@'&.@$%"B@4'?&@;'
@;$'B6%$'6E'@;$'=<N'17&]1&@&.'#&8"4$'B6D:#$V+'?;&#$'@;$'<L@$%D&."#'ACSH'%$:$"@'
36D"&.':%6-&3$4'4174@%"@$'%$B68.&@&6.'4:$B&E&B&@2'E6%'@;$'B6D:#$V'[^,QYK=>_'".3'
C,=*(_,='JHHT\)''A$'8$.$%"@$3'=(KLI'".3'=,/LIH'2$"4@'@?6';27%&3'B6.4@%1B@4'@6'
$V"D&.$'@;$'"7&#&@2'6E'@;$4$'@?6':%6@$&.4'@6'&.@$%"B@'[4$$'N&81%$'OF+'O<+'".3'ON'E6%'
:%6@$&.'$V:%$44&6.'&.'2$"4@\)''('4@%6.8'&.@$%"B@&6.'7$@?$$.'=(KLI'".3'=,/LIH'&4'
3$@$B@$3'72'$V:%$44&6.'6E'"'*_=O'%$:6%@$%'[*_='8%6?@;'"44"2+'3"@"'.6@'4;6?.\'".3'"'
."=N'%$:6%@$%'[!L8"#"B@64&3"4$'"B@&-&@2'"44"2+'N&81%$'O(\)''F2'@$4@&.8'@;$'NL76V'".3'@;$'
ACSH'36D"&.4'6E'=,/LIH'4$:"%"@$#2+'?$'E61.3'@;"@'@;$'ACSH'36D"&.'&4'41EE&B&$.@'@6'
&.@$%"B@'?&@;'=(KLI+'".3'"'4&.8#$'D&44$.4$'D1@"@&6.'&.'@;$'$&8;@;'ACSH'%$:$"@'6E'
=,/LIH'[0TeP,\'[9(0,Y-#+-".:'JHHS\'3&4%1:@4'@;&4'&.@$%"B@&6.'[N&81%$'O(\)''Q;$'0TeP,'
4174@&@1@&6.'D"2'3&%$B@#2'&.@$%E$%$'?&@;'=(KLI'n'=,/IH'&.@$%"B@&6.+'6%'&@'D"2'
&.3&%$B@#2':%$-$.@'=(KLI'&.@$%"B@&6.'72'3&4%1:@&.8'=,/LIH'3&D$%&b"@&6.'[>_//_(M-#+-".:'
JHHU\)''Q;$'=(KLI'&.@$%"B@&6.'"::$"%4'4:$B&E&B'E6%'@;$'ACSH'36D"&.'E%6D'=,/LIH+'"4'
Hale et al.
28
?$'3&3'.6@'3$@$B@'".'&.@$%"B@&6.'?&@;'@;$'ACSH'36D"&.'E%6D'".6@;$%'!:-#.#/"$,'N7?P'
:%6@$&.+'/_MLJO'[3"@"'.6@'4;6?.\)'''
_.'6%3$%'@6'"33%$44'?;$@;$%'@;$'=(KLI'L'=,/LIH'&.@$%"B@&6.'&4'%$#$-".@'@6'@;$'
,"%123*G28'41::%$44&6.'6E'"0'123452678+'?$'&.@%631B$3'@;$'*G2D1@"@&6.'[/TWN\'&.@6'
@;$'=(KLI'2$"4@'@?6';27%&3'B6.4@%1B@)''Q;$'*G2'D1@"@&6.'36$4'.6@'3&4%1:@'$V:%$44&6.'
6%'4@"7&#&@2'6E'@;$'=(KLI'E14&6.':%6@$&.'&.'2$"4@'[N&81%$'O<\+'71@'3%"D"@&B"##2'
3$B%$"4$4'@;$'"D61.@'6E'3$@$B@"7#$'&.@$%"B@&6.'?&@;'=,/LIH'[N&81%$'O(\)''('4&D&#"%'
3$B%$"4$'&.'&.@$%"B@&6.'&4'674$%-$3'?;$.'@;$'&46#"@$3'ACSH'36D"&.'6E'=,/LIH'&4'
@$4@$3'"8"&.4@'=(KLI[/TWN\'-$%414'?&#3L@2:$'=(KLI'[N&81%$'O(\)'''Q;$4$'%$41#@4'%"&4$'
@;$':644&7&#&@2'@;"@'&.@$%"B@&6.'7$@?$$.'=(KLI'".3'=,/LIH'B6.@%&71@$4'@6'.$8"@&-$'
%$81#"@&6.'6E'M6@B;'4&8."#&.8)'''
'
' Q;$'M'@$%D&."#'%$8&6.'6E'=(KLI'B6.@"&.4'@;$'05N'36D"&.'[%$4&31$4'IJLPO\+'
?;&#$'@;$'<'@$%D&."#'@?6'@;&%34'[%$4&31$4'PSLJJS\'6E'@;$':%6@$&.'&4'.6.3$4B%&:@)''
C$4:&@$'@;$'E"B@'@;"@'=,/LIH'7&.3&.8'?"4'3&4%1:@$3'72'@;$'*G2[/TWN\'D1@"@&6.'?;&B;'
%$4&3$4'&.'@;$'05N'36D"&.'6E'=(KLI+'?$'E61.3'@;"@'&@'?"4'"B@1"##2'@;$'<L@$%D&."#'
:6%@&6.'6E'=(KLI'@;"@'D$3&"@$4'=,/LIH'7&.3&.8'[N&81%$'OC\)''<6.4@%1B@4'$V:%$44&.8'
6.#2'@;$'<L@$%D&."#'IST'6%'IPS'%$4&31$4'6E'=(KLI'?$%$'41EE&B&$.@'@6'&.@$%"B@'?&@;'
&.@"B@'=,/LIH'6%'&@4'ACSH'36D"&.'"#6.$'[N&81%$'OC\)''N1%@;$%D6%$+'@;$'/TWN'
D1@"@&6.';"3'.6'$EE$B@'6.'=,/LIH'7&.3&.8'1.#$44'@;$'=(KLI'05N'36D"&.'?"4'&.@"B@)''
Q;$%$E6%$+'61%'3"@"'4188$4@'@;"@'=,/LIH'%$B68.&@&6.'&4'D$3&"@$3':%&D"%&#2'72'@;$'=(KL
I'<L@$%D&."#'36D"&.+'71@'@;"@'@;$'ML@$%D&."#'05N'36D"&.'B".'D631#"@$'@;$'=,/LIHL
Hale et al.
29
=(KLI'&.@$%"B@&6.'&.'"'?"2'@;"@'&4'"EE$B@$3'72'@;$',"%123*G28'/TWN'D1@"@&6.)''_@'&4'
B6.B$&-"7#$'@;"@'@;$'/TWN'D1@"@&6.'$.;".B$4'".'&.@%"D6#$B1#"%'&.@$%"B@&6.'@;"@'
D"4Z4'@;$'=,/LIH'7&.3&.8'4&@$'&.'=(KLI'[4$$'C&4B144&6.\)''_.'41DD"%2+'61%'2$"4@'@?6L
;27%&3'"."#24&4'3$D6.4@%"@$4'"'4:$B&E&B'&.@$%"B@&6.'7$@?$$.'=(KLI'".3'=,/LIH'@;"@'&4'
3&4%1:@$3'72'@;$',"%123*G28':6&.@'D1@"@&6.'@;"@'?$'&46#"@$3'"4'"'41::%$446%'6E'"0'1
23452678:'
3!)$%+,8!<B$+$(#*!%+*!=+>$(+,,1!'B==>.''!'./+,012,345!.<0>1#*(%!,.$&+,($1H!!!
Q;$':;24&B"#'&.@$%"B@&6.'7$@?$$.'=(KLI'".3'=,/LIH'#$3'14'@6'"4Z'?;$@;$%'
3&4%1:@&.8',#.129'E1.B@&6.'&.'@;$'$D7%26'D&8;@';"-$'"'4&D&#"%'$EE$B@'"4'%$D6-&.8',"%12'
E1.B@&6.d'."D$#2+'41::%$44&6.'6E'"0'123452678'$D7%26.&B'#$@;"#&@2)''Q?6'4#.129-#644L
6ELE1.B@&6.'D1@"@&6.4'?$%$'@$4@$3R'(=>6A'&4'"'.1##'"##$#$'[9(0,Y-#+-".:'JHHS\'".3'"&62'&4'
"'4@%6.8'#644L6ELE1.B@&6.'"##$#$'@;"@'%$@"&.4'46D$'%$4&31"#'"B@&-&@2[*`FF(YC-#+-".:'
IWWP\)''M$&@;$%',#.1293(=>6A8-.6%-,#.1293"&628'?"4'E61.3'@6'41::%$44'"0'123452678-
$D7%26.&B'#$@;"#&@2'[Q"7#$'T\)''0&-$.'@;"@'@;$4$',#.129'D1@"@&6.4'$#$-"@$'M6@B;'
4&8."#&.8'31%&.8'-1#-"#'3$-$#6:D$.@'[*`FF(YC-#+-".:'IWWP\'".3'8$%D#&.$'3$-$#6:D$.@'
[^,^^,Y-#+-".:'JHHO\+'61%'%$41#@4'4188$4@'"'3&EE$%$.@'4B$."%&6'&.'@;$'$D7%26R'$&@;$%'=,/L
IH'36$4'.6@'36?.'%$81#"@$'M6@B;'4&8."#&.8+'6%'&@'D"2'36'46'%$31.3".@#2'?&@;'6@;$%'
:%6@$&.4)''A$'@;$.'@$4@$3'@;$',#.129[$29778'"##$#$+'?;&B;'B"14$4'@;$'4&.8#$'"D&.6'"B&3'
4174@&@1@&6.'0TeP,'@$4@$3'&.'61%'2$"4@'@?6L;27%&3'"."#24&4)''Q;&4'D1@"@&6.';"4'7$$.'
4;6?.'@6':%$-$.@'=,/LIH'E1.B@&6.'".3'"#46'B"14$'"'36D&.".@'.$8"@&-$':;$.6@2:$'&.'
?;&B;'@;$'=,/LIH[0TeP,\':%6@$&.'&4'@;618;@'@6'&.@$%E$%$'?&@;'@;$'E1.B@&6.'6E'6@;$%'
%$31.3".@'=<N'NL76V'B6D:6.$.@4'[9(0,Y-#+-".:'JHHS\)''_.@$%$4@&.8#2+'@;$',#.1293$29778'
Hale et al.
30
"##$#$'&4'"7#$'@6':"%@&"##2'41::%$44'"0'123452678'$D7%26.&B'#$@;"#&@2'[Q"7#$'T\)''Q;$'
3$8%$$'6E'41::%$44&6.'&4'4@%6.8$4@'"@'IT°<+'B6.4&4@$.@'?&@;'@;$'6@;$%':;$.6@2:&B'
$EE$B@4'3$4B%&7$3'E6%'@;$',#.1293$29778-"##$#$'[9(0,Y-#+-".:'JHHS\)''Q;$'7$;"-&6%'6E',#.1
29'D1@".@'"##$#$4'&.'@;$'"0'123452678'D1@".@'7"BZ8%61.3'41::6%@4'@;$'D63$#'@;"@'&.'
@;$'$D7%26'=,/LIH'D"2'7$'&.-6#-$3'&.'M6@B;'36?.'%$81#"@&6.+'71@'@;"@'@;&4'%6#$'D"2'
7$'%$31.3".@'?&@;'6@;$%':%6@$&.4'?;64$':"%@&B&:"@&6.'&.'M6@B;'36?.'%$81#"@&6.'&4'
&.@$%%1:@$3'72'@;$':%$4$.B$'6E'@;$'=,/LIH[0TeP,\'36D&.".@'.$8"@&-$':%6@$&.)'''
'
0&-$.'@;$'4&D&#"%'41::%$44&-$'$EE$B@4'6E',#.1293$29778-".3',"%12'D1@"@&6.4'6.'
"0'123452678+'?$'$V"D&.$3'?;$@;$%',"%12'D&8;@'7$'&.-6#-$3'&.'6@;$%'36B1D$.@$3'
%6#$4'6E',#.129)''G1@"@&6.4'&.',#.129'$#$-"@$'/_MLIJ'4&8."#&.8'&.'?6%D4'@;"@';"-$'
D1@".@'.*$12>'%$B$:@6%'6%'B6D:%6D&4$3',#.12>':%$4$.&#&.'E1.B@&6.[*`FF(YC-#+-".:'
IWWPd'9(0,Y-#+-".:'JHHSd'=`MC(Y(G'".3'0Y,,MA(/C'IWWO7d'A`-#+-".:'IWWU\'71@'?$'
E61.3'.6'$-&3$.B$'E6%',"%12'E1.B@&6.'&.'@;$4$'%6#$4R',"%12'D1@"@&6.4'3&3'.6@'41::%$44'
6%'$.;".B$'@;$',8#'3$E$B@'6E'.*$12>'6%',#.12>'D1@"@&6.4'[Q"7#$'=I'".3'=J\'".3',"%12'
D1@"@&6.4'3&3'.6@'&D:%6-$'1@$%&.$'v'B$##'3$-$#6:D$.@'&.',#.12>'D1@".@4'6%'(<'
4:$B&E&B"@&6.'6E'.*$12>'D1@".@4'[Q"7#$'=I'".3'=O\)''=,/LIH'"#46'E1.B@&6.4'61@4&3$'6E'@;$'
M6@B;'4&8."#&.8':"@;?"2'@6'&.E#1$.B$'46D"@&B'4$V'3$@$%D&."@&6.'[9(0,Y-#+-".:'JHHS\+'
71@',"%12'D1@"@&6.4'36'.6@'B"14$'@;$'D"4B1#&.&b"@&6.'$EE$B@4'3$4B%&7$3'E6%',#.129'
D1@"@&6.4'[Q"7#$'=S\)''Q;$'E"B@'@;"@',"%12'D1@"@&6.4'36'.6@'D&D&B'"##',#.129'D1@".@'
:;$.6@2:$4'4188$4@4'@;"@'@;$'%6#$4'6E'=(KLI'".3'=,/LIH'D"2'6-$%#":'&.'46D$'B"4$4+'
41B;'"4'&.'@;$'36?.'%$81#"@&6.'6E'0/^LI'4&8."#&.8'&.'@;$'8$%D'#&.$'".3'$"%#2'$D7%26+'
Hale et al.
31
71@'.6@'&.'6@;$%'B"4$4+'41B;'"4'-1#-"#'".3'1@$%&.$'E"@$'4:$B&E&B"@&6.+'?;$%$'=,/LIH'D"2'
E1.B@&6.'?&@;61@'=(KLI)'
C_=<`==_KM'
'
_.'6%3$%'@6'1.B6-$%'@;$'B$##1#"%'D$B;".&4D4'@;"@'%$81#"@$'M6@B;'4&8."#&.8'
B6D:6.$.@4'&.'@;$'$"%#2'!:-#.#/"$,'$D7%26+'?$'14$3'"'8$.$@&B"##2'4$.4&@&b$3'424@$D'&.'
?;&B;'6.$'D$D7$%'6E'@;$'8"DD"'4$B%$@"4$'B6D:#$V+'(^*LI+'?"4'B6D:%6D&4$3'".3'
B61#3'D$3&"@$'6.#2'-$%2'#&D&@$3'$D7%26.&B'M6@B;'4&8."#&.8)''A$'&3$.@&E&$3',"%12'"4'"'
.$?'8$.$'@;"@'.$8"@&-$#2'&D:&.8$4'6.'M6@B;'4&8."#&.8'31%&.8'$"%#2'$D7%268$.$4&4'
".3'"#46'31%&.8'#"%-"#a"31#@'8$%D#&.$'3$-$#6:D$.@)''G1@"@&6.4'@;"@'B6D:%6D&4$'
M6@B;'4&8."#&.8'"@'@;$'#$-$#'6E'%$B$:@6%'[/.012\+'8"DD"'4$B%$@"4$'["0'12\'6%'
36?.4@%$"D'@%".4B%&:@&6.'E"B@6%'[."/12\'"##'4;6?'4$.4&@&-&@2'@6',"%12'"B@&-&@2)''Q;$'
"0'123452678'$D7%26.&B'#$@;"#&@2'&4'E1##2'41::%$44$3'?;$.'=(KLI'E1.B@&6.'&4'%$D6-$3'
72'YM(&'6%'72'8$.$'3$#$@&6.+'".3'$-$.'%$31B&.8',"%12'8$.$'364"8$'72';"#E'%$41#@4'&.'
:"%@&"#'41::%$44&6.)''Q;$'8$.$@&B'7$;"-&6%'6E',"%12'D1@"@&6.4'4188$4@4'@;"@'@;$'?&#3L
@2:$'8$.$':%631B@'".@"86.&b$4'6.$'6%'D6%$'B6D:6.$.@4'6E'@;$'M6@B;'4&8."#&.8'
:"@;?"2'31%&.8'$D7%268$.$4&4'".3'8$%D#&.$'3$-$#6:D$.@)'
'
(':644&7#$'D$B;".&4D'72'?;&B;'=(KLI'D"2'36?.'%$81#"@$'M6@B;'4&8."#&.8'&4'
4188$4@$3'72'&@4'4:$B&E&B'&.@$%"B@&6.'?&@;'@;$'N7?P':%6@$&.'=,/LIH+'?;&B;'E1.B@&6.4'"4'
"'4174@%"@$'%$B68.&@&6.'4171.&@'[=Y=\'E6%'=<NL@2:$',O'17&]1&@&.'#&8"4$4)''=,/LIH';"4'
7$$.'4;6?.'@6'3&%$B@#2'7&.3'M6@B;'".3':%$4$.&#&.4+'".3'@6'E"B&#&@"@$'@;$&%'
17&]1&@&."@&6.'".3'3$8%"3"@&6.'72'@;$':%6@$646D$'[%$-&$?$3'&.'/(_'JHHJ\)''Q;$'
Hale et al.
32
%$#$-".B$'6E'@;$'=(KLI'n'=,/LIH'&.@$%"B@&6.'@6'@;$'"7&#&@2'6E'=(KLI'@6'".@"86.&b$'
M6@B;'4&8."#&.8'&4'41::6%@$3'72'@;$'674$%-"@&6.'@;"@'D&44$.4$'D1@"@&6.4'&.'=(KLI'6%'
&.'=,/LIH'@;"@'3&4%1:@'@;$'=(KLIL=,/LIH'&.@$%"B@&6.'"%$'"#46'E61.3'@6'B"14$'"0'1
23452678'41::%$44&6.'[N&81%$'O'".3'Q"7#$4'I'".3'T\)'''
'
0&-$.'@;"@'@;$'ACSH'36D"&.'6E'N7?P':%6@$&.4'&4'%$4:6.4&7#$'E6%'4174@%"@$'
%$B68.&@&6.'72'=<N=,/LIH'17&]1@&.'#&8"4$4+'@;$'E"B@'@;"@'@;&4'&4'@;$'36D"&.'6E'=,/LIH'@;"@'
&.@$%"B@4'?&@;'=(KLI'&4'B6.4&4@$.@'?&@;'@?6'"#@$%."@&-$'D63$#4R'$&@;$%'=(KLI'&4'
%$B68.&b$3'"4'"'4174@%"@$'6E'=<N=,/LIH'LD$3&"@$3'17&]1&@&."@&6.'".3'3$8%"3"@&6.+'6%'
=(KLI'"B@4'"4'"'B6LE"B@6%'?&@;'=<N=,/LIH'@6':"%@&B&:"@$'&.'@"%8$@'%$B68.&@&6.'".3a6%'
17&]1&@&."@&6.)'Q;$'4$B6.3'D63$#'&4'41::6%@$3'72'@;$':"%"##$#':;$.6@2:&B'$EE$B@4'6E'
,"%12'".3',#.129)''N&%4@+'D1@"@&6.4'&.'$&@;$%',#.129'6%',"%12'B".'$#$-"@$'M6@B;'4&8."#&.8'
&.'"0'123452678'$D7%264'[@;&4'?6%Z\)''=$B6.3+'%$D6-&.8'=(KLI'6%'=,/LIH'"B@&-&@2'
"::$"%4'@6'$#$-"@$'M6@B;'4&8."#&.8'&.'/.0123"&>9>8'D1@".@4'[@;&4'?6%Z'".3'^,^^,Y-#+-
".:'JHHO\)''Q;&%3+'%$31B$3'M6@B;'%$B$:@6%'"B@&-&@2'B".'7$'&.B%$"4$3'72'%$D6-&.8'=(KLI'
6%'=,/LIHR'@;&4'&4'3$D6.4@%"@$3'72'@;$'41::%$44&6.'6E'/.0123.H8'4@$%&#&@2'72',"%12'
D1@"@&6.4'[@;&4'?6%Z\'".3'@;$'41::%$44&6.'6E'.*$12>3.H8'-1#-"#'3$E$B@4'72',#.129'
D1@"@&6.4'[=`MC(Y(G'".3'0Y,,MA(/C'IWWO7\)''(#@;618;'61%'%$41#@4'"%$'B6.4&4@$.@'
?&@;'@;$'1#@&D"@$'$EE$B@'6E'=(KLI'"B@&-&@2'7$&.8':%6@$646D"#'3$8%"3"@&6.'6E'
17&]1&@&."@$3'@"%8$@4+'&@'%$D"&.4':644&7#$'@;"@'".2'%6#$'E6%'=(KLI'&.'=<N=,/LIHLD$3&"@$3'
17&]1&@&."@&6.'D"2'%$81#"@$'M6@B;'4&8."#&.8'@;%618;'6@;$%'D$B;".&4D4+'41B;'"4'
$EE$B@4'6.'%$B$:@6%'$.36B2@64&4+':%6@$&.'@%"EE&BZ&.8+'6%'#24646D$LD$3&"@$3'
3$8%"3"@&6.'[4$$'NKYQ_M_'".3'F_/C,Y'JHHWd'G`>*K^(C*5(5'".3'Y_,fG(M'JHHP\)'
Hale et al.
33
'
(BB$446%2':%6@$&.4';"-$'.6@'2$@'7$$.'3&4B6-$%$3'E6%'4174@%"@$'%$B68.&@&6.'72'
N7?PL7"4$3'=<N'B6D:#$V$4d';6?$-$%+'$V"D:#$4'6E'41B;'B6LE"B@6%4'36'$V&4@'"D6.8'
6@;$%'@2:$4'6E'17&]1&@&.'#&8"4$4)''N6%'$V"D:#$+'@;$'=<N=Z:J'B6D:#$V'$D:#624'@;$'
#$1B&.$L%&B;'%$:$"@'6E'=Z:J'E6%'4174@%"@$'%$B%1&@D$.@+'71@'"#46'%$]1&%$4'@;$'"BB$446%2'
:%6@$&.'<Z4I'E6%'%$B68.&@&6.'6E':"%@&B1#"%'4174@%"@$4[0(MKQ*-#+-".:'JHHId'=^Y`<>-#+-".:'
JHHI\+'".3'4174@%"@$'%$B68.&@&6.'72'@;$'N,GLI'B6D:6.$.@'6E'@;$'<`/JL7"4$3',O'
17&]1&@&.'#&8"4$'%$]1&%$4'@?6'"BB$446%2':%6@&.$4+'N,GLJ'".3'N,GLO+'@;"@'"%$'.6@':"%@'
6E'@;$'B6%$'#&8"4$'B6D:#$V'[=Q(YK=Q_M(-#+-".:'JHHP\)''K1%'"."#24&4'&.@%631B$4'=(KLI'"4'
"':644&7#$'N7?P'B6LE"B@6%d'E1@1%$'D6#$B1#"%'?6%Z'?&##'@$4@'@;&4'&3$"'".3'@;$'D6%$'
8$.$%"#'.6@&6.'@;"@'N7?P':%6@$&.4'D&8;@'$D:#62'B6LE"B@6%4'@6'3&-$%4&E2'@;$&%'
4174@%"@$'4$#$B@&-&@2)'
'
A;&#$'61%'%$41#@4'4188$4@'@;"@'=(KLI'".@"86.&b$4'M6@B;':"@;?"2'"B@&-&@2+'&@4'
&DD$3&"@$'@"%8$@4'"%$'1.Z.6?.)''=,/LIHLD$3&"@$3'17&]1&@&."@&6.';"4'7$$.'4;6?.'@6'
3&%$B@#2'@"%8$@'M6@B;'%$B$:@6%4'".3':%$4$.&#&.4'&.'6%3$%'@6':%6D6@$'@;$&%'3$8%"3"@&6.d'
@;$'"BB$44&7&#&@2'6E'6.$'6%'76@;'6E'@;$4$'@"%8$@':%6@$&.4'&.'@;$'$D7%26'D"2'7$'"EE$B@$3'
72'=(KLI'"B@&-&@2+'".3'@;$'4@"7&#&b"@&6.'6E'$&@;$%'6E'@;$4$':%6@$&.4'B61#3'"BB61.@'E6%'
@;$'674$%-$3'8$.$@&B'&.@$%"B@&6.4'7$@?$$.',"%12'D1@"@&6.4'".3'"0'12+'/.012+'6%'."/12'
D1@"@&6.4)''*6?$-$%+'"@':%$4$.@'&@'&4'$]1"##2':#"14&7#$'@;"@'"#@$%."@&-$'6%'"33&@&6."#'
M6@B;'4&8."#&.8'B6D:6.$.@4'"%$'@"%8$@4'6E'=(KLI'".3a6%'=,/LIH)''(.'&.@%&81&.8'
:644&7&#&@2'&4'@;$'Y,NLI'E"D&#2'6E'@%".4B%&:@&6.'E"B@6%4'@;"@'"%$'$V:%$44$3'1:6.'M6@B;'
"B@&-"@&6.'&.'$"B;'6E'@;$'$"%#2'$D7%26.&B'4&8."#&.8'$-$.@4)''/6?'#$-$#4'6E'$D7%26.&B'
Hale et al.
34
M6@B;'4&8."#&.8'B61#3'7$'"D:#&E&$3'72'4@"7&#&b&.8'@;$'Y,NLI':%6@$&.4)''_.@$%$4@&.8#2+'
Y,NLI':%6@$&.4';"-$'7$$.'4;6?.'@6'%$:%$44$4'@;$'@%".4B%&:@&6.'6E'/_MLIJ+'/(0LJ+'".3'
@;$'QFgLOP'".3'QFgLOU'@%".4B%&:@&6.'E"B@6%4+'@;$%$72':%$-$.@&.8'B$##4'E%6D'
:"%@&B&:"@&.8'&.'4174$]1$.@'M6@B;'4&8."#&.8'$-$.@4[M,!,='".3'^Y_,=='JHHT\)'''M$-$4'
".3'^%&$44';"-$'4:$B1#"@$3'@;"@'%":&3'3$8%"3"@&6.'6E'Y,NLI':%6@$&.4'D14@'6BB1%'&.'
6%3$%'@6'3$L%$:%$44'/_MLIJ'$V:%$44&6.'&.'3$4B$.3".@'B$##4)''('%6#$'E6%'=(KLI'&.'Y,NLI'
3$8%"3"@&6.'B61#3'$V:#"&.'@;$'$D7%26.&B'#$@;"#&@2'B"14$3'72',"%12'D1@"@&6.4'&.'/.012L
3$E&B&$.@'$D7%264R'=(KLI'%$D6-"#'?61#3'4@"7&#&b$'Y,NLI':%6@$&.4+'".3'@;$%$72'
:%$-$.@'.6%D"#'$D7%26.&B'.*$12>'$V:%$44&6.+'B%$"@&.8'"'4&@1"@&6.'@;"@'?61#3'.6@'7$'
3$@%&D$.@"#'@6'$D7%264'[7$B"14$'6E'@;$'%$31.3".B2'6E'.*$12>-".3-/.012\'1.#$44'@;$'
$D7%26'"#46';"4'%$31B$3'/.012'E1.B@&6.+'"4'&.'@;$'B"4$'6E'@;$'/.0123.H8R-,"%12'D1@".@4'
"."#2b$3';$%$)'
'
=(KLI'36$4'.6@'"::$"%'@6'7$'".'67#&8"@$':"%@.$%'6E'=,/LIH'7$B"14$'#644'6E'=(KL
I';"4'.6'&D:"B@'6.'4$-$%"#'6@;$%':%6B$44$4'@;"@'"%$'"EE$B@$3'72'=,/LIH+'41B;'"4'(<a!`'
".3'1@$%&.$'v'B$##'4:$B&E&B"@&6.+'"4'?$##'"4'46D"@&B'4$V'3$@$%D&."@&6.'[4$$'Q"7#$4'=IL
=S\)''_.'@;$4$'B6.@$V@4+'@;$'"74$.B$'6E'=(KLI'6%'@;$'&."7&#&@2'6E'=,/LIH'@6'&.@$%"B@'?&@;'
=(KLI'36$4'.6@':%$-$.@'=,/LIH'E%6D'36?.'%$81#"@&.8'&@4'@"%8$@4)''Q;$'3&EE$%$.B$'
7$@?$$.'$-$.@4'@;"@'%$]1&%$'=,/LIH'".3'=(KLI'-$%414'@;64$'@;"@'%$]1&%$'6.#2'=,/LIH'
B61#3';"-$'@6'36'?&@;'@;$'4:$B&E&B'4174@%"@$+'41B;'"4'0/^LI'-$%414'/_MLIJ+'6%'?&@;'@;$'
4:$B&E&B'B$##1#"%'B6.@$V@+'41B;'"4'8$%D#&.$'-$%414'46D"@&B'@&441$+'4&.B$'?$';"-$'
674$%-$3'".'$EE$B@'6E'=(KLI'6.'0/^LILD$3&"@$3'8$%D#&.$'$-$.@4+'71@'.6@'6.'/_MLIJL
D$3&"@$3'46D"@&B'$-$.@4)''_E'=(KLI'E1.B@&6.4'"4'"'=,/LIH'B6LE"B@6%+'&@'D&8;@'"B@'@6'
Hale et al.
35
7%6"3$.'@;$'%$:$%@6&%$'6E'=,/LIH'4174@%"@$4+'41B;'@;"@'"'3&4@&.B@'"%%"2'6E'4174@%"@$4'
"%$'%$B68.&b$3'72'=(KLILB6.@"&.&.8'@&441$4'B6D:"%$3'@6'@&441$4'@;"@'#"BZ'=(KLI)'
'
=(KLI'D"2'"#46'7$'"7#$'@6'&.E#1$.B$'M6@B;'4&8."#&.8'?&@;61@'=,/LIH+'-&"'
&.@$%"B@&6.4'?&@;'"#@$%."@&-$':%6@$&.4)''Q;&4'.6@&6.'&4'41::6%@$3'72'@;$'E"B@'@;"@'
$D7%26.&B'M6@B;'4&8."#&.8'B".'7$'&.B%$"4$3'&.'"0'123452678'$D7%264'72'B6D:#$@$#2'
%$D6-&.8',"%12'"B@&-&@2+'71@'.6@'72'B6D:#$@$#2'%$D6-&.8',#.129'"B@&-&@2+'4188$4@&.8'
@;"@'=(KLILD$3&"@$3'36?.'%$81#"@&6.'B".'4@&##'6BB1%'&.'@;$'"74$.B$'6E'=,/LIH)''F2'
B6.@%"4@+'@;$'36D&.".@'.$8"@&-$'=,/LIH[0TeP,\':%6@$&.+'$.B63$3'72',#.1293$29778+'
&.@$%E$%$4'?&@;'M6@B;'36?.'%$81#"@&6.'&.'@;$'$D7%26)''Q;&4'$EE$B@'&4'4&D&#"%'@6'@;"@'6E'
,#.129'D1@"@&6.4'&.'@;$'B6.@$V@'6E'*=M'3$-$#6:D$.@+'?;$%$',#.1293$29778<-71@'.6@',#.1
293$5..8+'B"14$4'"'D1@".@':;$.6@2:$+'".3'B6.@%"4@4'?&@;'@;$'$EE$B@'6E',#.129'D1@"@&6.4'
6.'-1#-"#'3$-$#6:D$.@+'?;$%$',#.1293$29778'B"14$4'@;$'4"D$'3$E$B@&-$':;$.6@2:$'"4'
B"14$3'72',#.1293$5..8'[9(0,Y-#+-".:'JHHS\)''9"8$%'".3'B6##$"81$4':%6:64$3'@;"@'@;$'*=M'
:%$B1%46%'B$##4'D&8;@'B6.@"&.'"#@$%."@&-$'NL76V':%6@$&.4'@;"@'B61#3'4174@&@1@$'E6%'=,/L
IH'&.'&@4'"74$.B$+'71@'@;"@'.6.LE1.B@&6."#'=<N=,/LIH[0TeP,\''B6D:#$V$4'D&8;@':%$-$.@'
41B;'4174@&@1@&6.+'%$41#@&.8'&.'"'D6%$'4$-$%$':;$.6@2:$'E6%',#.1293$29778'@;".',#.1
293$5..8)''K1%'%$41#@4'"%$'B6.4&4@$.@'?&@;'@;&4'D63$#+'".3'4188$4@'@;"@'@;$'$D7%26'D"2'
B6.@"&.'NL76V':%6@$&.4'@;"@'B".'"B@'%$31.3".@#2'?&@;'=,/LIH)''Q;14'=,/LIHL3$E&B&$.@'
$D7%264'D"2'7$'"7#$'@6'"44$D7#$'"#@$%."@&-$'=<N'B6D:#$V$4'@;"@'B".'&.@$%"B@'?&@;'
=(KLI+':%6-&3&.8'"'3&EE$%$.@'%61@$'@6'=(KLILD$3&"@$3'36?.'%$81#"@&6.'6E'M6@B;'
4&8."#&.8)'
'
Hale et al.
36
(#@;618;'?$';"-$'&3$.@&E&$3'=(KLI'".3'4@13&$3'&@4'%6#$'&.'%$#"@&6.4;&:'@6'M6@B;'
4&8."#&.8+'4$-$%"#'B#1$4'4188$4@'@;"@'=(KLI'E1.B@&6.'&4'.6@'#&D&@$3'@6'@;$'M6@B;'
:"@;?"2)''N&%4@+'?6%D4'@;"@'"%$'3$E&B&$.@'&.',"%12'E1.B@&6.'".3'%"&4$3'"@'JTq<'$V;&7&@'
4$-$%"#'&.B6D:#$@$#2':$.$@%".@':;$.6@2:$4+'46D$'6E'?;&B;'36'.6@'%$4$D7#$'Z.6?.'
M6@B;'D1@".@':;$.6@2:$4'[41B;'"4';&8;'&.B&3$.B$'6E'D"#$4'".3'$88'%$@$.@&6.\)''
=$B6.3+'&.'"'4@132'6E'8$%D#&.$L$V:%$44$3'8$.$4+',"%12'?"4'B#"44&E&$3'"D6.8'@;$'ITP'
D64@';&8;#2'$V:%$44$3'8$.$4'[61@'6E'"'@6@"#'6E'SeWW'8$%D#&.$L$V:%$44$3'8$.$4d'A(M0-
#+-".:'JHHW\)''K@;$%';&8;#2'$V:%$44$3'8$.$4'&.'@;&4'4@132'&.B#13$'8$.$4'&.-6#-$3'&.'
@%".4#"@&6.+'YM('7&.3&.8+'".3'17&]1&@&.a:%6@$646D$':%6@$&.'3$8%"3"@&6.d'&.'B6.@%"4@'
M6@B;'4&8."#&.8'B6D:6.$.@4'"%$'$V:%$44$3'"@'D1B;'D6%$'D63$4@'#$-$#4)''('@;&%3';&.@'
@;"@',"%12'D"2'7$'&.-6#-$3'&.':%6B$44$4'6@;$%'@;".'M6@B;'4&8."#&.8'B6D$4'E%6D'&@4'
&3$.@&E&B"@&6.'"4'6.$'6E'JI'YM(&'@"%8$@4'@;"@'B".'41::%$44'@;$'B2@6Z&.$4&4'3$E$B@4'6E'
&H.12'D1@".@'$D7%264'[CKYNG(M-#+-".:'JHHW\)''_.@$%$4@&.8#2+'@;&4'4B%$$.'"#46'&3$.@&E&$3'
,#.129[YM(&\'"4'"'D631#"@6%'6E'&H.12'"B@&-&@2+';6?$-$%'@;$'$EE$B@'6E',#.129'YM(&'?"4'
6::64&@$'@6'@;"@'6E',"%12'YM(&)''Q;$'YN/LI'8$.$':%631B@'&4':"%@'6E'@;$'M$33U':%6@$&.'
B6.m18"@&6.':"@;?"2'@;"@'.$332#"@$4'B1##&.L7"4$3',O'17&]1&@&.'#&8"4$4+'".3'B61#3'
%$:%$4$.@'".6@;$%'@"%8$@'6E'=(KLIL=,/LIH'B6D7&.$3'"B@&-&@2)'
'
'
'' =(KLI':%6-&3$4'"'14$E1#'D63$#'?&@;'?;&B;'@6'4@132'@;$'%6#$4'6E'05N'36D"&.'
:%6@$&.4'&.'"'8$.$@&B"##2'@%"B@"7#$'".&D"#'424@$D)''05NL36D"&.'B6.@"&.&.8':%6@$&.4'
8$.$%"##2'36'.6@'B6.@"&.'"33&@&6."#'4;"%$3'4$]1$.B$'D6@&E4+'4188$4@&.8'@;"@'@;&4'
36D"&.'&4'"'4$#ELB6.@"&.$3'D631#$'14$3'&.':%6@$&.4'6E'-"%&614'E1.B@&6.4)''Q;$'!:-
Hale et al.
37
#.#/"$,'8$.6D$'B6.@"&.4'@;%$$'"33&@&6."#'8$.$4'@;"@'$.B63$'?$##LB6.4$%-$3'05N'
36D"&.4+'71@'.6.$'4;6?'".2'4$]1$.B$'4&D&#"%&@2'?&@;'=(KLI'61@4&3$'6E'@;$'05N'
36D"&.'[>KN/,Y'".3'NY,`MC'JHHe\)''_.'2$"4@+'05NL36D"&.':%6@$&.4';"-$'7$$.'
"446B&"@$3'?&@;'"'-"%&$@2'6E'B$##1#"%'E1.B@&6.4+'41B;'"4'<K^__'-$4&B#$'
E6%D"@&6.[*_0(=*_K-#+-".:'JHHU\+'D$D7%".$'@%"EE&B[0,KY0_,!-#+-".:'JHHU\+'8$.$L4:$B&E&B'
&.;&7&@&6.'6E'@%".4#"@&6.'&.&@&"@&6.[=,f,M-#+-".:'JHHW\+'".3'DYM('4:#&B&.8[F_(/>KA=>('
".3'>`Y/(MCf>('JHHJ\)''_3$.@&E&B"@&6.'6E'7&.3&.8':"%@.$%4'E6%';1D".'05N'36D"&.4'
"#46'41::6%@4'&.-6#-$D$.@'&.'-$4&B#$'@%".4:6%@'".3'DYM(':%6B$44&.8[(=*-#+-".:'JHIHd'
>KN/,Y-#+-".:'JHHW\)'Q;$'05N'36D"&.'6E'=(KLI'D64@'B#64$#2'%$4$D7#$4'@;64$'6E'@;$'
=G5LJ'B#"44'6E'05N'36D"&.4+'?;&B;'&.B#13$4'@;$'D"DD"#&".'0_05NI'".3'0_05NJ'
:%6@$&.4)''C&4%1:@&6.'6E'@;$'D614$'8$.$'0_05NJ'B"14$4'"'.$1%63$8$.$%"@&-$'
:;$.6@2:$'"4'?$##'"4'"#@$%$3'&.41#&.'%$B$:@6%'E1.B@&6.[0_K!(MMKM,-#+-".:'JHHW\+'71@'
@;$'D6#$B1#"%'B6..$B@&6.'7$@?$$.'@;$4$':;$.6@2:$4'".3'0_05NJ'E1.B@&6.'%$D"&.4'
1.Z.6?.)'!!!
'
_.'"33&@&6.'@6'&@4'05N'36D"&.+'=(KLI'B6.@"&.4'"'<L@$%D&."#'$V@$.4&6.'@;"@'&4'
B6.4$%-$3'"D6.8'.$D"@63$'4:$B&$4'".3'&4'B6.4:&B1614#2'$.%&B;$3'&.':%6#&.$'
%$4&31$4+'&.B#13&.8'"'^^0'4$]1$.B$'@;"@'&4'"'B6DD6.'%$B68.&@&6.'4$]1$.B$'E6%'05N'
36D"&.4[>KN/,Y-#+-".:'JHHT\)''Q;$':%$4$.B$'6E'"'05N'%$B68.&@&6.'D6@&E'&.'6@;$%'05N'
36D"&.':%6@$&.4';"4'7$$.';2:6@;$4&b$3'@6'E"B&#&@"@$'&.@%"D6#$B1#"%'
&.@$%"B@&6.4[NY,`MC-#+-".:'JHHO\)''(.'&.@%"D6#$B1#"%'&.@$%"B@&6.'7$@?$$.'@;$'05N'".3'
@;$'<L@$%D&."#':6%@&6.'6E'=(KLI'B61#3'$V:#"&.';6?'@;$'=(KLIL=,/LIH'&.@$%"B@&6.'&4'
3&4%1:@$3'72'@;$',"%123*G28'D1@"@&6.'&.'@;$'05N'36D"&.+'3$4:&@$'@;$'05N'36D"&.'
Hale et al.
38
7$&.8'3&4:$.4"7#$'E6%'=(KLIL=,/LIH'&.@$%"B@&6.)''N6%'$V"D:#$+'@;$'*G2'D1@"@&6.'D&8;@'
$.;".B$'@;$'&.@%"D6#$B1#"%'7&.3&.8'6E'=(KLI+'@;$%$72'%$31B&.8'@;$'"7&#&@2'@6'7&.3'
6@;$%':%6@$&.4'41B;'"4'=,/LIH)''KE'B61%4$+'@;$'=(KLI'05N'36D"&.'&@4$#E'D"2'"#46'7$'
&.-6#-$3'&.';$@$%6@2:&B'&.@$%"B@&6.4'?&@;'"4'2$@'1.&3$.@&E&$3':"%@.$%4)'''
'
(#@;618;'@;$':%$B&4$'D6#$B1#"%'E1.B@&6.'6E'=(KLI'%$D"&.4'1.Z.6?.+'61%'
"."#24$4'6E'-"%&614':;24&B"#'".3'8$.$@&B'&.@$%"B@&6.4'41::6%@'"'4&D:#$'D63$#'&.'?;&B;'
=(KLI'"B@4'"4'"'B6LE"B@6%'"#6.8'?&@;'@;$'=,/LIH'4174@%"@$L%$B68.&@&6.'D6&$@2'6E'@;$'
=<N'17&]1&@&.'#&8"4$'@6'36?.'%$81#"@$'6.$'6%'D6%$'B6D:6.$.@4'6E'M6@B;'4&8."#&.8)''
Q;$'$EE$B@'6E':%$-$.@&.8'@;&4'&.@$%"B@&6.'[$)8)+'72'3$B%$"4&.8'=(KLI'#$-$#4'6%'72'
3&4%1:@&.8'@;$'=(KLIL=,/LIH'&.@$%"B@&6.'@;%618;':6&.@'D1@"@&6.4'&.'$&@;$%':%6@$&.\'
"::$"%4'@6'7$'".'&.B%$"4$'&.'M6@B;'4&8."#&.8'&.'B$%@"&.'B6D:%6D&4$3'4$@@&.84)''A;&#$'
@;$'=,/LIH':%6@$&.'".3'&@4'%6#$'&.'M6@B;'36?.'%$81#"@&6.'&4'4@%1B@1%"##2'".3'
E1.B@&6."##2'B6.4$%-$3+'@;$'=(KLI':%6@$&.'36$4'.6@';"-$'67-&614'6%@;6#684'&.'6@;$%'
6%8".&4D4)''Q;&4'#"BZ'6E'B6.4$%-"@&6.'D"2'%$E#$B@'@;$'3&-$%4&@2'6E'4174@%"@$4'@;"@'"%$'
%$B68.&b$3'72'=<NN7?P'B6D:#$V$4'".3'@;$'-"%&$@2'6E':%6@$&.4'@;"@'D"2':%6-$'@6'
"446B&"@$'?&@;'B6.4$%-$3'=<NN7?P'B6D:#$V$4'&.'6%3$%'@6'"#@$%'@;$&%'&.@$%"B@&6.'?&@;'
4174@%"@$4)''A;&#$'=(KLI'&4'&D:6%@".@'31%&.8'8$%D#&.$'".3'$"%#2'$D7%26.&B'
3$-$#6:D$.@+'1.%$#"@$3'71@'E1.B@&6."##2'"."#68614':%6@$&.4'D&8;@'B6.@%&71@$'@6'N7?P'
4174@%"@$'4:$B&E&B&@2'&.'6@;$%'@&441$4'".3'4@"8$4)''*$.B$+'E1%@;$%'$V:#6%"@&6.'6E'@;$'
D6#$B1#"%'%6#$'6E'=(KLI'?&##':%6-&3$'"'14$E1#'D63$#'E6%'05N'36D"&.'E1.B@&6.+'=<N'
"BB$446%2'E"B@6%4+'".3'M6@B;':"@;?"2'%$81#"@6%4'&.'"'-"%&$@2'6E'".&D"#'424@$D4)''
'
Hale et al.
39
'
'
(<>MKA/,C0,G,MQ='
A$'"%$'8%"@$E1#'@6'9&D'^%&$44'&.'?;64$'#"7'@;$'E$"4&7&#&@2'6E'".'"0'12'41::%$446%'
4B%$$.'?"4'E&%4@'$V:#6%$3)''A$'@;".Z'F"%7"%"'<6.%"3@+'_-"'0%$$.?"#3+'A$.32'*".."L
Y64$+'K#&-$%'*67$%@+'9".$'*177"%3+'913&@;'>&D7#$+'9&D'^%&$44+'".3'^$@$%'^%2B&"Z'E6%'
4@%"&.4'".3'%$"8$.@4)''=6D$'4@%"&.4'14$3'&.'@;&4'?6%Z'?$%$'41::#&$3'72'@;$'
<"$.6%;"73&@&4'0$.$@&B4'<$.@$%+'?;&B;'&4'E1.3$3'72'@;$'M"@&6."#'_.4@&@1@$4'6E'*$"#@;'
[M_*\'M"@&6."#'<$.@$%'E6%'Y$4$"%B;'Y$461%B$4'[M<YY\)''Q;$',"%123%GAAAB8'"##$#$'?"4'
8$.$%"@$3'".3':%6-&3$3'72'@;$'<)'$#$8".4'0$.$'>.6BZ61@'<6.46%@&1D'[`)'6E'
G&..$46@"\+'?;&B;'&4'E1.3$3'72'@;$'M_*)''A$'@;".Z'=;&.'5&'/&.+'964$@@$'G".b".6+',##$.'
/$EE#$%+'<"%6#2.'>61#"%&4+',#&.6%'/$$+'/$&8;'*"%%&4+'(##246.'Y&.3$%#$+'Y$7$BB"'Y$4.&BZ+'
".3'<#"%$'*6?"%3'E6%'B6.@%&71@&6.4'@6'@;&4'?6%Z+'".3'".6.2D614'%$-&$?$%4'E6%'
@;618;@E1#'B6DD$.@4'6.'@;$'D".14B%&:@)''Q;&4'%$4$"%B;'?"4'41::6%@$3'72'@;$'
M"@&6."#'=B&$.B$'N61.3"@&6.'[0%".@'p_FMLHIOIeTP\)'
'
'
Hale et al.
40
'
LITERATURE CITED
ASH, M. R., K. FAELBER, D. KOSSLICK, G. I. ALBERT, Y. ROSKE et al., 2010

