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T]:IE G E N E T I C BASIS OF THE ~ASSIMILA.TED

BITHOI~AX ' STOCK

BY C. I-I. WADDINGTON
I'r~stit~te qf i~d~)~a[, Genetics, Edi~zburgh

(1Received I Mc~y 1956)

INTf%ODUCTION
Eggs of a wild-type Oregon-K strain of D~'osophila~nda~oyastersubj coted to ether vaponr
at 289189hr. after laying frequently develop a bithorax-like phenotype, as described by
Gloor (19'~7). By selection, for some thh'ty generations, of those individuals which reacted
in this way, a strain has been produced in which bRhorax-tike individuals appear in high
frequency even in the absence of any ether treatment (Waddii~gton, 1956). This 'assimi-
lated bRhorax' stock is known as He*. (In this paper, in which none of the other bithorax-
like stocl~s are involved, it will be referred to simply as He, omitting the asterisk.) If one
tries to evaluate the importance of such processes of genetic assimilation for evolution as it
occurs in nature, it becomes important to know whether strains m which an environmental
modification has been assimilated contain only genes -which were alrBady present in the
initig! population, or whether there is glay reason to suppose tha~ the environmental stress
has stimulated the occurrence of an appropriate mutation, i t is diNeult to see how the
occurrence of a mutation could be completely disproved. On the other hand, if it could be
shown that the assimilated race contains a gene or genes which produce an abnormal
phenotype, and which would have done so even in the initial population from which that
phenotype is known to have been absent, then one would have to conclude that mutation
had made an. important co~3ribution to the assimilation process. It was from this point of
view that a further analysis of the genetic basis of the assimilated bithorax stock was
undertaken.
~XPEBIMENTAL P~ESULTS
The breeding results wit]] the He stock which have been recorded previously (Waddington,
1956) suggested that, although .the character undoubtedly has a polygeic basis and is
affected by genes on all three maj or chromosomes, a particularly importazat role is played
by a recessive maten:~al-effsct genetic condition. Females homozygous for this throw a
large proper@ca of bithorax phenotypes in their offspring_ The previous data did not allow
one to decide either wheth.er tlhe maternal effect is produced by one or by several gelies, or
on which chromosome or chromosomes it is carried. In order to elucidate this, He males
were crossed with CZ @B Me females, a.:a.dthe female off;spring baekcrossed to He males.
The next generation will include three classes of females, Cy M.e, CtB Me and OlB Cy, in
which ~he first, second and third chromosomes, respectively, will be homozygous (if we
neglect) the small amount of crossmpover in the F:t CZB Cg 21"Isfemales). These @aree types
of females were crossed separately to He males. The percentages of bithorax phenotypes in
the various classes arising in the first backcross generation~ a:nd ia the second bs,ekcross of
the three types of females, are shown in Table 1.
242 Genetic basis of the 'assimilated bithorax' .steele

Table 1. Percentages of He phenoty_pes (females o,~ly)

T h e firs~ baekeross was derDed from 6'U?/He; Cy/ft~; Me~He9 • tts~. From fheir offspring, three second back-
crosses were bred: (1) Cy/YYe; Me~Be9 x Hod'; (2) Ol.B/Ne; Me~He9 x Zle6'; (3) C~B/Hs; OF~He 9 x l~e~.
FirsL baekeroas Second backcross

r Which He a Mother Cy Me ~':[iother O/B ATe i~iofher C1B Cy

chromosome~ Percentage Ha (chromosome (chromosome (chromosome
Type os offspring homozygous phcnotypes t homozygous) 2 homozygous) 3 homozygoue]
CIB Cy Me 0 6 . . . .
O y M~ 1 2 75 -- --
Cl]3 Me 2 IS -- 0 --
O ~ B Cy 2 1,% -- -- 2
Ms 1, 2 g 57 2 --
Cy 1, 3 :24 92 -- I

