Lecture 12:

Discovering modularity in
skulls across amniote
phylogeny – focus on birds

Paedomorphosis again.
 The evolutionary context of premaxilla and maxilla...

Upper jaw: premaxilla and maxilla carry

teeth Studying the Pmax
Lower jaw: only dentary carries teeth – In birds and mammals
Tells us about the common
In basal tetrapods there were more dermal Amniote condition.
bones carrying teeth – and also an internal
and external tooth arcade – an Studying this across to zebrafish
osteichthyan feature. The inner arcade of Woudl tell us about teh common
teeth was lost within the amniotes. Osteichthyan condition...
Where does the change happen?

What tissue is the ‘carrier’ of species specific

Information (and thus change)?

What is the source of modularity?

Inside the neural crest mesenchyme.

 A new Head, neural crest as 4th germ layer,
Hox genes inside neural crest! NC forming skeleton,
Somitic vertebrae
tunicates

enteropneusts amphioxus Craniates

Early Echinoderms First beginnings of neural

evolution of chordates (sea urchins, Crest (pigment cells)
from minute 4 onwards! Sea stars)
chordates
Central notochord, neural
Tube dorsal to it, somitic
ecdysozoa Gill slits
mesoderm
lophotrochozoa
deuterostomes
cnidaria

porifera bilateria Gastrulation, mesoderm along body AXIS,separate from

endoderm
trichoplax Hox cluster organization, colinear organization of body axis

metazoa
Remarkable similarities in bone structure

Remarkable similarities in the patterns of bones across

Vast phylogenetic distances.

TWO DIFFERENT FORMS OF BONE FORMATION IN SKULL
Two different types of bone formation:
1. Dermal bone formation – bone sheet formation directly from mesenchyme
The oldest ossification form – in the gnathostome stem: Astraspis, Heterostracans.

Diploe: two compacta

Layers L1,3 sandwiching
A spongiosa L2

2. Endochondral ossification - CRANIAL BASE+ CAPSULES

Cartilage formation first – to be replaced by bone
The last form of ossification evolving – in placoderms:
The first gnathostomes with proper jaws.
The head of jawed vertebrates

Chondrocranium – originally made of cartilage, later either replaced by endochondral

ossification
or covered by bone (through dermal ossification)
Viscerocranium or Splanchnocranium – cartilaginous and dermal bone parts surrounding
The mouth.

CAPSULES: NOSE, EYE, EAR

 Evolution of skeletogenesis:
Stepwise evolutionary sequence of bone formation
processes

Secondary dermal bone loss

placoderm
Bony fish

endochondral
Bone
Osteostracans are the (bone replacing
most advanced jawless cartilage)
vertebrates in the
gnathostome stem ?
Head vs shoulder
group girdle
heterostracans
Cellular dermal bone
Astraspis, click on film Pectoral fins
Dermal bone, acellular (no cells
Inside), with enamel caps
arthropod apex predators: 510MYA Cartilage, no bone
 Mouse, 2yrs ago

Juvenile placoderm 380 mya

heterostracan fish, 430mya

Remarkably similar dermal bone ontogeny –

from 2 to 3 layers across several hundred Million years....
Remarkable similarity of bone patterns across
Vertebrates – here placoderms with osteichthyans
Not much changed with regards to jaw and suspensorium (hyomandibula) until the tetrapod
Stem group

Acanthostega
elasmobranch
Eusthenopteron
actinopterygians Crown tetrapods

Tetrapod stem group

sarcopterygians
osteichthyans

Jawed vertebrates
How can we reconcile similarity of pattern
Conservation of developmental processes

With differences in sizes/shapes of parts?

Differences in signalling within these

Developmental units.

