Vertebrate embryonic patterning 6

Vertebrate Embryonic Patterning 6

Limb Formation

Tetrapod Limbanterior

posterior

proximal distal

ventral

dorsal

Limb Bud Formation

Establishment of Limb Fields

Hox Gene Expression in Trunk During Limb Bud Development

Fig 23.21 Analysis of Biological Development 2nd ed, Kalthoff

FGF10 Induces FGF8

supernumerary limbs from implanting FGF10 beads

Positioning of the Limb Field• Forelimb position lies near anterior expression

boundary of hox b8, b9, c6, c9, & d9 • FGF8 expression in intermediate meso + hox

genes induces FGF10 in the lateral plate meso• Wnts in the lateral plate maintain FGF expression

in regions of hoxd9 expression• Between the fore & hindlimb buds, the

developing kidney (mesonephros) inhibits FGF expression

• Exogenous FGF can induce supernumerary limbs

Forelimb vs Hindlimb

Forelimb vs Hindlimb

Correlates with Tbx5 & Tbx4

Expression Respectively

Tbx5

Tbx4

Tbx4 Gain of Function Study

Tbx4 expressing retrovirus infected embryo

Proximal Distal Limb Axis

• AER– overlying ectoderm– responsible for maintaining outgrowth of limb

• Progress zone– underlying mesoderm– region of cell division– maintained in active mitotic state by AER

AER – Apical Ectodermal Ridge

AER Necessary for Limb Bud Outgrowth

Removal of AER at various times prematurely truncates limb

Reimplantation of older AER into younger bud causes loss of proximal structures

AER Necessary for Limb Bud Outgrowth

Reimplantation of younger AER into older bud causes duplication of distal structures

Interactions of AER and PZ

FGF8 in the AER

FGF in ectoderm AER3 day embryo

limb buds

Hox Code of the Limb

Proximal Distal

Hoxa & d = forelimb; Hoxc & d = hindlimb

Mammalian Hox Genes

Hox Genes Required for Proper Limb Development

Hoxa-11/Hoxd-11 double knockout

wild-type

human with synpolydactyly resulting from homozygous

HOXD-13 null mutation

Proximal-Distal Structures

Anterior-Posterior Limb Axis

Zone of Polarizing Activity

ZPA

Classic Experiments: Retinoic Acid Induced Limb Duplication

• Implanting RA soaked beads caused duplication of zeugopod/autopod structures

• Depletion of RA reduces the normal structures

Shh is Expressed in ZPA

Demonstration that Sonic Hedgehog is Primary ZPA Component

Dorsal-Ventral Gene Patterning in Limb Bud

Fig 23.23 Analysis of Biological Development 2nd ed, Kalthoff

Dorsal-Ventral Limb Axis

Lmx

1. Fgf-8 induces Shh2. Shh & Fgf-8 induce Fgf-43. All these induce Lmx-14. Wnt-7a in the ectoderm

represses Lmx-1 and induces Shh

BMPs

Molecular Interactions in Limb Bud

Dorsal-to-Ventral Transformations

WT mouse autopod

Wnt7a-/- mouse autopod

Homology of Process: A-P Axis

Homology of Process: D-V