OlB 2, 3 33 --- 20 6
+ 1, Z, 3 61 91 3t 9
To~M flies -- 312 r 592 7,t7

The numbers involved are not ve,'y large, since this was a preliminary and exploratory
experiment. They are sufficient for the first backeross to show clearly that all three
chromosomes contribute to produce the bithorax phenobype, the third having a particu-
larly strong effect, and the first seeming to have rather little when it operates directly on
the animal containhag it. The second backeross demonstrates anmistskably t h a t the first
chromosome is the seat of the maternal-effect condition, since the females in which it is
homozygous produce a considerably greater percentage of bithorax phenotypes than do
the others. The females in which either the second or the third chromosome is homozygous
give offspring which,.in every class of the table, contain a lower proportion of bithoraxes
than the corresponding class of the first baokeross. It is not very obvious why t~is should
be so. Perhaps the reason is that in ancestry of the second baekeross there has been an
extra generation in which crossingoover with the inversion chromosomes m a y have aliowed
some of the autosomat genes of the original He stock to have been lost.
The next question to be settled is whether the X-chromosome maternal-effeet condition
can produce any bithorax ph~noty]?es is i~ is made homozygous in a genotype which, in
other respects, is the same as that of the initial Oregon-K strain before any selection
occurred. This was tested by ti~e crosses shown at the top of Table 2. I t will be seen that
half the female parents of-the fourth generation would be homozygous for the He X-
chromosome, though containing only Oregon K second and third chromosomes. If the
maternaLeffeet condition is effective in the Oregon K background, some of the pair
rantings shoNd produce bithoraxdike offspring. It will be seen that one-fifth of them did
so. There is only rather slight possibility that second- or third-chromosome He genes
were carried over into the fifth generation. They would have had to cross over ;nto the Cy
or Me chromosomes in the second generation and out of them again in the third; and even
if they did so, they wotdd be present onIy as heterozygotes in ehe fom'th-generation
individuals. It therefore seems certain that the appearance of bithoraxes at the end of this
series of crosses is mai]nly due to the maternal effect of the He X-chromosome.
No bithorax phenotypes have ever been seen in the Oregon K stock when this is kept
under normal conditions. In the light of the facts just described, this seems to call for one
or other of the three following hypotheses : either we must sappose that the maternal-effect
genetic condition arose (presumably as a single gone) by m.ueadon during the progress of
the selection experiment; or we can suppose that a single maternal-eCfec~ gene was present
 C. If. W~DDXNGTO~ 243
in the initial population at such a / o w frequency that homozygo~es are exceedingly rare,
but that a heterozygote was included in the sample of the population from which the
selected line was derived ; or, finally, it is possible t h a t the maternal effect is determined
not by a single gene bu~ by two or more linked genes, each of them rather rare, so that the
homozygote for aI1 the set was initially very rare il~deed. It will be convenient to disettss'
the possibilities in the reverse order to tha~ in which they have jast been Iis%d.
Table 2, [nse~t'io~z of. He ~nate~'ncd-effect conditio~ i~to O~'ego~ K 9enoty2~e
C~B Cy ,]~reo Or. K.
He H s H~ ~ x

He OF Me Cy 31e
Or. K; Or. K' O-E-.IX9xge; Or. X' Or. t( 4
$
TndividuM pairs
He Or.K Or. Ko Or. Kd
t / e ' O r . K ' Or.ls ~•
H~ Or. K Or. K
or Or. I ( ; Or. Is Or. K 9 x O~'. K d'
No. of He

P~h" no, To~l

1 77 1 5
2 102 1 0
3 14 1 2
4 65 ! i
5 2I 0 1
6 92 2 0
7 4~ I 1
8 S9 ~ 3
9 iS 5 7
].0 15 I 0
35 other families, eont~izfing 28.94 •es, contained no/:7e.

