Limb Development Notes PDF

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Development of the Tetrapod Limb Limb Anatomy -

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Tetrapods: four-limbed vertebrates (i.e. amphibians, mammals, reptiles, birds) The bones of any tetrapod consist of a proximal stylopod (humerus/femur) adjacent to the body wall, a zeugopod (radius-ulna/tibia-fibula) in the middle region, and a distal autopod (carpals-digits/tarsals-toes) Limbs grow using three-dimensional coordinate systems o Proximal-distal (“close-far”)  Stylopod (humerus) = most proximal bone  Autopod (Carpal-digits) = most distal bone o Anterior-posterior axis (“thumb-to-pinkie”)  Thumb marks the anterior end  Pinkie marks the posterior end o Dorsal-ventral axis (“knuckles-to-palm”)  Knuckles = dorsal side

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Palm = ventral side

The Limb Bud -

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The first visible sign of limb development is the formation of bilateral bulges called the limb buds at the presumptive forelimb and hindlimb locations o The limb bud is created by cells from the lateral plate mesoderm Emergence of the Limb Bud = Signaling occurs between lateral plate mesoderm and ectoderm to set up the limb bud o Proliferation of mesenchymal cells from the somatic region of the lateral plate mesoderm causes the limb bud in the embryo to bulge outward

The cells that make up the limb bud are derived from the posterior lateral plate mesoderm, adjacent somites, and the bud’s overlying ectoderm o Lateral plate mesenchyme and somite mesenchyme (in that location) migrate within the limb fields to form the limb bud The limb bud is regionalized into three functionally distinct domains:

o Progress zone (PZ) = the highly proliferative mesenchyme that fuels limb bud growth  Aka the undifferentiated zone o Zone of polarizing activity (ZPA) = the cells found within the most posterior region of the progress zone  It patterns cell fates along the posterior-anterior axis o Apical ectodermal ridge (AER) = a thickening of the ectoderm at the apex of the developing limb bud

Hox Gene Specification of Limb Skeleton Identity -

Hox genes = Homeobox transcription factors o Gives limb bud cells its identity along the proximal-distal axis  Specifies whether a particular mesenchymal cell will become the stylopod, zeugopod, or autopod o Proximal-distal axis patterned by Hox genes through spatial & temporal linearity

From proximal to distal: Hox genes in the limb -

The 5’ portions of the Hoxa and Hoxd gene complexes are active in the limb buds of mice Hox genes specify the identity of a limb region o Hox9 and Hox10 = stylopod o Hox11 paralogues = zeugopod o Hox12 and Hox13 paralogues = autopod

From fins to fingers: Hox genes and limb evolution -

Changes in the spatial and temporal pattern of distal Hox genes may be responsible for the evolution of the tetrapod hand o Hox genes are critical for specifying fates along each axis of the limb

Determining What Kind of Limb to Form and Where to Put It Specifying the limb fields -

The forelimbs are found at the most anterior expression region of Hoxc6, the position of the first thoracic vertebra o Limb field can regulate for lost or added parts; very regulative  Splitting limb disc results in two adjacent limbs growing  i.e. the result of infestation of the tadpole-stage frogs developing

limb buds by trematode cysts

Induction of the early limb bud Initiation of limb bud formation = (1) making mesoderm permissive for limb formation; (2) specifying forelimb and hindlimb; (3) inducing epithelial-to-mesenchymal transitions; (4) establishing two positive feedback loops for limb bud formation (1) Making Mesoderm Permissive for Forelimb Formation (2) Forelimb and Hindlimb Specification by Tbx5 and Islet1, respectively -

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Tbx genes controls the identity of the limbs (limb type in chick is specified by Tbx genes) o Downstream of hox genes o Determines if limb bud forms the forelimb or hindlimb  Tbx5 = forelimb formation (wings)  Tbx4 = hindlimb formation (legs) When a FGF bead is injected in-between the forelimb and hindlimb limb bud formation regions, an additional limb grows displaying phenotypic characteristics of both the forelimb and hindlimb o Forelimb structure expresses Tbx5 anteriorly o Hindlimb structure expresses Tbx4 posteriorly