=> Deviation – modification of later devl. processes is the norm in skull evolution.
 Cartilage,bone,
Muscle connective
Key embryological components tissue in head
Of the head And shoulders

hindbrain

Mesodermal
Branchial arches Limb buds
Mesodermal somites
 The Neural Crest
The vertebrate neural crest
Conserved neural crest modules:

Discrete cell populations act as developmental modules

They come from specific places and go to specific places

And are instructed in specific ways before migration

 7 rhombomeres
(brain segments
Organized by Hox genes)
1st
2nd
3rd

Branchial arches

Each cell group is one

Genetic module, and
Migrates into one place:
A branchial arch.

Quail-chick
Chimera
transplantation
Remarkable conservation of the early crest
emigration pattern in lampreys – it is the same
as in gnathostomes!
Same early expression of Dlx genes in branchial arches in agnathans
AND gnathostomes

placoderm
It is in later phases of development where
The differences emerge – similar to von Baer’s rule
Big anatomical differences
Later on in agnathans In jawless
Fish the
Velar cartilage
Is considered
the jaw homologue
Neural Crest segmentation retained in muscle attachment system

• 1. Neural Crest

Matching between hindbrain

Segment (rhombomere),
Neural crest in branchial arch
And its innervation by hindbrain
segment –
a gnathostome invention!
A shark head shows nicely the chondrocranial parts of the skull
Cartilage is major shape determinant for dermal skeleton/bones

Nasal
Capsule - cartilage
Otic
capsule

palatoquadrate
quadrate

articular

Meckel’s cartilage
Branchial
basket
 Placoderms

Bothriolepis (antiarch)

Dunkleosteus (arthrodire)
Jawed members of the gnathostome
stem group, characterised by a unique
dermal bone pattern, a dermal shoulder
girdle that forms a complete hoop, and
(usually) gnathal plates in the jaws.
Lunaspis (petalichthyid)
bothriolepis - our earliest placoderm ancestors
Earliest placoderms containing babies fossilized
Growth of the oldest jaws:
a telescoping out process
from the inside…can this
be found in today’s jawed
vertebrates?
How are cells migrating that
form the jaw?

First teeth in placoderms

Individual neural crest populations as ‘modules’
Carry species specific anatomical information!

THIS IS HARD-WIRED INTO THESE LINEAGES

‘information of Neural Crest segmentation/modules’
retained in muscle attachment specificity

Matching between hindbrain

Segment (rhombomere),
Neural crest in branchial arch
And its innervation by hindbrain
segment –
a gnathostome invention!
 Evolution of beak shapes – neural crest is the key determinant to convey
Species specific beak patterns
premaxilla

maxilla

Fig. 1 Quail–duck chimeric system to study jaw muscle development. (A) Head skeleton of adult Japanese quail in lateral view. (B)
Head skeleton of adult white Pekin duck. (C) Quail head with jaw muscles (pink dashed lines). (D) Duck head with jaw muscles. ...
Quail and duck specific
Anatomical features can be
‘transplanted’ by transplanting
The neural crest into the
Host of the other species..

A Quck and a duail

Þ Species-specific features
Hard-wired into neural crest
Populations.
Sources of change

a. Brain growth – effects on surrounding chondrocranium

b. Paedomorphosis – big eyes vs rest of head
The head of jawed vertebrates

Chondrocranium – originally made of cartilage, later either replaced by endochondral

ossification
or covered by bone (through dermal ossification)
Viscerocranium or Splanchnocranium – cartilaginous and dermal bone parts surrounding
The mouth.
Chondrocranium modularity – invisible to naked eye – but genetically separable
And they also evolve separately. The blue part houses the forebrain, the yellow part
The rest of the brain!

Blue
Neural crest

Yellow:
Mesoderm
Spot the difference?
vertebrate

lamprey
gnathostomes

Ascidians
vertebrates

Amphioxus

echinoderms
chordates

deuterostomes

‘basic’ chordate
Craniates – Neural crest a novel tissue generating a
Branchial apparatus

Yellow CNS-brain
Green notochord
Blue cartilage hagfish

lamprey

shark
Rostrum
snout

jaw
Branchial basket w gills
Chondrocranial skull differences explained
By brain differences!
When adjacent tissues grow:

Brain growth due to

Novel neuronal stem cell types for massive encephalization of the cortex in
mammals...
Significant increase in encephalization / cortical growth

Growth signal
From brain!

