BIOL 346 Developmental Biology Lesson 26 PDF

BIOL 346 Developmental Biology Lesson 26 Chapter 21: Development of the Tetrapod Limb Fig. 21.1: Tetrapod limb anatomy _Tetrapods___________: Four-limbed vertebrates such as amphibians, reptiles, birds, and mammals Limb anatomy: Stylopod: Most proximal...

BIOL 346 Developmental Biology Lesson 26 Chapter 21: Development of the Tetrapod Limb Fig. 21.1: Tetrapod limb anatomy _Tetrapods___________: Four-limbed vertebrates such as amphibians, reptiles, birds, and mammals Limb anatomy: Stylopod: Most proximal (closest to body) In humans: humerus in arm, femur in leg Zeugopod: Middle region of limb In humans: radius + ulna in arm, tibia + fibula in leg Autopod: Most distal (furthest from body) In humans: carpals of fingers and tarsals of toes Fig. 21.1: Tetrapod limb anatomy Limb field: Area of embryo including all cells capable of forming a limb on their own Limb bud: Bulge of cells at sites of limb development, precursor to full limb Progress Zone (PZ): Highly proliferative mesenchyme fueling limb bud growth Apical Ectodermal Ridge (AER): Thickening of ectoderm at apex of limb bud Zone of Polarizing Activity (ZPA): Posterior cells of the progress zone, patterns anterior- posterior axis of limb Fig. 21.2: The limb field and limb bud (A) Prospective Forelimb Field: In salamander, the central area forms the limb (free limb), while surrounding cells form peribrachial flank tissue and shoulder girdle. Outer cells can form a limb if central tissues are removed. (B) Limb Bud Emergence: Mesenchymal cell proliferation from the lateral plate mesoderm causes the limb bud to bulge, forming skeletal elements. Myoblasts from the lateral myotome contribute to musculature. (C) Myoblast Entry: Stereogram of Myf5 mRNA shows developing muscle cells (purple) entering the limb bud. AER (D) Chick Forelimb Bud: Scanning electron micrograph highlights the apical ectodermal ridge (AER) in an early chick Limb bud forelimb bud. Fig. 21.3: Deletion of limb bone elements by the deletion of paralogous Hox genes Limb Region Specification: Hox genes specify limb regions: Hox9/10: Stylopod (proximal) Hox11: Zeugopod (middle) Hox12/13: Autopod (distal) Experimental Evidence: Hoxa11/Hoxd11 Knockout: In mice, lack of Hoxa11 and Hoxd11 results in reduced or missing ulna and radius (zeugopod). Human Polysyndactyly: Homozygous HOXD13 mutation causes fused digits and malformations in the urogenital system. Fig. 21.5: Transplantation of different regions of the presomitic mesoderm (PSM) to the limb field causes changes in limb size Determining limb type and location: Morphogenetic Rules: Limb formation follows universal rules across tetrapods, with limb bud grafts able to induce limb formation in other species. Limb Fields: Limbs form at specific body positions, defined by predetermined mesodermal regions (somitic and lateral plate). Limb Bud Positioning: Limb buds align with specific Hox gene expression patterns along the body axis, such as Hoxc6 for forelimbs. Hox Genes: Hox genes provide positional cues; mesoderm in limb regions promotes limb formation, while non-limb mesoderm represses it. Fig. 21.8: Model for forelimb field initiation _FGF8 and Retinoic Acid (RA) signaling are antagonistic_ Where RA is expressed, but no FGF8, a limb bud will form RA induces Tbx5, Tbx5 by way of Wnt2b induces Fgf10 Tbx5 induces epithelial to mesenchymal transitions Fig. 21.9: Fgf10 expression and action in the developing chick limb FGF10 expression can induce limb development Type of limb is determined by expression of either Tbx4 or Tbx5 Fig. 21.11: Manipulation of the apical ectodermal ridge (AER) Key roles of AER: Maintain mesenchyme beneath it in a proliferative state to promote growth of limb Maintain expression of molecules that generate A-P axis Interact with proteins specifying A- P and D-V axes so each cell is given instructions on how to differentiate Key roles of mesenchyme: Induce and sustain AER Determine type of limb to be formed Fig. 21.12: Control of proximal-distal specification of the limb is correlated with the age of the progress zone (PZ) mesenchyme (A) _Early PZ grafted onto late wing bud forms extra ulna and radius. (B) Late PZ grafted onto early wing bud results in missing intermediate structures. Conclusion: PZ age determines proximal-distal limb structure formation. Fig. 21.13: (A) Model for limb patterning, (B) transplantation