Two experiments have been made to try to discover whether the maternal-effect con-
dition can be broken down by crossing over. In the first of these, ]:Ie females were crossed
with Oregon K males, and two ba,ckerossss made, of F 1 males fo He females and of F t
females to He males. In ~he first of ~hese there could be no orossing~over between the tle
and Oregon X-chromosomes; the Y 1 daughters should be either He~He, or He/Oregon 1<251
their sex chromosomes, and if crossed individually with fare shales should fall into two
sharply distinct classes giving higher or lo~er percentages of bithorax offspring. On the
other hand, in the second. 1~'1 there could be crossing-over between the He and Oregon K
X~ehromosomes, and if this separated t,wo components of the ma~ernaLeffeet condition,
the dis~baetio_n between the/~'~ daughters w e n d be blurred. This experimen~ failed to
provide ehe information sought from it, since although fifty-s F z dauglr~ers from the
fi.rse type of backeross were tested, the percentages of bithorax types among their offspring
did not fail into two clearly defined groups, bat varied more or less uniformly :f~'om20 to
100 ~ . Under these circumstances it, was hopeless 5o expect to decide whether the crossing-
over in. ~ffteother tylt)e of baokcross has any effect on the matsrnal-effecl; eondi.tion. I-low-
ever, it is interesting to note Chat those daughters of the He/Oregon K females which
produce any bighorax offsl?ring at all do so in a lower percentage than the daughters of the
other baekm'oss do; this is evidence that crossing-over.in the former type reduces the
effectiveness o f t h e He chromosomes in their action in reinforcing the maternal effect. The
16 Gene~. 58
244 Ge'netic basis of the 'assi~ikctcd bitAo~"c~x' s~oclr
#all He assimilated, genotype therefore probably contains several eo-operati~lg genes on
each chromosmne.
In the second attempt to deteo~ crossing-over between comt]onents of the maternal-effect
condition, females heterozygous for the ~[s X-ehrom.osome and for a chromosome con-
taining so vfsc~r were crossed widz ,Fie males, and their daughters again m'ossed with
similar males. Ilalf of these daughters should be homozygou.s :for the maternal effect
condition and should throw a high percentage of bithoraxes ; if" they also produce males
showing any of the marker mul~ant genes, crossing-over m~l.s~ ?nave occurred in theh.
mothers. Actually among forty-two such families, fen'teen showed high percentages of
bit~horax. Seven of these did not produce any mutant mal~s. Of the other seven, all
contained f e a r , and one ao as well. This is consistent with the maternal-effect condition.
lying somewhere betweenf and so (the position of these genes is so, 0; v, 33;f, 57; cry'r, 62),
By utilizing a Mtffle>5 chromosome to stabilize the various X-chromosomes, it was easy to
build up stocks homozygous for the maternal-effec~ condition linked to so or to f car. On
the other hand, an at~empe was made to link it to v, starting from a family which had
given a very low percentage of bithoraxes along wi~h v males, but this was quite unsuc-
cessful, it becoming elear that the family did not contain, any maternal-effect condition.
One must conclnde that there is no evidence that crossing-over can break down the
maternal-effect condition into separate components. All the evidence is consistent with its
being a single gone located in the neighbourhood of vermilion. Bnt the testa are of coarse
nothing like adequate to disprove the hypothesis that it is a fairly closely linked group of
genes in this region.
The above experiments provide no positive grounds for believing that the existence of
the maternal-effect condition in the initial population can be 'explained away' by the
supposition that the condition is composed of a number of.linked genes. One turns, then,
to examine the suggestion that the condition is due to a single gone which was present,
%hough rare, in the foundation stool<. I f the frequency of such a gone were ,~, it would be
homozygous in u2 individuals iu the whole population, of which half would be females,
and ~hese should make Choir presence noticeable by producing bithoraxdype offspring. I~
was previously estimated (Waddington, 1958) that about 150,000 'wild-type' flies were
inspected before any spent.snoots bithoraxes were found, and this includes a majority
which belonged to the" upward' line ia which selection was being made for phenoGopies of
this type. Even if we allow g factor of two for the effects of this selection in facilitating
the penetranee of the gone, we should find 89 = I in 150,000, whence u =c. 1 in 270. Since
the first generation of the upward selection line certainly contained a Nw hundred flies, it
would not be at all surprising if a gone with this order of frequency in the initial popula-
tion were included in i& Thus the da~a are z>ot inconsistent with the suggestion that the
mabernal~effeet condition is due to a single gone which was present in low frequency from
the beginning.
The calcalati.on just given has been based on the hypothesis that the maternabeffeet gone
eventually became homozygons by chance, having regained its initial frequency throughout
the course of selection. An examination of ghe effect of a standard ether treatment on
various crosses involving the matemml~effect condition does in fact show that *,his effect
is one which infl~tences the fl'equency with which untreated eggs produce bithorax types,
but which does not al~er their sensitivi V to the ether stimulus. This is demons~rated in
Table 3, in which figures are given, both for pe:eeentages of bithorax types among e~aerged
 C. H. WADDX~Gr 245
adults, and among pupae examined before emergence so as to avoid any complication due
to the difficulty He types may find in ik.eemg themselves from the pupal integuments.

