Biology 4361 Developmental Biology Tetrapod Limb Development July 29, 2009
Tetrapod Limbs Merlin D. Tuttle Vicki Lockard and Paul Barry Father Alejandro Sanchez Anne Fischer
Limb Development - Overview Patterning Early development Specification Establishing limb axes Morphogenic models Cross-talk / regulation
Limb Patterning Humerus Carpals Metacarpals Dorsal Anterior Human arm Radius Ulna Phalanges Proximal Posterior Ventral Distal Chicken wing Knuckle Thumb Shoulder Finger Chicken leg Pinkie Palm Francesca V. Mariani and Gail R. Martin 2003 Nature 423:319-325 doi:10.1038/nature01655
Limb Field
Limb Bud Formation
Somites Vertebrate Limb Buds Hox expression determines limb bud location Hox5 Hox6 Limb bud Ectoderm Lateral plate mesoderm Mesodermal mesenchyme
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 Dorsal Anterior Proximal Distal Posterior Ventral
Apical Ectodermal Ridge Proximal-Distal Axis Apical Ectodermal Ridge (AER) forms at boundary between dorsal and ventral ectoderm lateral plate mesoderm Fgf10 Fgf10 Fgf8 Apical Ectodermal Ridge (AER) Lateral plate mesoderm expresses Fgf10 Fgf10 initiates AER via Wnt3a, β-catenin AER expresses Fgf8, Fgf4; maintains Fgf10 expression
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
Fgf10 Progress Zone Progress Zone (PZ) Fgf8 Fgf4 AER establishes Progress Zone Fgf8 PZ ~200 μm Apical Ectodermal Ridge (AER) Progress Zone mesodermal mesenchyme; receives AER signals: - promotes proliferation (mitosis) - prevents differentiation into cartilage - maintains expression of A/P and D/V-related signals PZ mesenchyme specifies proximal-distal axis - transplantation experiments demonstrated that positional information was carried by PZ cells - PZs conveyed age-appropriate specification instructions
Proximal Distal Proximal-Distal Specification Models Progress zone model: Identity established by residence time in PZ Early allocation and progenitor expansion: Elements specified early Specifying mechanism -??
Anterior-Posterior Specification Progress Zone (PZ) Morphogen Shh Apical Ectodermal Ridge (AER) Zone of Polarizing Activity (ZPA) 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 1 2 Shh gradient ZPA 5 3 4 High [Morphogen] Low 5 4 3 Posterior 2 Anterior
ZPA Transplantation Posterior tissue transplant to anterior = duplicated autopod Mirror-image duplication effects can be replicated by transplanting Shh bead 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 4 3 2 2 3 4 new posterior
Drosophila Hedgehog Pathway Shh (vertebrates) / Gli1, 2, 3 (vertebrates) / Gli1, 2, 3 (vertebrates)
Shh/Gli Interactions Without Shh: Gli3 proteolytic fragment acts as a transcriptional repressor (Gli3R) - represses e.g. dhand, Gremlin, Fgf4, Hoxd13 Gli3R Shh gradient Gli3A ZPA With Shh: Gli s retained in long form acts as a transcriptional activator (Gli3A) - e.g. Gli1 activates Shh
Shh/Gli Interactions Anterior high Gli3R Posterior low Gli3R/ high Gli3A Gli3 -/- = polydactyly ~ 8 digits - unpatterned - Shh prevails C. Tickle, Nature Molecular Biology 7(2006)45-53 Shh -/- = 1 digit; - Gli3R prevails Shh main function may be to relieve Gli3R repression in posterior region
Shh Specifies Digit Identity digit 4 progenitor cells digit 5 precursors (paracrine) (autocrine & paracrine) (autocrine)
BMPs Regulate Digital Identity Shh initiates BMP2 and BMP7 gradients - BMPs in interdigital mesoderm specifies identity of digits anteriorly - BMP targets unknown NOTE BMP effects probably only on tissue primed by Shh 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 Insert BMP antagonist into interdigital webbing Noggin BMP antagonist
Hox Genes in Early Limb Bud
5 Hox Genes Pattern Limb Elements Forelimb Hindlimb
Dorsal-Ventral Specification Dorsal Progress Zone (PZ) Ectoderm Apical Ectodermal Ridge (AER) Zone of Polarizing Activity (ZPA) Ventral Wnt7a necessary and sufficient to dorsalize limb bud - Wnt7a knockouts = ventral footpads on both surfaces - induces Lmx1 in dorsal mesenchyme - Lmx1 knockouts = ventralized phenotype
Apoptosis in Limb Primordia BMP signals