Except where mentioned, all figures are scanned from

“Developmental Biology”, By Scot F Gilbert

At early stages, any half of field can generate entire
limb
 Hox 6 _ forelimb
bud

Hox 10 _ hind
limb

Fgf10 expression in lateral

Tbx required for maintaining Fgf10 in limb bud
plate mesoderm
regions .
Mis-expression of Tbx
can convert from one
limb to other. In
mouse hindlimb also
requires Pitx1.

Not clear if Hox can turn on

Tbx5
insitu

Tbx4
insitu
Apical Ectodermal Ridge
(AER)

Stage 23 Stage 26
HH
Fernandez-Teran HHJ Dev Biol, 52, 85
and Ros (2008), Int
 Progress
zone

Summerbell and Lewis (1975) J Embroyol. Exp. Morph. 33,

 23HH, stained for cell 26HH, green dye marks cartilage
division marker formation
Stage 28 onwards cell division index drops sharply in progress zone, 300 μm
from tip
Fernandez-Teranand Ros (2008), Int J Pearse, et al., A cellular lineage analysis of the chick limb bud, (2007) Developmental
Dev Biol, 52, 857-871 Biology, 310, 388-400
AE
R

Distal structures
missing

When transplanted to more proximal region, can produce

supernumerary limb

When more proximal mesoderm is placed below AER, it contributes to terminal

limb parts

INTERPRETATION: AER is the

 + Normal
wing
Stage 23 to Stage 16 to
25 18

Normal
+ wing

win
Le
g
g Stage 23 to Stage 23 to
25 25
Ectodermal-Mesodermal Interactions in the Growth of Limb Buds in the
Chick Embryo: Constancy and Temporal Limits of the
Ectodermal Induction’ LEO RUBIN~ AND JOHN W. SAUNDERS, JR.
(1972), Developmental Biology, 28, 94-112
 + Only distal
structures
Stage 23 to
Stage 16 to
25
18

+ Normal wing, success decreased with age of

Ectoderm
Stage 26 to Stage 16 to
30 18
Ectodermal-Mesodermal Interactions in the Growth of Limb Buds
in the Chick Embryo: Constancy and Temporal Limits of the
Ectodermal Induction’ LEO RUBIN~ AND JOHN W. SAUNDERS,
JR. (1972), Developmental Biology, 28, 94-112
 Ectodermal donor Number of cases Complete wings Wings truncated or
stage absent
26 15 9 6
27 13 13 0
28 7 6 1
29 (-) 11 8 3
29 22 4 18
30 9 0 9

Ectodermal-Mesodermal Interactions in the Growth of Limb Buds in the

Chick Embryo: Constancy and Temporal Limits of the
Ectodermal Induction’ LEO RUBIN~ AND JOHN W. SAUNDERS, JR.
(1972), Developmental Biology, 28, 94-112
In chick
Psuedostratified
.

In mouse,
Polystratified

Stage
Stage 29
28

LEO RUBIN~ AND

JOHN W.
SAUNDERS, JR.
(1972),
Developmental
Biology, 28, 94-
112
 • Apical mesoderm from stage 25 limb bud when transplanted to
shoulder will form toes

• If proximal leg mesoderm is placed directly under wing AER, toes

develop at the end of the wing.

• However, if this mesoderm is placed farther from the AER, leg

mesoderm gets integrated with wing proximal structures and directs
the formation of typical thigh feathers

• If limb mesoderm is replaced by non-limb mesoderm, AER regresses

and development ceases.
• Mesoderm gives instructive signals that specify the proximo-distal axis

• AER required for sustained growth of limb _ keeps mesoderm cells

proliferating, prevents them from forming cartilage
Mesoderm makes FGf10. FGF10 induces
Wnt3a (in chick, but Wnt 3 in mouse and
human), in overlying ectoderm. Wnt
induces through canonical signalling
pathway, FGF8 in ectoderm. This keeps
underlying mesoderm proliferating. Thus,
feedforward loop. Both FGFs required to
keep each other expressed.

fgf8
How does mesoderm give rise to proximo-distal
axis?
Results
in
deletion
s

Donor: stage 19 or 20, thick

tip
Host: Stage 24

Results in
duplications
 The apparently proximo-distal heirarchy of
information:
• If proximal leg mesoderm is placed directly under wing AER, toes develop at the end
of the wing.

• However, if this mesoderm is placed farther from the AER, leg mesoderm gets
integrated with wing proximal structures and directs the formation of typical thigh
feathers
When more proximal mesoderm is placed below AER, it contributes to terminal limb
parts

This is not because of signals from the AER, but because of patterning within the
mesoderm.

Normal wing
+ This suggests that proximal regions can
”induce” or regenerate missing distal
win information, when this distal information is
Le
g removed.
g Stage 23 to Stage 23 to
25 25
38 somite stage,
Approx HH19 to
20

The proximo-distal sequence of origin of the parts of the chick wing and the role of the
ectoderm, (1948), J Exp Zool, 108, 363-403.
Bermejo-Sanchez, et al 2011, Am J Med Genet C Semin Med Genet, 4,
 Models proposed emphasize time, space or generation of periodicity

X • Progress zone model (time) Longer the time spent dividing in progress zone, more distal the
structures contributed to. PROBLEMS: cell division all over not only at progress zone, when
individual cells labelled, progeny found in specific zones.