of limb bud tips, and (C) FGF and Wnt treatment of transplanted mesenchyme Opposing gradients of FGF/Wnt from the AER and Retinoic Acid from the proximal flank pattern the cells of the growing limb Treating grafts with RA or FGF+Wnt can alter limb development – supporting model Fig. 21.18: When a ZPA is grafted to anterior limb bud mesoderm, duplicated digits emerge as a mirror image of the normal digits _A-P axis specified early in limb development Zone of Polarizing Activity Small block of mesodermal tissue near posterior junction of young limb bud and body wall Specifies A-P axis Fig. 21.19: Sonic hedgehog is expressed in the ZPA Shh mRNA is expressed in ZPA Shh is sufficient to serve the function of the ZPA Fig. 21.20: The descendants of Shh-secreting cells form digits 4 and 5 and contribute to the specification of digits 2 and 3 in the mouse limb _Shh specifies digit identity by preventing apoptosis Shh signal can be received in autocrine or paracrine manner Digit identity is influenced by length of time expressing Shh and length of time receiving paracrine Shh signal Only Digit 1 (most anterior) is specified independently of Shh signal Shh works two ways at different times: First, specifies digit identity (as described above) Next, works as mitogen to stimulate proliferation and expansion of limb bud mesenchyme Fig. 21.26: Lmx1b-dependent dorsal-ventral patterning by Wnt7a The overlying ectoderms epcifies D-V axis Wnt7a: _Expressed in epidermis (ectoderm) Wnt7a knockout mice have ventralized paws Activates Lmx1b transcription factor Lmx1b: Expressed in dorsal mesenchyme Lmx1b knockout mice have ventralized paws Fig. 21.27: Model of dorsal-to-ventral patterning in the limb bud by Wnt and BMP signaling Dorsal Patterning: Wnt7a promotes dorsal limb cell fates via Lmx1b. Ventral Patterning: BMP signaling drives ventral patterning through Engrailed-1 (En1).  Apoptosis and formation of the digits _Role of Apoptosis: Programmed cell death (apoptosis) is essential for forming separated digits and functional joints in tetrapod limbs. Necrotic zones undergo apoptotic cell death Species differences: Chick embryos show significant interdigital cell death, resulting in separate digits, whereas ducks have minimal interdigital cell death, leading to webbed feet. _BMP signaling in Apoptosis: BMP proteins (2, 4, 7) trigger apoptosis in the interdigital mesenchyme, while Noggin protein suppresses BMP activity in digit cartilage, preventing cell death and preserving structure. Fig. 21.28: Patterns of cell death in leg primordia of duck (A) and chick (B) embryos  Forming the joints Fig. 21.29: Possible involvement of BMPs in stabilizing cartilage and apoptosis BMP has a context dependent role in joint formation: Depending on the cell responding to the signal and the cell’s developmental stage Dual Role of BMP: BMP signaling leads to apoptosis in the presence of FGFs or induces bone formation with Wnts. Dickkopf (Dkk) Role: Dkk mediates apoptosis and inhibits Wnt to prevent skeleton formation when FGFs are present. Noggin’s Effect on Joint Formation_: Noggin is crucial to prevent excessive cartilage formation; its absence results in no joint formation and excessive BMP-driven cartilage.  Evolution of the autopod BMP4 Induces Apoptosis: Both chick and duck webbing express BMP4, promoting apoptosis. Gremlin Prevents Duck Apoptosis: Gremlin inhibits BMP4 in duck webbing, preventing cell death, unlike in chicks. Changes in expression of BMPs and BMP inhibitors over time can lead to changes in autopod structure Fig. 21.30: Autopods of chick (upper row) and duck (lower row) are shown at similar stages  Evolution of cetacean limbs from land mammals Cetaceans: whales, dolphins, porpoises Evolved from hoofed land mammals Required conversion of forelimb into a flipper and elimination of hindlimb Steps required: _Forelimb modification: Extended FGF signaling in the forelimb AER led to elongated flippers with more phalanges. Interdigital Preservation: BMP inhibition Fig. 21.31: An approximately 110-day embryo of a pantropical spotted dolphin (Stenella attenuata), prevented interdigital apoptosis, similar to duck stained to show bones (red) and cartilage (blue) feet, aiding flipper formation. Hindlimb loss: Early cessation of Shh signaling in the hindlimb ZPA halted hindlimb development. Without ZPA, AER is not sustained and hindlimb does not develop