Conserved beta-hairpin recognition by the GYF domains of Smy2
and GIGYF2 in mRNA surveillance and vesicular transport
complexes. Structure 18: 944-954.
BERRY, L. W., B. WESTLUND and T. SCHEDL, 1997 Germ-line tumor formation
caused by activation of glp-1, a Caenorhabditis elegans member of
the Notch family of receptors. Development 124: 925-936.
BIALKOWSKA, A., and A. KURLANDZKA, 2002 Proteins interacting with Lin 1p,
a putative link between chromosome segregation, mRNA splicing
and DNA replication in Saccharomyces cerevisiae. Yeast 19: 1323-
1333.
BOSU, D. R., and E. T. KIPREOS, 2008 Cullin-RING ubiquitin ligases: global
regulation and activation cycles. Cell Div 3: 7.
BRENNER, S., 1974 The genetics of Caenorhabditis elegans. Genetics 77:
71-94.
DALFO, D., J. R. PRIESS, R. SCHNABEL and E. J. HUBBARD, 2010 glp-1(e2141)
Sequence Correction, pp. 4 in The Worm Breeder's Gazette.
DE SOUZA, N., L. G. VALLIER, H. FARES and I. GREENWALD, 2007 SEL-2, the C.
elegans neurobeachin/LRBA homolog, is a negative regulator of lin-
12/Notch activity and affects endosomal traffic in polarized
epithelial cells. Development 134: 691-702.
DESAI, C., and H. R. HORVITZ, 1989 Caenorhabditis elegans mutants
defective in the functioning of the motor neurons responsible for egg
laying. Genetics 121: 703-721.
DORFMAN, M., J. E. GOMES, S. O'ROURKE and B. BOWERMAN, 2009 Using RNA
interference to identify specific modifiers of a temperature-sensitive,
embryonic-lethal mutation in the Caenorhabditis elegans ubiquitin-
like Nedd8 protein modification pathway E1-activating gene rfl-1.
Genetics 182: 1035-1049.
FORTINI, M. E., 2009 Notch signaling: the core pathway and its
posttranslational regulation. Dev Cell 16: 633-647.
FORTINI, M. E., and D. BILDER, 2009 Endocytic regulation of Notch
signaling. Curr Opin Genet Dev 19: 323-328.
FRANCIS, R., G. MCGRATH, J. ZHANG, D. A. RUDDY, M. SYM et al., 2002 aph-1
and pen-2 are required for Notch pathway signaling, gamma-
secretase cleavage of betaAPP, and presenilin protein accumulation.
Dev Cell 3: 85-97.
FREUND, C., R. KUHNE, S. PARK, K. THIEMKE, E. L. REINHERZ et al., 2003
Structural investigations of a GYF domain covalently linked to a
proline-rich peptide. J Biomol NMR 27: 143-149.
Hale et al.
41
GANOTH, D., G. BORNSTEIN, T. K. KO, B. LARSEN, M. TYERS et al., 2001 The

cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-
mediated ubiquitinylation of p27. Nat Cell Biol 3: 321-324.
GEORGIEV, A., A. LEIPUS, I. OLSSON, J. M. BERREZ and A. MUTVEI, 2008
Characterization of MYR1, a dosage suppressor of YPT6 and RIC1
deficient mutants. Curr Genet 53: 235-247.
GIOVANNONE, B., W. G. TSIARAS, S. DE LA MONTE, J. KLYSIK, C. LAUTIER et al.,
2009 GIGYF2 gene disruption in mice results in neurodegeneration
and altered insulin-like growth factor signaling. Hum Mol Genet 18:
4629-4639.
GOUTTE, C., M. TSUNOZAKI, V. A. HALE and J. R. PRIESS, 2002 APH-1 is a
multipass membrane protein essential for the Notch signaling
pathway in Caenorhabditis elegans embryos. Proc Natl Acad Sci U S
A 99: 775-779.
GRANT, B., and I. GREENWALD, 1996 The Caenorhabditis elegans sel-1 gene,
a negative regulator of lin-12 and glp-1, encodes a predicted
extracellular protein. Genetics 143: 237-247.
GREENWALD, I., 2005 LIN-12/Notch signaling in C. elegans. WormBook: 1-
16.
GUPTA-ROSSI, N., O. LE BAIL, H. GONEN, C. BROU, F. LOGEAT et al., 2001
Functional interaction between SEL-10, an F-box protein, and the
nuclear form of activated Notch1 receptor. J Biol Chem 276: 34371-
34378.
HANNA-ROSE, W., and M. HAN, 1999 COG-2, a sox domain protein
necessary for establishing a functional vulval-uterine connection in
Caenorhabditis elegans. Development 126: 169-179.
HIGASHIO, H., K. SATO and A. NAKANO, 2008 Smy2p participates in COPII
vesicle formation through the interaction with Sec23p/Sec24p
subcomplex. Traffic 9: 79-93.
HOLLENBERG, S. M., R. STERNGLANZ, P. F. CHENG and H. WEINTRAUB, 1995
Identification of a new family of tissue-specific basic helix-loop-helix
proteins with a two-hybrid system. Mol Cell Biol 15: 3813-3822.
HUBBARD, E. J., G. WU, J. KITAJEWSKI and I. GREENWALD, 1997 sel-10, a
negative regulator of lin-12 activity in Caenorhabditis elegans,
encodes a member of the CDC4 family of proteins. Genes Dev 11:
3182-3193.
HUTTER, H., and R. SCHNABEL, 1994 glp-1 and inductions establishing
embryonic axes in C. elegans. Development 120: 2051-2064.
JAGER, S., H. T. SCHWARTZ, H. R. HORVITZ and B. CONRADT, 2004 The
Caenorhabditis elegans F-box protein SEL-10 promotes female
development and may target FEM-1 and FEM-3 for degradation by
the proteasome. Proc Natl Acad Sci U S A 101: 12549-12554.
KILLIAN, D. J., E. HARVEY, P. JOHNSON, M. OTORI, S. MITANI et al., 2008 SKR-
1, a homolog of Skp1 and a member of the SCF(SEL-10) complex,
regulates sex-determination and LIN-12/Notch signaling in C.
elegans. Dev Biol 322: 322-331.
Hale et al.
42
KIM, S. K., J. LUND, M. KIRALY, K. DUKE, M. JIANG et al., 2001 A gene