‘passive/reactive
Increase in skull growth”
Vast
Expansion of
Olfactory
Space..
PAEDOMORPHOSIS AS MOTOR OF CHANGE
Paedomorphosis at the fish tetrapod transition (FTT)

tetrapod
up
gro
s tem
rph
omo
pod
e tra
T
A significant paedomorphic event happened at the fish-tetrapod transition:
Late adult forms retain features from early larval forms fo ancestors
Best indicator: eye/head relationships.
 Even stem reptiles such as seymouriamorphs had an aquatic larval (tadpole) phase…
Adult skull anatomy but with gills!

Paedomorphosis
Is a widespread event,
Not just at the fish-tetrapod
transition
avialans
 Significant paedomorphosis
In theropod dinosaur avialans

avialans
 Look at eye size! Look at Premaxilla
1 2,3
marginally heterodont Teeth…separately

homodont

theropod avialan Modern paleognathous bird

Teeth as developmental modules

1. Serial repetition, early similarity/odontodes

2. Redeployment of programme from outside to
Inside/jaw region in placoderms

3. Differences of teeth – heterodonty/homodonty.

4. Loss of teeth – gradual and complete.

Finely choreographed epithelio-mesenchymal interaction sequences shape tissue
morphogenesis, the BMP and shh pathways

Amelogenin
Enamelin
Ameloblastin
 Protein family evolution driving structural evolution –
from enamel proteins to milk proteins – evolution of the epithelial placodal layer...

Odontode,
Common gnathostome stock –
Formation of enameloid.

Amelogenin-Ameloblastin-Enamelin
Are part of a wider family
Of Ca binding proteins
Neural crest makes denticle odontoblast and tooth odontoblasts –
cooption of body odontode programmes to the head/mouth region.

neural crest
labelling
Odontode programme:
3 tissues – 1.ectodermal enamel, 2.dentine (neural crest), bone (neural crest)
Its cooption to new location Placoid
denticles/scales on
body+teeth
Agnathan cyclostomes sharks
osteichthyans

placoderms

Crown
Gnathostomes =

‘Agnathan stem Novelty of jaws +

Gnathostomes’: teeth= denticle
osteostracans programme used
Keratinous ‘denticles’ inside mouth
People avoid calling them heterostracans (cooption)
Denticles as this implies hard
Tissue differentiation o no
f Denticles on body surface,
i t i
Add stage (with enamel caps+bone)
End
gnathostomes

craniates
 Placoderms

Bothriolepis (antiarch)

Dunkleosteus (arthrodire)
Jawed members of the gnathostome
stem group, characterised by a unique
dermal bone pattern, a dermal shoulder
girdle that forms a complete hoop, and
(usually) gnathal plates in the jaws.
Lunaspis (petalichthyid)
bothriolepis - our earliest placoderm ancestors
Earliest placoderms containing babies fossilized: the origins of sex, internal fertilization
 Placoderm

Growth of the oldest jaws:

a telescoping out process
from the inside

bidirectional tooth addition

 TRANSITIONS FROM
HETERODONTY TO
HOMODONTY

Seymouria

Stem amniotes
New: Molariform teeth
Amniote heterodonty Incisiform teeth
+thecodonty
Primitive condition of Limnoscelis
Amniotes: teeth along
One tooth row.
cotylosauria
Diadectes

mouse
synapsids

sauropsids Heterodonty lost –> homodonty.

 Stem amniotes evolve thecodonty and heterodonty

LeBlanc ARH, Reisz RR (2013) Periodontal Ligament, Cementum, and Alveolar Bone in the Oldest Herbivorous Tetrapods, and Their
Evolutionary
Significance. PLoS ONE 8(9): e74697. doi:10.1371/journal.pone.0074697
 Skull of insectivorous mole – 1 lower jaw bone = dentary bone

Synapsids and mammals keep this

heterodonty - largely

Secondary homodonty in whales (dolphins)

Then tooth loss (humpbacks)
 In fact, tooth loss
happened
Many times
independently within
the tetrapod tree...

