8.2 Limb Development in Chick Flashcards
Models for limb patterning (Proximal-distal)
Opposing gradients of
retinoic acid in proximal
flank and Fgf and Wnt in the
distal AER.
Fgf nd Wnt hight in distal region. Depending on what [ ] the cells see, they form •stylopod, that results in Meis gne to form humorous (high ret and low wnt).
•Med of boht forms zeugopod by making Hox11 forms R/U. •High Fgf/Wnt and low retinoic activated Hox13 forms digits in the autopod
Theory of posterior distal axis formaition.
Progressing zone model. All three zones that differentiate as cells leave the progress zone. As limb bud grows, the cell fate is established. Other models of limb bud gormation. First is progress zone model.
Thoeyr Early allocation and progenitor expansion model
All regions are pre-established. As time progresses, limb bud grows and regions (refer to pic) expand. Early stage small, late sage large in size.
Retinoic acid proximalizes the bone
- If you sub retenoic acid, it results in promixalization of the bone structures.
- Shaded area trasplated onto young AER. No RAcid for 4 digits. If treat with Racid results in other proximal structures as well as the digits.
- Age of mesenchye determines type of bones formed.
- More proximal structures formed if treated with RA along with Wnt and Fgf. Distal structures formed if treated with Fgf and Wnt.
Experiment: Zone of Polarizing activity (ZPA) transplanted onto a normal limb bud
ZPA transplanted onto a normal limb bud results in the formation of mirror-image digits. ndicate that ZPA whatever it contains its responcible for anterior posterior digit formation. Molecule of relevance was SSH.
Experiment: Shh gene was introduced into chicken fibroblast cells. These cells were pelleted and implanted onto the anterior margin of limb bud.
Mirror-image digits were formed, indicating that Shh
protein secreted had polarizing activity. HH is expressed only in small region of fore limb buds (the 4 black arrows is where SHH is expressed exculsivley). Can reproduce result by traspering ZPA or virus infected SSH. Indicating that SHH is a secretory protein. Has ability to polarise digit formation.
ZPA express SHH
- Sonic hedgehog (shh) gene is expressed in the precise location of the ZPA-it is induced to be expressed by retinoic acid and ectodermal FGF8
- cells transfected with virus containing the sonic hedgehog gene acquire the properties of a ZPA (see next slide)
- beads soaked in sonic hedgehog protein will act in place of a ZPA in transplants
- in humans, polydactyly (extra digits) may be related to ectopic expression of sonic hedgehog
ZPA formation
•Stg 17: Induced AER secretes Fgf8 into the underlying mesenchyme.
•Underlying mesenchyme expressing Hoxb8 is induced to express SHH forming ZPA in the posterior margin
.Stg 18: SHH maintains FGF in the AER and FGF maintains SHH in + feedback loop.
Early interactions between AER and limb-bud : SHH
- As limb bud grows, the ZPA region is induced.
- ZPA region is where SHH is active.
- SHH turns on activation of Grem1 which is inhibitor of BMP pathway.
- Once Bmp is inhibited, Fgf8 can be expressed and that’s where we have AER secluded to small region of ectoderm. •Posterior is En1 and anterior is Wnt7a expressed. That’s how you get borders of AER. Dorsal has Wnt7a but not ventral side.
- First is proximal-distal established, then anterior posterior, then dorsal ventral.
Digit Formation
Digit 5
Seceterory proetin, exsoxitosed outside the cell.
Autocrine fashion, secretted and effects the same cells. These cells see SHH for longest period of time and highest [ ] .
Digit 4
Cells that consitute digit 4, the amount of time they spend with SHH for less time than digit 5. Less time period. Cells also express SHH, but they see if for less time.
Digit 3
Some cells secrete SHH but mostly reply on ZPA to produce SHH. They encounter lower [ ] of SHH and for even less of time.
Digit 2
The cells don’t secrete SHH must rely on concetration produced from ZPA cells. Rely of difusion of SHH from Zpa
Digit 1
Cells see zero [ ] of SHH, this is the defalut fate of cells. If cell doesn’t see SHH they will automatically form a thumb. Independent of SHH.
AER and limb-bud mesenchyme interactions : SHH
- Fgf10 from mesenchyme generated by the lateral plate mesoderm activates Wnt.
- Wnt activates β-catenin that induces the synthesis of Fgf8 in AER.
- Fgf8 activates Fgf10 in positve feedback loop.
- BMP expressed in the ventral region causes the expression of engrailed-1, which represses Wnt signaling.
3-zones of cell-death within the undifferentiated mesoderm of limb bud of chicken embryo:
- Anterior necrotic zone (ANZ)
- Posterior necrotic zone (PNZ)
- Opaque patch of mesenchyme (OP)
OP in chick embryo
- OP = opaque Patch
- The first appearance of OP is at stage 23.
- At stage 24 OP reaches the maximum length.
- At stage 25 dead cells lie between the radius and ulna.
- At stage 26 proximal part of radius and ulna are separate and dead cells become smaller.
- By stage 27 and 28 the dead cells shrink to small no’s and are located in the triangular area bound by the base of radius and ulna and head of humerous.
- At stage 29 the OP disappears.
Formation of Radius and Ulna
When digits are formed, there’s tissue inbetwee nthe bones. For fingers to separate and get rid of tissue inbetween therere’s the INZ (interior necrotic zone) cells undergo apoptosis in between the digits fo that fingers form and are free. Ducks have a flap inbetween the digits due to cells failing to undergo apoptosis. Web is because cells fail to undergo apoptosis. We don’t because our cells have died so we have free moving digits.
To begin R/U is one pice of bone. There there’s this OP that forms and resilts in seperation of radious from ulna. Also NB in digit formation.