expression map for Caenorhabditis elegans. Science 293: 2087-
2092.
KIMBLE, J., and S. L. CRITTENDEN, 2005 Germline proliferation and its
control. WormBook: 1-14.
KODOYIANNI, V., E. M. MAINE and J. KIMBLE, 1992 Molecular basis of loss-of-
function mutations in the glp-1 gene of Caenorhabditis elegans. Mol
Biol Cell 3: 1199-1213.
KOFLER, M., K. MOTZNY and C. FREUND, 2005 GYF domain proteomics
reveals interaction sites in known and novel target proteins. Mol Cell
Proteomics 4: 1797-1811.
KOFLER, M., M. SCHUEMANN, C. MERZ, D. KOSSLICK, A. SCHLUNDT et al., 2009
Proline-rich sequence recognition: I. Marking GYF and WW domain
assembly sites in early spliceosomal complexes. Mol Cell Proteomics
8: 2461-2473.
KOFLER, M. M., and C. FREUND, 2006 The GYF domain. FEBS J 273: 245-
256.
KOPAN, R., and M. X. ILAGAN, 2009 The canonical Notch signaling pathway:
unfolding the activation mechanism. Cell 137: 216-233.
KUSHNIROV, V. V., 2000 Rapid and reliable protein extraction from yeast.
Yeast 16: 857-860.
LAI, E. C., 2002 Protein degradation: four E3s for the notch pathway. Curr
Biol 12: R74-78.
LAMBIE, E. J., and J. KIMBLE, 1991 Two homologous regulatory genes, lin-
12 and glp-1, have overlapping functions. Development 112: 231-
240.
LI, J., A. M. PAULEY, R. L. MYERS, R. SHUANG, J. R. BRASHLER et al., 2002
SEL-10 interacts with presenilin 1, facilitates its ubiquitination, and
alters A-beta peptide production. J Neurochem 82: 1540-1548.
LI, S., C. M. ARMSTRONG, N. BERTIN, H. GE, S. MILSTEIN et al., 2004 A map of
the interactome network of the metazoan C. elegans. Science 303:
540-543.
MANGO, S. E., E. M. MAINE and J. KIMBLE, 1991 Carboxy-terminal
truncation activates glp-1 protein to specify vulval fates in
Caenorhabditis elegans. Nature 352: 811-815.
MOURIKIS, P., R. J. LAKE, C. B. FIRNHABER and B. S. DEDECKER, 2010
Modifiers of notch transcriptional activity identified by genome-wide
RNAi. BMC Dev Biol 10: 107.
MUKHOPADHYAY, D., and H. RIEZMAN, 2007 Proteasome-independent
functions of ubiquitin in endocytosis and signaling. Science 315:
201-205.
NEVES, A., and J. R. PRIESS, 2005 The REF-1 family of bHLH transcription
factors pattern C. elegans embryos through Notch-dependent and
Notch-independent pathways. Dev Cell 8: 867-879.
PARKS, A. L., and D. CURTIS, 2007 Presenilin diversifies its portfolio. Trends
Genet 23: 140-150.
Hale et al.
43
PEPPER, A. S., D. J. KILLIAN and E. J. HUBBARD, 2003 Genetic analysis of