(orange partial tooth

loss, red total tooth
loss)...

Modularity of tooth loss

Find phylogenetic signatures

Modularity in tooth loss – associated tooth loss within premaxilla vs maxilla bone
This suggests separate patterning units to be at work: a source of diversity....
 Ichthyornis

(c) Sharkey Trike

Hesperornis
http://www.guokr.com/article/94104/

Longipteryx
Nobu Tamura
Jeholornis
How often loss?

Tooth loss (edentulism)

Happened many times
Independently within the
Tree of the birds (Aves)..
Structural loss by Independent gene losses
Lead to new morphologies

Teeth leftovers
without enamel

edentulism

Loss of :
Amelogenin AMGN

Ameloblastin AMBN
Enamelin ENAM
 Retrograde loss of entire tooth programme sequences as a result of a terminal loss

Selective pressures!

Ontogenetic timing

X X
Ancestor: A B C
Tooth initiation Formation of hard enamel to bite.

Enamel structural gene loss!

Descendant: A B X
C

Selective pressure to keep Incomplete teeth cannot function

programme is gone. Þ Retrograde loss of the entire programme
Þ Formation of bird/turtle beaks/bills.
 Look at eye size! Look at Premaxilla
1 2,3
marginally heterodont homodont Teeth…
adult adult

Juvenile: edentulous
Juvenile:
homodont
homodont

theropod avialan Modern paleognathous bird

Summary

A. Anatomical conservation – explained by:

Early developmental conservation of cryptic modules,
Stereotypic migration, lineage specific ‘knowledge’ of behaviour

Deviation

B. Sources of change
Signalling differences later on
Brain growth
Paedomorphosis

C. Teeth as modules:
Temporal sequence of odontode development
Co-option to new places
Making differences between modules: hetero-homodonty
Tooth loss – retrograde tooth losses as result of gene losses.
Lecture 13 – beak shape evolution

Within mesenchymal neural crest

modules.

Darwin finches and beyond

 The evolutionary context of premaxilla and maxilla...

Upper jaw: premaxilla and maxilla carry

teeth Studying the Pmax
Lower jaw: only dentary carries teeth – In birds and mammals
Tells us about the common
In basal tetrapods there were more dermal Amniote condition.
bones carrying teeth – and also an internal
and external tooth arcade – an Studying this across to zebrafish
osteichthyan feature. The inner arcade of Woudl tell us about teh common
teeth was lost within the amniotes. Osteichthyan condition...
Birds are theropod dinosaurs....

Avialae!
The big
Transitions
Within birds..

premaxilla

maxilla

dentary

Figure 2. Bird skulls compared. Here are Archaeopteryx, Struthio (ostrich), Gallus (chicken) , Anser (goose) and
an unnamed Early Cretaceous enantiornithine nestling as large as Archaeopteryx (Sanz et al. 1997).
Archaeopteryx by Greg Paul used with permission. From:
https://pterosaurheresies.wordpress.com/2015/02/05/bird-skull-evolution/
Exploring sources of
Morphological differences

Within neural crest

Genetically.
Neural Crest segmentation retained in muscle attachment system

• 1. Neural Crest

Matching between hindbrain

Segment (rhombomere),
Neural crest in branchial arch
And its innervation by hindbrain
segment –
a gnathostome invention!
Darwin finches beak morphology and phylogeny
Finding key genes of beak shape in Darwin finches by comparative microarray
analysis
 Find key genes

Differential gene expression of

Differently looking animals at the *same* stage, same cells.