(3) Induction of Epithelial-to-Mesenchymal Transitions by Tbx5 (4) Establishment of Two Positive Feedback Loops for Limb Bud Formation by FGF-WNT

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Hox genes from the somatic region of the lateral plate mesoderm activates a FGF positive feedback signaling sets up the growth of the limb bud Fgf10 signals from the mesoderm are sent to the epithelium (ectoderm) o this causes the ectoderm to send planar signals (Fgf8) back into the mesoderm  creates a positive feedback loop for continuing signal cycle Fgf10 is sufficient for limb development o Because it creates the positive feedback signaling that initiates the limb bud formation o (A) Fgf10 becomes expressed in the lateral plate mesoderm precisely at the regions where limbs normally form

o (B) when cells expressing Fgf10 are transplanted into another region, Fgf10 causes formation of another limb (arrow)

Outgrowth: Generating the Proximal-Distal Axis of the Limb The apical ectodermal ridge -

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AER = the ectoderm o Induced by Fgf10 signals sent from within (mesoderm) o Expresses Fgf8 AER: Promotes outward limb growth o (1) maintaining the Progress Zone mesenchyme beneath it as a  PZ enables proximal-distal growth of the limb o (2) maintaining the expression of those molecules that generate the anteriorposterior axis o (3) interacting with the proteins specifying the anterior-posterior and dorsalventral axes so that each cell is given instructions on how to differentiate AER Experiments o Removal of Forelimb AER  limb growth ceases distally o Addition of extra Forelimb AER grafted on existing limb bud  duplication of a limb o Place Leg (hindlimb) mesenchyme under AER of forelimb  distal hindlimb structures develop at the end of the forelimb o Limb mesenchyme is replaced by nonlimb mesenchyme  AER regresses and limb development ceases

The AER is responsible for the sustained outgrowth and development of the limb o The mesenchyme cells induce and sustain the AER and determine the type of limb to be formed

- Fgf8 = major active factor in AER - Fgf8-secreting beads can substitute for the AER functions in inducing limb growth Specifying the limb mesoderm: Determining the proximal-distal polarity - If the AER is removed from an early-stage limb bud, only the humerus forms - If the AER is removed slightly later, humerus, radius, and ulna form - Explaining how this happens has not been easy. First it had to be determined whether the positional information for proximal-distal polarity resided in the AER or the PZ mesenchyme o Through a series of reciprocal transplantations, this specificity was found to reside in the mesenchyme - Progress Zone Experiments o Early PZ into late limb  proximal structures developed

o Late PZ into early limb  distal structures developed such that digits were seen to emerge from the humerus without an intervening ulna and radius

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PZ: Regulates the proximal-distal develop of a limb

Specifying the Anterior-Posterior Axis -

In the chick, this axis is specified shortly before a limb bud is recognizable

Sonic Hedgehog defines a zone of polarizing activity -

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When the ZPA is taken from a young limb bud and transplanted to a position on the anterior side of another limb bud, a mirror-image duplication occurs

o Information is coming from both an anterior and posterior direction ZPA = zone of polarizing activity In 1993, Riddle and colleagues showed by in situ hybridization that Sonic hedgehog (Shh) (a vertebrae homolog of Drosophila hedgehog) was expressed specifically in the ZPA o As evidence that this association between the ZPA and Shh was not just a mere coincidence, they demonstrated that the secretion of Shh protein is sufficient for polarizing activity (gain of function experiment)  They transfected embryonic chick fibroblasts with a viral vector containing the Shh gene  The gene became expressed, translated, and secreted in these fibroblasts, which were then inserted under the anterior ectoderm of an early chick limb bud  Result: mirror-image digit duplications like those induced by ZPA transplants were resulted o Thus, Sonic hedgehog appears to be the active agent of the ZPA...