Caenorhabditis elegans glp-1 mutants suggests receptor interaction
or competition. Genetics 163: 115-132.
PETROSKI, M. D., and R. J. DESHAIES, 2005 Function and regulation of
cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 6: 9-20.
PRIESS, J. R., 2005 Notch signaling in the C. elegans embryo. WormBook:
1-16.
PRIESS, J. R., H. SCHNABEL and R. SCHNABEL, 1987 The glp-1 locus and
cellular interactions in early C. elegans embryos. Cell 51: 601-611.
PRYCIAK, P. M., and L. H. HARTWELL, 1996 AKR1 encodes a candidate
effector of the G beta gamma complex in the Saccharomyces
cerevisiae pheromone response pathway and contributes to control
of both cell shape and signal transduction. Mol Cell Biol 16: 2614-
2626.
QIAO, L., J. L. LISSEMORE, P. SHU, A. SMARDON, M. B. GELBER et al., 1995
Enhancers of glp-1, a gene required for cell-signaling in
Caenorhabditis elegans, define a set of genes required for germline
development. Genetics 141: 551-569.
REINKE, V., H. E. SMITH, J. NANCE, J. WANG, C. VAN DOREN et al., 2000 A
global profile of germline gene expression in C. elegans. Mol Cell 6:
605-616.
SEZEN, B., M. SEEDORF and E. SCHIEBEL, 2009 The SESA network links
duplication of the yeast centrosome with the protein translation
machinery. Genes Dev 23: 1559-1570.
SPIETH, J., G. BROOKE, S. KUERSTEN, K. LEA and T. BLUMENTHAL, 1993
Operons in C. elegans: polycistronic mRNA precursors are processed
by trans-splicing of SL2 to downstream coding regions. Cell 73: 521-
532.
SPRUCK, C., H. STROHMAIER, M. WATSON, A. P. SMITH, A. RYAN et al., 2001 A
CDK-independent function of mammalian Cks1: targeting of
SCF(Skp2) to the CDK inhibitor p27Kip1. Mol Cell 7: 639-650.
STAROSTINA, N. G., J. M. LIM, M. SCHVARZSTEIN, L. WELLS, A. M. SPENCE et al.,
2007 A CUL-2 ubiquitin ligase containing three FEM proteins
degrades TRA-1 to regulate C. elegans sex determination. Dev Cell
13: 127-139.
SUNDARAM, M., and I. GREENWALD, 1993a Genetic and phenotypic studies of
hypomorphic lin-12 mutants in Caenorhabditis elegans. Genetics
135: 755-763.
SUNDARAM, M., and I. GREENWALD, 1993b Suppressors of a lin-12
hypomorph define genes that interact with both lin-12 and glp-1 in
Caenorhabditis elegans. Genetics 135: 765-783.
SZE, J. Y., M. VICTOR, C. LOER, Y. SHI and G. RUVKUN, 2000 Food and
metabolic signalling defects in a Caenorhabditis elegans serotonin-
synthesis mutant. Nature 403: 560-564.
Hale et al.
44
VERHEYEN, E. M., K. J. PURCELL, M. E. FORTINI and S. ARTAVANIS-TSAKONAS,

