Vertebrate Limb Development

Tetrapod Limb Development

Biology 4361 – Developmental Biology

July 29, 2009

Tetrapod Limbs

© Vicki Lockard and Paul Barry

© Father Alejandro Sanchez

© Merlin D. Tuttle

© Anne Fischer

Limb Development - Overview

Patterning

Early development

Specification

Establishing limb axes

Morphogenic models

Cross-talk / regulation

Francesca V. Mariani and Gail R. Martin 2003 Nature 423:319-325 doi:10.1038/nature01655

Limb Patterning

Humerus

Radius

Ulna

Carpals Metacarpals

Phalanges

Human

arm

Chicken wing

Chicken

leg

Shoulder Finger

Pinkie

Thumb

Knuckle

Palm

Proximal Distal

Posterior

AnteriorDorsal

Ventral

Limb Field

Limb Bud

Formation

Vertebrate Limb Buds

Limb bud

So

mite

s

Ectoderm

Mesodermal

mesenchymeLateral

plate

mesoderm

Hox expression

determines limb

bud locationHox5

Hox6

Hoxc6 Specification of Limb Buds

Forelimb initiation:

- anterior-most point of

Hoxc6 expression

Tbx Genes Specify Limb Type

Tbx expression initiated by Wnts, FGFs

NOTE – Tbx genes are not the first step in forelimb/hindlimb specification

(initial step(s) unknown)

Fgf/Wnt - Limb Bud Initiation

Limb Bud Axes

Distal

Proximal

Anterior

Posterior

Dorsal

Ventral

Apical Ectodermal Ridge

Apical Ectodermal

Ridge (AER)

Fgf10 initiates AER via Wnt3a, β-catenin

AER expresses Fgf8, Fgf4; maintains Fgf10 expression

Proximal-Distal Axis

Apical Ectodermal Ridge (AER) forms at boundary

between dorsal and ventral ectoderm

Fgf8

lateral

plate

mesoderm

Fgf10Fgf10

Lateral plate mesoderm expresses Fgf10

Apical Ectodermal Ridge Manipulation

Extent of development depends

on time of AER removal

Distal structures

are duplicated;

- note structure

Degree of “legness” of wing

depends on placement of

leg mesenchyme

Progress Zone

Progress Zone – mesodermal mesenchyme; receives AER signals:

- promotes proliferation (mitosis)

- prevents differentiation into cartilage

- maintains expression of A/P and D/V-related signals

Progress Zone

(PZ)

Apical Ectodermal

Ridge (AER)

Fgf8

Fgf4

PZFgf8

Fgf10

PZ mesenchyme specifies proximal-distal axis

- transplantation experiments demonstrated that positional

information was carried by PZ cells

- PZs conveyed age-appropriate specification instructions

AER establishes

Progress Zone

~200 μm

Proximal-Distal Specification ModelsProgress zone model: Identity established by residence time in PZ

Pro

xim

al

Dis

tal

Early allocation and progenitor expansion: Elements specified early

Specifying mechanism - ??

Anterior-Posterior Specification

Zone of Polarizing

Activity (ZPA)

Apical Ectodermal

Ridge (AER)

Progress Zone (PZ)

Morphogen

Shh

Shh necessary and sufficient for establishing ZPA

(NOTE – Shh not necessary for polarity of styolpod)

Shh induced by dHAND and Hoxb8

ZPA maintained by feedback loop with AER

ZPA/AER Feedback Loop Model

1. dHAND - bHLH transcription factor

and Fgf8 from AER stimulate Shh

- Fgf8 (and Fgf4) maintains Shh

expression

2. Shh up-regulates Gremlin1 in

posterior mesenchyme

- Grem1 antagonizes BMP ligands

(BMPs repress Fgf expression in AER)

3. Wnt7a maintains Shh

Wnt7a determines the size of AER

Loss-of-function mutants (both Shh and Grem1) = syndactyly, loss of digits

ZPA Morphogen Gradient

ZPA

Shh gradient

12

3

4

5

[Morphogen]

High

Low

Posterior Anterior

5

4

32

ZPA Transplantation

Mirror-image duplication effects can be replicated by transplanting Shh bead

Posterior tissue transplant to anterior = duplicated autopod

Retinoic acid operates upstream of Shh

- implant RA-soaked bead =mirror-image duplication

- possible Hox gene involvement

C. Tickle, Nature Molecular Biology 7(2006)45-53

2

34

43

2

“new” posterior

Drosophila Hedgehog PathwayShh

(vertebrates)

/ Gli1, 2, 3

(vertebrates)

/ Gli1, 2, 3

(vertebrates)

Shh/Gli Interactions

Gli3 – proteolytic fragment – acts as a transcriptional repressor (Gli3R)

- represses e.g. dHAND, Gremlin, Fgf4, Hoxd13

Without Shh:

With Shh:

Gli’s retained in long form – acts as a transcriptional activator (Gli3A)

- e.g. Gli1 activates Shh

ZPA

Shh gradient

Gli3A

Gli3R

Shh/Gli Interactions

C. Tickle, Nature Molecular Biology 7(2006)45-53

Shh main function may be to relieve Gli3R repression in posterior region

Anterior – high Gli3R

Posterior – low Gli3R/ high Gli3A

Shh-/- = 1 digit;

- Gli3R prevails

Gli3-/- = polydactyly

- Shh prevails

~ 8 digits

- unpatterned

Shh Specifies Digit Identity

digit 4 progenitor cells

digit 5 precursors

(paracrine)

(autocrine & paracrine)

(autocrine)

BMPs Regulate Digital IdentityShh initiates BMP2 and BMP7 gradients

- BMPs in interdigital mesoderm specifies identity of digits anteriorly

Noggin – BMP

antagonist

Insert BMP antagonist

into interdigital webbing

NOTE – Fgfs from AER control phalange development;

Shh bead inserted between digits can add phalange; Shh

sustains Fgf signal; Fgf inhibitor = lack of phalange

Remove interdigital

mesoderm

- BMP targets unknown

NOTE – BMP effects probably only on tissue “primed” by Shh

Hox Genes in Early Limb Bud

5’ Hox Genes Pattern Limb ElementsForelimb

Hindlimb

Dorsal-Ventral Specification

Zone of

Polarizing

Activity

(ZPA)

Apical Ectodermal

Ridge (AER)

Progress Zone (PZ)

Ectoderm

Wnt7a – necessary and sufficient to dorsalize limb bud

- induces Lmx1 in dorsal mesenchyme

- Lmx1 knockouts = ventralized phenotype

- Wnt7a knockouts = ventral footpads on both surfaces

Dorsal

Ventral

Apoptosis in Limb Primordia

BMP signals