Vertebrate limb development

Describe how limbs form at specific positions of the main body axis

Recall how limb-type identity is determined

Tbx4, Tbx5 and Pitx1 are involved in determining limb-type identity: forelimb (Tbx5), hindlimb (Tbx4 & Pitx1), Tbx4 and Tbx5 trigger limb bud initiation by activating a Fgf10/Fgf8 feedback loop between the mesenchyme and AER.

Understand how pattern is specified along the antero-posterior, proximo-distal and dorso-ventral axes

What are the 3 axes of the vertebrate limb (forelimb and hindlimb)?

stylopod, zeugopod and autopod

What are 3 signalling centres that the limb bud is equipped with?

ZPA, AER and dorsal epithelium

What does the ZPA express?

Shh

What does the AER express?

Fgf8

What is expressed in the dorsal epithelium?

Wnt7a

Where is the ZPA localised and what does it express?

posterior axis - Shh

Where is the AER localised and what does it express?

distally located, expresses FGF8

Where is the dorsal epithelium localised and what does it express?

expresses Wnt7a

How do limb buds appear along the AP axis of the embryo?

as protrusions from the flank at precise positions along the AP axis of the embryo

At which verterbrae position are Hox4, 5 expressed across different species? In which germ layer/plate do hox genes interpret positional information along the AP axis?

Typically C2-8.within the paraxial mesoderm and the lateral plate mesoderm

What specifies limb type identity ?

Hox proteins along and other factors induce limb-specific transcription factors that specify limb type identity

Name some limb specific transcription factors?

Tbx4, Tbx5 and Pitx1

What would be the result of ectopic Tbx5 expression in the leg?

feathers appear in leg

What would be the result of ectopic Tbx4 expression in the wing?

adopt hindlimb features eg knee-like articulation

What would be the result of ectopic Pitx1 expression in the forelimb?

knee-like articulation

Where are Tbx4, Tbx5 and Pitx1 typically expressed?

forelimb, wing, leg

What does the Fgf10-Fgf8 feedback loop drive? Describe it.

limb bud outgrowth

Where are Fgf-10 vs Wnt3a and Fgf-8 expressed?

Fgf10 in LPM and limb bud mesenchyme

Fgf8 and Wnt3a in apical ectodermal ridge

Is Fgf sufficient for limb bud outgrowth? How can this be tested?

yes! ectopic source of Fgf in chick interlimb flank = limb bud outgrowth

How does an Fgf source lead to limb bud outgrowth?

expands Tbx4 and Tbx5 expression domains

What would a limb bud expressing both Tbx4 and Tbx5 lead to the development of? And each individually?

wing/leg chimera

Tbx4 = leg, Tbx5 = wing

In what manner are skeletal elements specified?

in proximal to distal sequence

How would skeletal development be impacted by experimentally removing the apical ectodermal ridge at 3 vs 4 days of development?

wing buds would be truncated less vs more distally to the stylopod (humerus)

Between which stages of skeletal development are skeletal elements specified?

between HH19-28 seven elements are specified

Describe the progress zone model of proximo-distal patterning

progress zone model is time dependent - the undifferentiated proliferating cells in the progress zone within the AER are progressively patterned, cells spending the longest time in the zone form the most distal structures ie autopod

Which bones correspond to the stylopod, zeugopod and autopod respectively?

humerus, ulna/radius, carpals/metacarpals/phalanges

What is the hox gene code for proximodistal patterning?

hox10 for humerus/stylopod, hox11 for zeugopod/ulna/radius and hox12 for carpals and hox13 for digits (autopod by hox12/13)

What would be lost in a hox13 double mutant? What does this inform on?

loss of autopod ie digits and carpals - role of hox10 in limb specification and development

Are hox paralogues necessary for limb segment specification and patterning?

yes! they are essential and knockouts = absence of the structures eg loss of hox 11 = loss of zeugopod ie ulna/radius

What is a way of experimentally determining the role of hox11 in limb segment specification and patterning?

triple knockout in mouse, whatever is missing is what hox11 is responsible for patterning ie zeugopod

Loss of Hox13 = loss of which limb segment?

carpals and digits

Loss of Hox11 = loss of which limb segment?

zeugopod ie ulna/radius

Describe the 2-signal model of proximo-distal patterning

proximal patterning by high RA vs distal patterning by high FGF

Describe the signal-progress zone model of proximodistal patterning

modern concept - originally patterning depends on competitive signals, with high RA at the start inhibited by FGF, like the 2-signal model, then shifts to a time dependent model like the progress zone model

What is the role of the signal that the ZPA produces?

specifies AP position and digit identity

What would be the result of additional polarising region being grafted to the anterior margin of a chick limb?

complete mirror image of zeugopod and autopod ie ulna and digits

What would be the result of a small number of polarising region cells being grafted to the anterior margin of a chick limb?

digit one duplicated

Which morphogen does the ZPA release?