1996 Analysis of dominant enhancers and suppressors of activated
Notch in Drosophila. Genetics 144: 1127-1141.
WALHOUT, A. J., R. SORDELLA, X. LU, J. L. HARTLEY, G. F. TEMPLE et al., 2000
Protein interaction mapping in C. elegans using proteins involved in
vulval development. Science 287: 116-122.
WANG, M., and P. W. STERNBERG, 2000 Patterning of the C. elegans 1
degrees vulval lineage by RAS and Wnt pathways. Development 127:
5047-5058.
WANG, X., Y. ZHAO, K. WONG, P. EHLERS, Y. KOHARA et al., 2009 Identification
of genes expressed in the hermaphrodite germ line of C. elegans
using SAGE. BMC Genomics 10: 213.
WELCKER, M., and B. E. CLURMAN, 2008 FBW7 ubiquitin ligase: a tumour
suppressor at the crossroads of cell division, growth and
differentiation. Nat Rev Cancer 8: 83-93.
WEN, C., and I. GREENWALD, 1999 p24 proteins and quality control of LIN-
12 and GLP-1 trafficking in Caenorhabditis elegans. J Cell Biol 145:
1165-1175.
WU, G., E. J. HUBBARD, J. K. KITAJEWSKI and I. GREENWALD, 1998 Evidence
for functional and physical association between Caenorhabditis
elegans SEL-10, a Cdc4p-related protein, and SEL-12 presenilin.
Proc Natl Acad Sci U S A 95: 15787-15791.
WU, G., S. LYAPINA, I. DAS, J. LI, M. GURNEY et al., 2001 SEL-10 is an
inhibitor of notch signaling that targets notch for ubiquitin-mediated
protein degradation. Mol Cell Biol 21: 7403-7415.
Hale et al.
45
N_0`Y,'/,0,MC='
;()B>.!6H!!)'*+,0*7999:5!'B==>.''.'!'./+,012,345#+*/#'./+,012,;95"'
Y$:%$4$.@"@&-$'@$%D&."#'4@"8$'$D7%264'-&$?$3'72'#&8;@'D&B%64B6:2d'
"%%6?;$"3'&.3&B"@$4':64@$%&6%':;"%2.V'".3'"%%6?4'&.3&B"@$'".@$%&6%':;"%2.V'&E'
:%$4$.@d'$D7%264'"%$'6%&$.@$3':64@$%&6%'@6'@;$'%&8;@'".3'B6##$B@$3'E%6D'D6@;$%4'?&@;'
@;$'E6##6?&.8'8$.6@2:$4R'[(\'5$=1>?3#297>8-"0'123452678d'[F\-5$=1>?3#297>8-"0'1
23452678R-,"%123%GAAAB8R-[<\'5$=1>?3#297>8-"0'123452>A8R-[C\-5$=1>?3#297>8-"0'1
23452>A8R-,"%123%GAAAB8:--Q;$'$D7%26'4;6?.'&.'[<\';"4'E"&#$3'@6'1.3$%86'7632'
D6%:;68$.$4&4d'&.4@$"3'6E'E1##2'$.B#64&.8'@;$'$D7%26+'@;$';2:63$%D"#'B$##4'"%$'
B#1D:$3'6.'6.$'4&3$+'#$"-&.8'@;$':64@$%&6%':;"%2.V'$V:64$3'6.'@;$'6@;$%'4&3$'6E'@;$'
$D7%26)'''=$$'Q"7#$'J'E6%'@;$'E%$]1$.B2'6E'@;$4$':;$.6@2:$4'"D6.8'$D7%264)'
'
;()B>.!RH!!)'*+,!).*.!,#%B'!+*/!=>#$.(*!'.CB.*%.S'
[(\'=B;$D"@&B'%$:%$4$.@"@&6.'6E',"%12':64&@&6.'6.'B;%6D646D$'!'".3'@;$'
%$#$-".@'B;%6D646D"#'3$#$@&6.4'[$;H6>'".3'*+;H>'@;"@'%$D6-$'@;$',"%12'#6B14\+'
B64D&34'[<HeFO'".3'YIHCIJ\+'".3'=M^4'[NTU,IHlOh'&4':64&@&6.'!RIOWWSSWU'".3'
NTTFIJlIh'&4':64&@&6.'!RIOUIWSWU\'@;"@'"%$'D$.@&6.$3'&.'@;$'@$V@)''[F\'^%$3&B@$3'
:%6@$&.'4$]1$.B$'6E'=(KLI'?&@;'05N'36D"&.'4;"3$3'".3'"'76V'"%61.3'@;$'%$4&31$'@;"@'
&4'"#@$%$3'72'@;$'*G2'D1@"@&6.)''[<\'(#&8.D$.@'6E'=(KLI'05N'36D"&.'?&@;'D64@'%$#"@$3'
05N'36D"&.4'E%6D'6@;$%'4:$B&$4R'V%E%-,"0*#$,'M^HHIHWeeIU'0YFIH'&.@$%"B@&.8'05N'
:%6@$&.J'[0_05NJ\+'Y"..5,-/"..5,'((FeePIP+';"$*%-&#&*%'M^HHIHJTOOJ+'X#$%05,-."#Z*,'
((*PPJWI+'V%E%-,"0*#$,'((_JWWWJ'0YFIH'&.@$%"B@&.8'05N':%6@$&.I'[0_05NI\+'".3'
Hale et al.
46
;&%,%0'*."-E#."$%/",+#&'MêTIWTH)''Q$.'"4@$%&4Z4'B6%%$4:6.3'@6';"##D"%Z'05N'
36D"&.'%$4&31$4'[>KN/,Y'".3'NY,`MC'JHHe\d'@;$'$&8;@;'%$4&31$'6E'@;$'=(KLI'05N'
36D"&.'[0#2IW\'36$4'.6@'D"@B;'@;$';"##D"%Z'"4:"%@&B'"B&3'6E'@;$'=G5J'B#"44'.6%'@;$'
;"##D"%Z'@%2:@6:;".'6E'@;$'<CJF^JLB#"44)'',"%123*G28'D&44$.4$'D1@"@&6.'&4'&.3&B"@$3'
"76-$'@;$'=(KLI'4$]1$.B$)''[C\'(#&8.D$.@'6E'!:-#.#/"$,'=(KLI'?&@;'6%@;6#684'E%6D'
6@;$%'.$D"@63$'4:$B&$4'4;6?4'B6.4$%-"@&6.'"%61.3'05N'36D"&.'"4'?$##'"4'7$26.3R'
<F0IISIe'E%6D'!:-(&*//,"#+'<Y,HTSTS'E%6D'!:-&#E"$#*+'".3'(UM*/T'E%6D'[&5/*"-
E"."F*)''KBB1%%$.B$4'6E'^^0'4$]1$.B$4'"%$'76V$3)''_3$.@&B"#'%$4&31$4'"%$'&.3&B"@$3'&.'
?;&@$'6.'7#"BZ+'".3'B6.4$%-$3'%$4&31$4'&.'7#"BZ'6.'8%"2)''=$]1$.B$'"#&8.D$.@4'?$%$'
:$%E6%D$3'?&@;'<#14@"#A'[???)B#14@"#)6%8\'".3'"3m14@$3'72';".3)'
'
'
;()B>.!AH!!3*+,1'('!#N!23456!T!2@D56E!=>#$.(*5=>#$.(*!(*$.>+%$(#*!01!1.+'$!$?#!
&10>(/!+*+,1'('S!!!
[(\'0%":;'6E'!L8"#"B@64&3"4$'1.&@4'D$"41%$3'E6%'3&EE$%$.@':"&%?&4$'B6D7&."@&6.4'6E'
E14&6.'B6.4@%1B@4R'CM('F&.3&.8'C6D"&.'[CFC\'=,/LIH'N14&6.'B6.4@%1B@4'"%$'&.3&B"@$3'
6.'@;$'#$E@'".3'@$4@$3'"8"&.4@'(B@&-"@&6.'C6D"&.'[(C\'"#6.$'[-$B@6%+'?;&@$'7"%4\'6%'(C'
E14$3'@6'$&@;$%'?&#3'@2:$'=(KLI[7#"BZ'7"%4\'6%'=(KLI[&ZI\'[8%$2'7"%4\)''Y$4&31$4'&.'
=,/LIH'E14&6.4'"%$'ISLTUP'[E1##'#$.8@;'".3'0TeP,\d'IPULTUP'[ACSH\d'ISLIPW'[N'76V\)'
[F+'<'".3'N\'C$@$B@&6.'6E'E14&6.':%6@$&.4'$V:%$44$3'&.'3&:#6&3'2$"4@'4@%"&.4'14$3'&.'!L
8"#"B@64&3"4$'$V:$%&D$.@4)''5$"4@'$V@%"B@4'?$%$':%67$3'?&@;'".@&4$%1D'"8"&.4@'/$V('
E6%'CFC'E14&6.'3$@$B@&6.'[F'".3'N\+'6%'"8"&.4@'0"#S'E6%'(C'E14&6.'3$@$B@&6.'[<\+'6%'
"8"&.4@'<3BJU'"4'"'B6.@%6#'E6%':%6@$&.'#$-$#4'[<\)''(##'4@%"&.4'@$4@$3'&.'[F'".3'N\'"#46'
Hale et al.
47
$V:%$44$3'@;$'(CL=(KLI'E14&6.+'".3'4&D&#"%'%$41#@4'?$%$'67@"&.$3'?&@;'4@%"&.4'@;"@'
3&3'.6@'$V:%$44'"'CFC'E14&6.)''(##'4@%"&.4'@$4@$3'&.'[<\'"#46'$V:%$44$3'@;$'CFCL=,/LIH'
E14&6.+'".3'4&D&#"%'%$41#@4'?$%$'67@"&.$3'?&@;'4@%"&.4'$V:%$44&.8'=,/LIH'ACSH'CFC'
6%'.6'CFC'E14&6.'"@'"##)''^64&@&6.4'6E'D6#$B1#"%'?$&8;@'D"%Z$%4'"%$'&.3&B"@$3d'@;$'D64@'
:%6D&.$.@'7".3'3$@$B@$3'B6%%$4:6.34'@6'@;$':%$3&B@$3'4&b$'E6%'@;$'&.3&B"@$3'E14&6.'
:%6@$&.)''Q;$'(C'36D"&.'"#6.$'&4'3$@$B@$3'"4'"'IT'>C'7".3+'".3'@;$'CFC'36D"&.'"#6.$'
&4'3$@$B@$3'"4'"'JO'>C'7".3'[.6@'4;6?.\)''N/+'E1##'#$.8@;)''[C\'0%":;'6E'!L8"#"B@64&3"4$'
1.&@4'D$"41%$3'E6%'3&EE$%$.@':"&%?&4$'B6D7&."@&6.4'6E'E14&6.'B6.4@%1B@4R'(C'E14&6.4'
?&@;'=(KLI'3$%&-"@&-$4'"%$'&.3&B"@$3'6.'@;$'#$E@'".3'@$4@$3'"8"&.4@'CFCL=,/LIH'E1##'
#$.8@;'[7#"BZ'7"%4\'6%'CFCL=,/LIH'ACSH'36D"&.'[8%$2'7"%4\'E14&6.'B6.4@%1B@4d'
t-$B@6%u'%$E$%4'@6'"'B6.@%6#'B6.4@%1B@'&.'?;&B;'.6@;&.8'&4'E14$3'@6'@;$'(C'[$V:%$44&6.'
6E'=(KLI'E%"8D$.@'(C'E14&6.4'&.'2$"4@'$V@%"B@'&4'4;6?.'&.'N&81%$'=I\)''!L8"#"B@64&3"4$'
D$"41%$D$.@4'"%$'%$:6%@$3'&.'4@".3"%3'!L8"#"B@64&3"4$'1.&@4'[4$$'D$@;634\d'$"B;'
-"#1$'&4'@;$'"-$%"8$'6E'"@'#$"4@'@;%$$'&.3$:$.3$.@'$V:$%&D$.@4)'',%%6%'7"%4'%$:%$4$.@'
4@".3"%3'3$-&"@&6.4'[@;64$'@;"@'"%$'#$44'@;".'@?6'1.&@4'36'.6@'4;6?'1:'6.'@;$'8%":;\)'
'[,\'=B;$D"@&B'%$:%$4$.@"@&6.'6E'@;$'=(KLI':%6@$&.'".3'@;$'3$%&-"@&-$4'@;"@'?$%$'
@$4@$3'&.'[C\)''Q;$'B6D:#$@$'=(KLI':%6@$&.'&4'%$:%$4$.@$3'6.'@;$'@6:'%6?'?&@;'@;$'05N'
36D"&.'76V$3'".3'@;$':64&@&6.'6E'@;$-*G2'D&44$.4$'D1@"@&6.'&.3&B"@$3'?&@;'".'
"4@$%&4Z)''M1D7$%4'&.3&B"@$'@$%D&."#'"D&.6'"B&3'.1D7$%4'%$#"@&-$'@6'@;$'&.@"B@'=(KLI'
4$]1$.B$)''
Hale et al.
48
'
TABLE 1: Reduction of sao-1 activity suppresses aph-1(zu147) embryonic lethality
Genotype of mother % Viable progeny (n)