Table 3. Effect of ether t~'ecttment (25 mi~. at 2-1. hr. after laying)
]Parents
"o _a , No. of pupae No. of ~dults
, c? examined % He ex~mmed % He
He He 100 100 790 91
Or. K Or. I( 100 29 '800 27
He Or. 1s2 89 75 800 ~
Or. I( ~e 100 76 800 ~tG

D I S CUSSION

The experiments just described amply confirm the suggestion that the assimilated bi~
thorax Ehenotype is dependent on two components: a collection of genes, located on all
~he chromosomes, which act directly on the individual containing them, and a genetic
condition of the first chromosome which causes a maternal effect. It cannot be asserted
positively tha~ this maternal-effect condition is a single locus, but on the other hand there
is no evidence that it consists of several genes which can be se:parated by crossing-over.
There is nothing which in any way militates against the hypothesis that all the directly
acting genes were present in the initial population before selection began. On the other
hand, the fact that the maternal-effect condition, when made homozygo~s in a wild-type
background, causes the appearance of bithoraxes in the next generation, might suggest
that it could not have been present initially. However, a consideration of the numbers of'
flies examined leads to the conclusion that it actually might have been there in a frequency
so low as to be undetectable by anything less than a very extensive investigation. There is,
then, no compelling reason to suppose that it originated by mutation during the course of
the experiment. :Even if such an origin were judged to be more probable than not, ~here is
certainly no reason to suppose t e a t we are confronted with anything Eke the directed
induction by gee emdronment of an appropriate mutation.

I. The phenotype of the ~assimflated bithorax; stock is due in ]?art to a number of

genes, on all the chromosomes, which act directly on the individuals containing them,
and partly to a. recessive X-chromosome condition which causes a maternal effect.
2. Attempfis to break down the maternal-effect condition by crossing-over were un-
suecess%l.
3. The maternal effect condieion, when made hom.ozygous in a wild4ype background,
causes the appearance of some bithoraxes in the ~aext generation.
{. The condition may have arism~ by mutation during the co~rse of the selection by
which the assimilated stock was built up, but it may have been present in very low
frecfaeney in the initial population.
5. The offspring of ~emales hmnozygous for the m.aternal-efl'ect condition do not pro-
dace a higher percentage of bithoraxes, following ether treatment, than do flies of similar
genotype from other mothers.
~F,]?'EgENCES
GLOOR, I-l. (lfl'f7). P h a e n o k o p l e u n i t Ae~her a3a ~'oao2/~.ila, ~?ev. suissr ZooL 54, 637-712.
W• C. t i . (1956). Genetic assimilations of the M H m r a x p h e n o t y p e . Evogution 10, 1-13.
16-2