Sonic hedgehog

What would be the consequence of trying to replicate a ZPA graft to the anterior margin of a chick wing bud? What could be used to do this?

• duplicated pattern of digits in a concentration-dependent manner

• bead soaked in Shh or Shh expressing cells implanted to the anterior margin

What is the consequence of a ZPA graft to the anterior//posterior margin of a limb bud of a chick?

posterior is the normal location so results in the development of a typical chick wing // anterior is abnormal, leads to a mirrored development of digits on the limb bud

How long does Shh signalling take to specify digit pattern in chick wing? What is a way of testing this process experimentally, producing 1, 2 or 3 digits?

12 hours in chick wing

Treating chick embryos with cyclopamine at four-hour intervals

What does the development of digits 1 through 3 vs 4 and 5 depend on in chicks? What is the concentration gradient therefore sufficient for?

digits 1 to 3 depend on concentration gradient of Shh morphogen // digits 4 and 5 depend on timing of the signal

sufficient for specifying 3 digit identities

How well is mouse digit formation understood? Where do ring and little fingers most likely come from?

unsatisfactory understanding of mouse digit patterning

ZPA

What transcription factors does Shh signalling operate through?

Gli tfs

What is the impact of no Shh signalling on Gli tfs ?

Ptch1 inhibits Smoothened allowing Gli activators (A) to be processed into Gli repressors (R)

What is the impact of Shh signalling on Gli tfs?

Shh relieves the Ptch1 inhibition of Smoothened preventing Gli activators being processed into repressors

What are target genes of Gli3A/R gradient across the limb bud?

Ptch1, Gremlin1, Tbx2/3

How is digit identity determined downstream of Shh signalling?

through specific activation of Hoxa13, Hoxd12, Tbx2/3

What does the gremlin gene, activated by Gli tfs downstream of Shh, encode? Where is it expressed and what is it known as?

Bone Morphogenetic Protein (BMP) antagonist

in limb bud mesenchyme, known as AERMF (Apical Ectodermal Ridge maintenance factor)

What morphogen expression requires AER-FGF4/8?

Shh

What feedback loop are Gremlin, BMP, Shh and AER-FGF4/8 all involved in?

positive feedback loop in which Shh ensures FGF4/8 activity by inducing the BMP inhibitor Gremlin, lifting BMP inhibition on FGF4/8

What controls specific expression in the limb bud?

ZRS (ZPA Regulatory Sequence)

Consequence of mutations in ZRS. In which species has this been studied?

anterior expression of Shh and polydactyly

Hemingway cats

What does this show about the relationship between ZRS (Zone of Polarizing Activity Regulatory Sequence) and Shh? What is the consequence of a mutation in the region?

Hand2 (bHLH) and HoxA/D transcription factors lead to Shh expression in posterior limb bud by binding ZRS cis-regulatory enhancer in the lmbr1 gene // Gli3 repressor interferes with ZRS to block induction of Shh in anterior margin

ZRS stops anterior signalling of Shh, mutation = anterior expression of Shh which leads to polydactyly

What tissue determines the dorsoventral pattern?

ectoderm (epithelium)

Describe ectodermal vs mesodermal dorsoventral patterns

nails vs pads // biceps vs triceps muscles

What would be the consequence of rotating the ectoderm by 180C?

ectoderm determines DV pattern so rotation = DV polarity of mesoderm reversed

Which transcription factors are induced by signalling molecules produced by the ectoderm?

Wnt7a → Lmx1b dorsally

Bmps → En1 -/ Wnt7a so also -/ Lmx1b ventrally

Which signalling molecule specifies dorsal pattern vs ventral pattern? Through the induction/inhibition of what molecule?

Wnt7a specifies D pattern // En1specifies V by inhibiting Wnt7a

What would a Wnt7a mutant lack?

dorsal patterning

What would an En1 mutant lack?

ventral patterning

What would a Lmx1b mutant lack?

dorsal pattern - reverts to default state ie ventral limb

What do mutations in mouse DV patterning genes result in the loss of?

result in dorsalisation or ventralisation

What does Wnt7a promote?

dorsal fate via Lmx1b

What does En1 promote?

ventral fate by inhibiting Wnt7a

Describe the phenotype of a Lmx1b double mutant mouse limb

double ventral pattern where ventral sweat glands (eccrine) and tendons form on both sides of the limb

Which hox genes specify forelimb vs hindlimb

Tbx5 vs Tbx4 and Pitx1

What is the AER essential for ? The ZPA? The ZRS? The ectoderm epithelium?

PD axis

AP position and digit identity

Shh transcription

DV pattern

What coordinates AP and DV patterning at ea

Which models describe PD patterning?

the progress zone, two-signal and signal-progress zone-models

How do Tbx4 and Tbx5 trigger limb bud initiation?

by activating a Fgf10/Fgf8 feedback loop between the mesenchyme and AER