aph-1(zu147) 1.2 (1835)
aph-1(zu147); sao-1(ik1) 99 (1645)
aph-1(zu147); sao-1(ok3335) 97 (1105)
a
aph-1(zu147); sao-1(RNAi) 99 (1210)
b
aph-1(zu147); sao-1(ik1)/itDf2 98 (1758)
c
aph-1(zu147); sao-1(ik1)/+ 22 (3381)
c
aph-1(zu147); sao-1(ok3335)/+ 53 (3901)
b
aph-1(zu147); itDf2/+ 50 (3623)
Embryos were collected from at least 10 mothers of the indicated genotype; all animals were
also homozygous for unc-29(e1072). Viable progeny are those that reach at least the L4 stage.
n, number of embryos scored.
a Embryos were collected 16 hrs after injection of L4s (see Methods).
b Percent viability was calculated after subtracting the expected one-quarter inviable
homozygous itDf2 progeny.
c Actual genotype: unc-29(e2072) aph-1(zu147); sao-1(ik1 or ok3335)/dpy-11(e224); embryos
of different genotypes [dpy-11(e224) or dpy-11(e224)/sao-1 or sao-1] had similar survival rates
(see Methods).
Hale et al.
49
TABLE 2: sao-1(ok3335) suppresses embryonic morphogenesis defect of weak aph-1 alleles
% Embryos with successful body

Genotype of morphogenesis (n )
mother sao-1(+) sao-1(ok3335)
aph-1 (zu147) 89% (767) 97% (325)
aph-1(zu123) 0.82% (968) 20% (712)
aph-1(ep140) 0% (401) 0% (445)
aph-1(or28) 0% (315) 0% (452)
Embryos were collected from at least 10 mothers that were homozygous for either sao-1(+) or sao-
1(ok3335) and also homozygous for the indicated aph-1 allele; all animals were also homozygous
for unc-29(e1072). n, number of embryos scored. Embryos were scored as having successful
body morphogenesis if the hypodermis successfully enclosed the embryo and elongation
proceeded; see Figure 1 for representative embryos.
Hale et al.
50
TABLE 3: Interaction of sao-1 with lag-1 and glp-1 embryonic mutants
Genotype of % Viable progeny (n)

mother Temperature sao-1(+) sao-1(ik1) sao-1(ok3335)
a o
lag-1(om13) 20 4.4 (480) 30 (502) ** 4.2 (1116)
o
25 4.0 (1289) 20 (868) ** 0.66 (911) **
a o
glp-1(e2144) 15 100 (161) 99 (282) 99 (928)
o
20 99 (881) 97 (800) * 41 (392) **
o
23 3.2 (693) 1.3 (634) 0 (430) **
o
25 0 (238) 0 (468) 0 (253)
a o
glp-1(e2142) 15 94 (599) 89 (555) * 38 (445) **
o
20 14 (273) 2.8 (324) ** 0 (306) **
o
25 0 (262) 0 (246) 0 (>500)
a o
glp-1(bn18) 20 93 (580) 84 (365) ** 17 (544) **
o
23 6.2 (499) 0 (477) ** 0 (>500) **
o
25 0 (>500) 0 (>500) 0 (>500)
a o
glp-1(ar202) 20 80 (426) 90 (479) ** 47 (403) **
o
23 68 (989) 70 (735) 22 (446) **
o
+ 15 100 (275) 100 (761) 99 (1044)
o
20 100 (288) 99 (718) 98 (588)
o
23 100 (309) 99 (873) 93 (1341) **
o
25 99 (569) 97 (646) * 60 (882) **
Embryos were collected from at least 10 mothers that were homozygous for either sao-1(+), sao-
1(ik1), or sao-1(ok3335) and also homozygous for the indicated glp-1 or lag-1 allele; mothers
represented in the last four rows have no mutation other than the indicated sao-1 allele. Mothers
o
were raised at 20 C, shifted to the designated temperature as mid L4s, and acclimated for at least
24 hours before embryos were collected and raised at the same temperature (see Methods for
details). Viable progeny are those that reach at least the L4 stage. n, number of embryos scored.
A two-tailed Fisher exact test was performed for each sao-1(ik1) or sao-1(ok3335) value relative to
the equivalent sao-1(+) value in the same row; asterisks indicate a statistically significant difference
between the sao-1 mutant and the corresponding sao-1(+) embryos with P values as follows: * P <
0.01, **P < 0.0001.
o
a These are temperature-sensitive alleles for which 25 C is the restrictive temperature
at which embryonic viability is severely compromised (KODOYIANNI et al. 1992; PEPPER et al. 2003;
PRIESS et al. 1987; QIAO et al. 1995).
Hale et al.
51
TABLE 4: sao-1 mutations increase glp-1 activity in the germ line
Genotype of % Fertile animals (n)

mother Temperature sao-1(+) sao-1(ik1) sao-1(ok3335)
a o
glp-1(q35) 20 0.9 (222) 8.6 (233) * 52 (266) *
o
23 13 (208) 99 (162) * 98 (262) *
b o
glp-1(bn18) 20 98 (122) 100 (>500) 99 (197)
o
23 31 (325) 50 (161) * 80 (215) *
o
25 0 (>500) 0 (>500) 0 (>500)
b o
glp-1(e2144) 20 100 (100) 100 (202) 99 (524)
o
23 0 (105) 0 (105) 3.4 (179)
o
25 0 (>500) 0 (>500) 0 (>500)
b,c o
glp-1(ar202) 20 98 (204) 95 (190) 85 (213) *
o
+ 20 ND 100 (>500) 100 (>500)
o
23 ND 100 (203) 100 (322)
o
25 ND 100 (324) 99 (193)
Hermaphrodites that were homozygous for either sao-1(+), sao-1(ik1) or sao-1(ok3335) and also
homozygous for the indicated glp-1 allele were raised at the specified temperature and scored for
fertility; the last four rows represent animals with no mutation other than the indicated sao-1 allele.
Animals that generated any embryos were scored as fertile. n, number of animals scored. ND, not
determined. A two-tailed Fisher exact test was performed on each sao-1(ik1) or sao-1(ok3335)
value relative to the equivalent sao-1(+) value in the same row; an asterisk indicates a difference
between the wild-type and sao-1 mutant that is statistically significant with a P value < 0.0001.
a The glp-1(q35) allele causes a loss-of-function germline phenotype that is cold-sensitive: animals
o o
are fertile at 25 C, but sterile at 15 C (MANGO et al. 1991). Full genotype: unc-32(e189) glp-1(q35).
o
b These are temperature-sensitive glp-1 alleles for which 25 C is the restrictive temperature
at which virtually all animals are sterile (KODOYIANNI et al. 1992; PEPPER et al. 2003; PRIESS et al.
1987).
c Fertile animals differed in appearance of gonads: for example aberrant gonads were visible under
the dissecting microscope in 17% of the fertile glp-1(ar202) mutants (n=201) versus 71% of the
fertile glp-1(ar202); sao-1(ok3335) double mutants (n = 182); such gonad abnormalities were not
observed among the sao-1(ok3335) single mutants.
Hale et al.
52
TABLE 5: Suppression of aph-1(zu147) by sel-10(n1077)
Genotype of mother Temperature % Viable progeny (n)

aph-1(zu147) 20° 0.7 (1013)
a
aph-1(zu147); sel-10(ar41) 20° 1.3 (1169)
aph-1(zu147); sel-10(bc243) 20° 0.5 (427)
aph-1(zu147); sel-10(n1077) 20° 6.7 (597) *
aph-1(zu147) 15° 2.1 (448)
aph-1(zu147); sel-10(n1077) 15° 12 (613) *
Embryos were collected from at least 10 mothers of the indicated genotype. All animals were also
homozygous for unc-29(e1072). Viable progeny are those that reach at least the L4 stage. n,
number of embryos scored. A two-tailed Fisher exact test was performed for each value obtained
with a sel-10 mutant relative to the single aph-1 mutant at the same temperature; an asterisk
indicates a difference between the single and double mutant values that is statistically significant
with a P value < 0.0001.
a These animals were also homozygous for lon-3(e2175)
FIGURE 1!
sao-1(+)! sao-1(ok3335)!
A! B!
aph-1!
(zu147)!
C! D!
aph-1!
(zu123)!
FIGURE 2!
A nDf42!
itDf2!
sqt-3! unc-76!
4.0 mu! 8.0 mu!
13800K! 14000K!
snp F55B12[1]! C06B3! snp F58E10[3]!
R10D12!
sao-1
B 1! GYF domain!
MHKNGNNGPVIDTKWHYLGPDSEKYGPYMSKDMLFWLQAGYFNDGLQLKTENEPNY!
HTLGEWSQLLGTHPFSMPVHSLDATIAQMNSMRPHGAMMMVPPGLQNQFQPPMPMR!
FPPFLPMPLLHQMNQNGPPMGAQMHSQPPSEPIDAGSLSHTPDSENEGHGRKPHHH!
QQILAHQHIPQMQHANVATDQVVMKSVECQTEPVEISKEQASRVLSELLGQMVIIN
224!
!
C
D
FIGURE 3!
A B
WD40(G567E)!
FL(G567E)!
DBD !
WD40(+)!
SEL-10 fusion! DBD-SEL-10 !
vector!
FL(+)!
fusion:!
full length!
anti-LexA!
100 Kd!
WD40! 60 Kd!
AD !
SAO-1 fusion!
SAO-1(ik1)!
F box! C
SAO-1(+)!
wt!
vector!
Ik1 (L59F)!
AD-SAO-1 !
G567E! fusion:!
vector!
anti-Gal4!
45Kd!
0! 20! 40! 60! 80! 100! 120!
!-galactosidase units! anti-Cdc28!
30Kd!
D E
AD ! SAO-1!
SAO-1 fusion! Fragments!
ik1!
vector!
full length!1!
GYFa! 1!
GYF!
* 65! 224!
GYFb! 1! 80!
full length wt!
GYFc! 1! 150!
full length ik1! Mid! 51! 150!
CTb! 51! 224!
GYFa!
DBD ! CTa 80! 224!
GYFb! SEL-10 fusion!
F
SAO-1(+)!
SAO-1(+)!
GYFc!
CTb(ik1)!
full length!
DBD !
vector!
GYFa!
GYFb!
GYFc!
Mid!
CTb!
CTa!
Mid!
WD40! fusion:!
CTa! anti-LexA!
CTb! 60Kd!
45Kd!
CTb ik1!
30Kd!
0! 20! 40! 60! 80! 100! 120! 140! 160!
!-galactosidase units!
Hale et al.
53
'
!"##$%&%'()*+,()-$%!,.,/)$%,01,234,
!"#$%&'()&!"#$%,56121789:,;8,981,2<<0=1,1>0,%?3,;0<0=1,8<,&'($%),56121789:&
9603:.16& !6;162-:<26& *&%++&,-./0+&1234/5/605.&708&