• Early allocation and progenitor expansion model (space). Progenitors of all regions present
right from the start. When AER is removed, 200μm region from tip undergoes apoptosis. Later
X this is done, more distal the structure that will be deleted. PROBLEMS with this model, too.

• Reaction diffusion model (periodicity)

 Tabin and Wolpert (2007),
Genes & Dev, 21, 1433-
1442
This model implies that if individual cells are labelled at
early stages, their progeny should be present throughout
the limb. But, the cell lineage analysis shown in slide 21,
does not comply with this expectation.

This model expects that distinct gene expression domains for each part of the limb
will be present in the early bud. But such gene expression patterns have not yet
been discovered despite at least two decades of research.
Hox genes are turned
on in sequence as the
bud grows. They are Tabin and Wolpert (2007),
not all there right from Genes & Dev, 21, 1433-
the beginning. 1442
 Progression of Time

Fgf at progress zone inhibits Fibronectin

synthesis
Fibronectin promotes cartilage formation, by
allowing cells to aggregate.

No. of nodules, depends on geometry of tissue

and strength of inhibition
Inhibitor
Newman and Bhat, not
2007 identified
Mesenchyme expressing Hoxb8 is induced to express sonic hedgehog (shh). Shh then
maintains Fgf8 expression in AER.
There is an error in this schematic!! Can you
spot it? Do Do
rs rs
al al

FGF levels
FGFs are increase. At
Ve Ve low, AER high levels
High BMPntr inhibit Gremlin
ntr active synthesis
Low BMP required
al to initiate makes AER
al
AER and induce En1 on regress
ventral side
Gremlin: secreted glycoprotein,
BMP antagonist, first identified as a
Fernandez-Teran and Ros (2008), Int J Dev Biol, 52, Dorsalizing factor in Xenopus
Specification of the Anterior –
posterior axis:

Grafting fewer cells Shh

Ectopic expression of Shh results in extra gave expression
digits 32234 or 2234 pattern
Chick mutant that does not
synthesize Shh in limb has only
digit 1

If Shh is not allowed to diffuse,

digits 2 and 3 are lost
 Shh GFP-Cre
promoter

Ubiquituous lac
promoter Z
Cre- Cre-
target target

+ Gets
lost

Ubiquituous lac
promoter Z
lacZ will now be expressed only in cells where GFP-Cre is
 Shh-
gfpCRE
shh RNA
insitu

anti- Shh
Antibody R26
R

anti- Shh
R26
Antibody
R

Harfe, et al, Cell, Vol. 118, 517–528, August 20,

2004
 23HH, stained for cell 26HH, green dye marks cartilage
division marker formation
Stage 28 onwards cell division index drops sharply in progress zone, 300 μm
Fernandez-Teranand Ros (2008), Int J from tip
Pearse, et al., A cellular lineage analysis of the chick limb bud, (2007) Developmental
Dev Biol, 52, 857-871 Biology, 310, 388-400
BMP signalling maintains Shh gradient. BMP signalling initially required to promote condensation and cartilage
formation. Afterwards required to induce cell death between digits

Bead soaked in Noggin, antagonist of Bmp

Deletion of interdigital region
Gli3 is a Kruppel like Zinc finger domain protein downstream of Shh. In absence of
gli3 shh double
Shh Gli3 represses transcription
Lmx1 homolog of apterous,
LIM domain protein,
expressed on Dorsal side,
induced by Wnt7a

Wnt7a
mutant
 Proliferating As
chondrocytes secrete chondrocytes
cartilage specific die, surrounding
extra-cellular matrix cells become
osteoblasts.

Hypertrophic
Compact chondrocytes
nodules Remodelling
secrete more into bone also
contain fibronectin,
chondrocyte releases VEGF.
Collagen X and
s CaCO3
containing
Developmental Biology by
vesicles,
mineralization. Gilbert
Also, VEGF,
Transplantation of growth plate to hosts of different ages and regions of
different growth potentials shows that the growth potential is intrinsic to the
donor growth plate, therefore not influenced by environment.
Low levels of Estrogen stimulates
skeletal growth

High levels at end of puberty

induce apoptosis of hypertrophic
chondrocytes

FGFs promote differentiation of

chondrocyte precursors

What phenotype would activated

FGFR have?
fgf Level of IGF1
expression
 y = Ae-be-kt

Miller and German (1999), Journal of nutrition, vol

129.
Reichling and German (2000), Journal of nutrition, vol
130.
 Number of
individuals siz
e

Size is a multigene trait and is also influenced

by the environment
 siz
e

time
Log
(size)
time

d(Log
(size))/dt
time
 Archeognatha:
bristletails
Zygentoma: silverfish,
Both are ametabolous

Hariharan, Wake and Wake, (2016), Indeterminate growth: could it represent the ancestral condition, Cold Spring Harb Perspect Biol
2016;8:a019174
 (size))/dt
(size)
siz

Log
e

d(Log
time time time

Growth and time

Growth and developmental

program
Dexamethason Epiphys
e slows is
Resting
proliferation zone
1 week after
Proliferatin
stopping
g zone
treatment in
rabbits Hyper-
trophic
zone
Metaphysis

16 weeks after
stopping
treatment in
rabbits
Extra slides
After A-P axis is dtermined, FGF8 is destroyed by RA in
somites