Gene expression list – verify by RNA ISH

Verify gene function by misexpression or knockdown

Assay:
1.How do genes found thus regulate each other? RNA ISH
2. Can we recover ancient morphologies or morphologies of related
Species by changing gene expression during this same ontogenetic stage?
Calmoduliin
Signalling pathways

Any of these components could be

Causative for structural difference

Razor!: differential gene expression

At the right time and space
Earlier: Levels and extent of Calmodulin (CaM) expression in the upper
beak correlates with upper beak shape/size across the Darwin finches

The more in the mesenchyme,

the more pointed the beak!

Early BMP4 expression
Correlates with deep and
wide
beaks
The BMP (4) signalling pathway – how it works?

Candidates for evol. Change.

Variation in expression of genes
TGFbeta signalling

Wnt pathway (beta catenin)

In upper beak (premaxilla)

Correlates with beak shape
Differences..
Wnt signalling

Beta catenin
DKK
Tissue specific
Gene expression
matters for shape:

Different effects
Of misexpression
In ectoderm vs

Mesenchyme!

Only the latter has

An effect on
chondrogenesis
Establishing some epistatic
relationships during beak devl – using
the chick model system...

Alk5 and DKK3 upregulates CaM,

BMP4 does not.

BMP4 overexpression downregulates

Itself.

Differential effects on bone and

cartilage
 Differential effect of different molecules upon width, length and depth of beak

TGFb depth,length
But not width

Alk5,Dkk4 D, L but
not width

www.pnas.org/cgi/doi/10.1073/pnas.1011480108 Molecular modularity

Synopsis of findings: different signalling systems affect length,width and depth
Small but probably very
Significant changes in the
Coding sequence of Alx1..
Between (magnirostris/blunt
And conirostris/pointed).

But the causal role of these

changes in Alx1
has not been tested
experimentally....

doi:10.1038/nature14181
Correlation of Alx1 region with pointed beaks (difficilis vs conirostris)
Now, what are the sources of variation in vivo??

Whole genome sequencing reveals Interspecific hybridization events!!!!

doi:10.1038/nature14181
 Interspecies mating as
Source of genomic
introgression and diversity

doi:10.1038/nature14181
Now we can start to understand the difference
Between paleognathous and neognathous birds
 The evolutionary context of premaxilla and maxilla...

Upper jaw: premaxilla and maxilla carry

teeth Studying the Pmax
Lower jaw: only dentary carries teeth – In birds and mammals
Tells us about the common
In basal tetrapods there were more dermal Amniote condition.
bones carrying teeth – and also an internal
and external tooth arcade – an Studying this across to zebrafish
osteichthyan feature. The inner arcade of Woudl tell us about teh common
teeth was lost within the amniotes. Osteichthyan condition...
A new FGF8 boost in the ancestry of the aves
Paleognathous and Neognathae) : to make the rostrum
 Expt FGF
inhibition

Does not just

Affect the
Premaxilla
(rostrum) but
Also the
Palatine bone

Evol
Hypothesis:
Co-evol. Of Structures
Via size of
Gene expression domains
 premaxilla

maxilla
Different bones are regulated by the same signalling centre – FGF8!

Show ostrich vs
Lateral bar
Cormorant.
Large Connection
Of Mx at back
to midline
bone

No connection
Differences in paleo vs neognathous birds… - accomplished by modification of FGF8 in
Lateral bar regions (blue), while the ‘rostrum’ got an early FGF8 boost in the ancestry of both.

It is not
Maintained
In paleognathous
birds
Short summary
• Outer vs inner tooth arcade
• Independent tooth losses within the osteichthyan and tetrpod trees (edentulism)
• Changes in birds – loss of teeth within the birds

• Changes in size and shapes of premaxilla/maxilla

• Molecular determinants

• Example of the Darwin finches, which signalling events are respondsible for
• Width , length and depth of the beak/bill?

• Sources of variation – interspecific hybridisaiton events in recent past asociated

changes in Alx1 gene regulatory regions, changes in coding sequences

• Look at real, in vivo populations,

• Different techniques used to find genes and regulatory regions fof interest that might
be responsible for changes in shape/anatomy....
Signalling pathways, expression in particular tissues at particular times.

Changes in signalling pathways as motors of anatomical diversity.