!"#$%*+,- !"#$%*'.%,- !"#$%*#.///0,-
&'($%)*(121(3/4,,2- @A8- ,,B4C,DB@BE, ,,@4B,DBFAE, ,,G,DFFE,
&'($%)*(121(3/4,,2- BA8- HA,,,,DIBHE, JI,,,,D@ABE, HA,DBB@E,
&'($%)*(/4),K-5-+- @G8- FA,,,,DBFHE, FB,,,,DBCIE, ,,'L,
, , *&=62;-1>23?/:6@&A/:>&B"C?&708&
!"#$%*+,- !"#$%*'.%,- !"#$%*#.///0,-
&'($%)*(121(3/4,,2M=-- @A8, IG,DJFE, IG,DBBIE, IC,DIIE,
-
/0N52O>N8;710:,1>21,P0N0,>858QR?86:,<8N,071>0N,!"#$%*+,6-!"#$%*'.%,-8N-!"#$%*#.///0,,29;,23:8,>2;,1>0,
79;7=210;,&'($%),?0981RO0,P0N0,N27:0;,21,1>0,:O0=7<70;,105O0N216N0,29;,:=8N0;,<8N,0??,32R79?,ON8<7=709=R,8N,
29=>8N,=033,D)SE,;0T038O50914--%??,32R79?,ON8<7=709=R,7:,;0<790;,2:,2=17T0,32R79?,<8N,21,302:1,1P8,=89:0=617T0,
;2R:4,9M,965K0N,8<,297523:,:=8N0;4,,'LM,981,;010N5790;4-
8
",,&'($%)*(121(3/4,-=26:0:,2,38::U8<U<69=1789,O>0981RO0,21,@A S,29;,2,?279U8<U<69=1789,O>0981RO0,21,21,
8
BA SD!"'L)*)&,29;,V*%%'W)$L,BJJI2E4,
7-,&'($%)*/4),,=26:0:,2,?279U8<U<69=1789,O>0981RO0,1>21,7:,:057U;8579291D!"'L)*)&,29;,V*%%'W)$L,BJJI2E4,
8,,!1N279:,23:8,=891279,1>0,7910?N210;,)SU:O0=7<7=,52NX0N,9:;$%<<=>;-P>7=>,P2:,6:0;,18,7;0917<R,)S:,79,02N3R,
$F,:12?0,>0N52O>N8;710:,DON0UT63T23,79;09121789E,DW)'V,29;,!(%*'-%*V,@GGGE4,
?,,Y8N,=85O2N7:89,&'($%)*(121(3/4,@-!A&$%4*"BC%,-R703;:,893R,IZ,@)SD!"'L)*)&,29;,V*%%'W)$L,BJJIKE4,
,
,
,
,
,
!"#$%&'B)&&!"#$%,56121789:,;8,981,:6OON0::,1>0,%?3,;0<0=1,8<,!A&$%),,
,
& *&%++&,-./0+&1234/5/605.&708&
9603:.16& !"#$%*+,- !"#$%*'.%,- !"#$
%*#.///0,-
!A%$%)*DE%%,-- ,,G,,DBGBE, ,,G,,DBGIE, ,,G,,DHJE,
"
!A%$%)*%2%, -- ,,G,,DJHE, ,,B,,DBGFE, ,,G,,DJAE,
"
!A%$%)*%/%, -- ,,G,,DJCE, ,,F4@,DBBJE, ,,G,,DJCE,
-
/0N52O>N8;710:,1>21,P0N0,>858QR?86:,<8N,071>0N,!"#$%*+,6-!"#$%*'.%,M-8N-!"#$%*#.///0,,29;,23:8,>2;,1>0,
79;7=210;,!A&$%),?0981RO0,P0N0,:=8N0;,<8N,0??,32R79?,ON8<7=709=R4,%??,32R79?,ON8<7=709=R,7:,;0<790;,2:,2=17T0,
32R79?,<8N,21,302:1,1P8,=89:0=617T0,;2R:4,9M,965K0N,8<,297523:,:=8N0;4,,'LM,981,;010N5790;4-
",,Y8N,=85O2N7:89[,%??,32R79?,ON8<7=709=R,7:,8K:0NT0;,2589?,@GZ,8<,!A&$%4*"BC%,@-!A&$%)*%2%,,297523:,29;,
\AZ,8<,!A&$%4*"BC%,@-!A&$%)*%/%,-297523:-DW"-AD-"&F,BJJHE4,
Hale et al.
54
!"#$%&'D)&&!"#$%*'.%,,;80:,981,75ON8T0,1>0,610N790,#,=033,;0<0=1,=26:0;,KR,!A&$%)*"B%2%,,
,
9603:.16& E:62/06&#&56,,@F@/?6&&708&
!"#$%*+,@-!A&$%)*+,- C4GA,DIHE,
2
!A&$%)*"B%2%, , G4BB,DACE,
2
!"#$%*'.%,@-!A&$%)*"B%2%, , G4GJ,DACE,
-
(>0,2T0N2?0,965K0N,8<,610N790,#&=033:,O0N,:7;0,P2:,;010N5790;,<8N,57;,$F,:12?0,>0N52O>N8;710:,
DS>N7:152:U1N00,:12?0E,8<,1>0,79;7=210;,?0981RO0:4,,)33,:1N279:,=8912790;,1>0,7910?N210;,#U=033,:O0=7<7=,52NX0N,
8#=$)<<=>;*GHIIH$JKLM-"(?-GHI-%333,4,,,9M,965K0N,8<,297523:,:=8N0;],893R,890,:7;0,P2:,=86910;,O0N,2975234,
"--!A&$%)*"B%2%,,297523:,P0N0,23:8,>858QR?86:,<8N,1>0,52NX0N,N(8$%*A0/O,F,
,
,
!"#$%&'G)&!"#$%*'.%,,;80:,981,>2T0,2,52:=63797Q79?,0<<0=1,:75732N,18,1>21,8<,!A&$%4,
56121789:,
,
9603:.16& !6;162-:<26& *&HH&I-,6&?6J6,31;60:&-&708&
!"#$%7K8& !"#$%&'(%)&
8
DB"$)*(%%41,- @G , @4A,DI@BE, @4F,DII@E,
- , , ,
- , *&%+,&L6465:M&&708&
- , !"#$%7K8& !"#$%&'(%)&
8
!A&$%4*(%422,5+- BA , @H,DBFBE, @H,DBICE,
8
!A&$%4*(%422,5+- @G , @@,DBGCE, B\,DBIAE,
- , , ,
- , *='N@&126@60:M&708&
- , !"#$%7K8& !"#$%&#(***+)&
8
:=P!C)-QD;R$%<<=>;S- @G , JG,DAAE, JI,DHGE,
,
/0N52O>N8;710:,1>21,P0N0,>858QR?86:,<8N,071>0N,!"#$%*+,6-!"#$%*'.%,M-#B-!"#$%*#.///0,,29;,23:8,>2;,1>0,
79;7=210;,DB"$)M,!A&$%46-#B-:=P!C),?0981RO0,P0N0,:=8N0;,<8N,5230U1RO0,;0T038O5091,2:,<8338P:[,,DB"$),
297523:,P0N0,:=8N0;,<8N,;0T038O5091,2:,5230:,K2:0;,89,1>0,ON0:09=0,8<,2,5230,12734,,!A&$%4*(%422,5+,
297523:,P0N0,:=8N0;,<8N,1>0,792K7371R,18,32R,0??:,D%?3E,2:,29,79;7=218N,8<,5230U:O0=7<7=,;0T038O5091,8<,1>0,
>0N52O>N8;710U:O0=7<7=,T63T23,906N89:,D/!'E,D^)V%*-AD-"&F,@GGFE4,,:=P!C)-297523:,P0N0,:=8N0;,<8N,1>0,
ON0:09=0,8<,/!'_:,KR,T7N160,8<,/!'U:O0=7<7=,D;R$%<<=>;,0ÒN0::789D!a%-AD-"&F,@GGGE],1>0,N0O8N10;,O0N=0912?0,
N0<30=1:,1>0,965K0N,8<,8K:0NT0;,/!'_:,;7T7;0;,KR,1>0,18123,0Ò0=10;,965K0N,8<,/!':M,P>7=>,7:,1P7=0,1>0,
965K0N,8<,8K:0NT0;,297523:,:79=0,1P8,/!':,2N0,0Ò0=10;,79,02=>,>0N52O>N8;7104,,9M,965K0N,8<,297523:,
:=8N0;4,,
",,Y8N,=85O2N7:89M,^2?0N,01,23,N0O8N1,@AZ,5230U37X0,;0T038O5091,2589?,DB"$)*(%%41,@-!A&$%4*78)C/,,
297523:D^)V%*-AD-"&F,@GGFEM,29;,L0:27,29;,/8NT71Q,N0O8N1,1>21,233,DB"$)*(%%41,@-!A&$%4*(%422,,297523:,2N0,233,
071>0N,5230U37X0,8N,7910N:0`623DL%!)b,29;,/c*db(a,BJHJE4,
7,,/858QR?86:,!A&$%4*(%422,,>0N52O>N8;710:,2N0,O2N17233R,52:=63797Q0;,79,1>21,1>0,/!':,<273,18,;0T038OM,
=26:79?,2,<633R,O0901N291,%?3,;0<0=1,29;,8K:0NT21789,8<,893R,HZ,8<,1>0,0Ò0=10;,/!':D^)V%*-AD-"&F,@GGFE4,

Notch Signaling Is Antagonized by SAO-1, A Novel GYF - Genetics (PDFDrive)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Notch Signaling Is Antagonized by SAO-1, A Novel GYF - Genetics (PDFDrive)

Uploaded by

Copyright:

Available Formats

!

ASH, M. R., K. FAELBER, D. KOSSLICK, G. I. ALBERT, Y. ROSKE et al., 2010

GANOTH, D., G. BORNSTEIN, T. K. KO, B. LARSEN, M. TYERS et al., 2001 The

KIM, S. K., J. LUND, M. KIRALY, K. DUKE, M. JIANG et al., 2001 A gene

PEPPER, A. S., D. J. KILLIAN and E. J. HUBBARD, 2003 Genetic analysis of

VERHEYEN, E. M., K. J. PURCELL, M. E. FORTINI and S. ARTAVANIS-TSAKONAS,

TABLE 1: Reduction of sao-1 activity suppresses aph-1(zu147) embryonic lethality

Genotype of mother % Viable progeny (n)

TABLE 2: sao-1(ok3335) suppresses embryonic morphogenesis defect of weak aph-1 alleles

% Embryos with successful body

TABLE 3: Interaction of sao-1 with lag-1 and glp-1 embryonic mutants

Genotype of % Viable progeny (n)

TABLE 4: sao-1 mutations increase glp-1 activity in the germ line

Genotype of % Fertile animals (n)

TABLE 5: Suppression of aph-1(zu147) by sel-10(n1077)

Genotype of mother Temperature % Viable progeny (n)

9603:.16& !6;162-:<26& *&%++&,-./0+&1234/5/605.&708&

You might also like