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Developmental Biology > Lecture 5 - Limb development and regeneration > Flashcards

Lecture 5 - Limb development and regeneration Flashcards

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1
Q

What is pattern formation in the developing embryo?

A

the formation of all the different cell types into functional in the developing embryo in a 3 dimensional pattern.

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2
Q

What are the three axis that limbs have?

A

Proximal to distal (shoulder to finger)
Anterior to posterior (thumb to pinky)
Dorsal to Ventral (knuckle to palm)

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3
Q

In the vertberate arm, what are teh 3 regions in the proximal to distal patterning?

A

From proximal to distal

  • Sytlopod
  • Zeugopod
  • Autopod
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4
Q

What is the region of the embryo capable of making a limb called?

A

The limb field

-forms at certain points along the anterior to posterior axis

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5
Q

How has the limb field been experimentally defined?

A

-fate mapping and grafting experiments

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6
Q

What is the boundary of the limb field defined by?

A

Boundary of hox gene expression

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7
Q

What is the process of formation of the limb bud?

A
  • in the somatic mesoderm in the lateral plate the mesenchyme cells proliferate to form a budge = limb bud
  • proliferation is initated by signals from the intermediate mesoderm which forms the pro-nephros
  • bus appear on the third day of chick development
  • consist of loose mesoderm surrounded by an ectodermal jacket
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8
Q

What is involved in establishing the proximal to distal polarity of a limb?

A

The apical ectodermal ridge

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9
Q

What is the AER (apical ectodermal rigde) and where does it form?

A
  • an area of thickened ectoderm at the distal end of the limb bud
  • forms at the margin of the dorsal and ventral ectoderm
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10
Q

How are FGF10 and the AER involved in the induction of limb outgrowth?

A
  • FGF10 expressed in the lateral plate mesoderm at the right time is capable of inducing limb forming interactions between the mesoderm and the ectoderm
  • to do this, FGF10 initially induces the formation of the AER in the ectoderm component (dorsal-ventral junction)
  • the AER is essential for the proximal distal outgrowth of the limb
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11
Q

What have experiments that have removed the AER shown?

A

Removal of the AER results in further proximal to distal development stopping, resulting in limb truncation
-level of truncation depends on the time that the AER is removed
-also demonstrated that the limb forms in a proximal to distal direction
-the AER is responsible for the sustained outgrowth of the limb
Experiment:
1. If the AER is left intact, normal development occurs
2. If the AER is removed early, then only the stylopod develops
3. If the AER is removed later, then the stylopod and zeulopod develop

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12
Q

What is the AER responsible for ?

A
  • the sustained outgrowth of the limb

- keeping the underlying mesoderm in a proliferative, undifferentiated state

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13
Q

What is the loosley associated mesenchyme located beneath the AER responsible for?

A

-inducing competent ectoderm to form an AER
-determines the type of limb that will form
-signals to maintain the AER
Progress zone

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14
Q

What experiment showed the purpose of the AER underlying mesoderm? Progress zone

A

-took underlying mesenchyme cells from the leg bud and placed in the area that would become wing
-resulted in the formation of toes in the wing area
Showed the AER underlying mesoderm is responsible for:
-inducing competent ectoderm to form an AER
-determines the type of limb that will form
-signals to maintain the AER

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15
Q

What is the progress zone?

A

The mesodermal component of proximal to distal polarity

-proliferative, undifferentiated plastic mesoderm underlying the AER

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16
Q

Where is muscle derived from?

A

Migration in from the somites

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17
Q

What is the ZPA (zone of polarising activity)?

A
  • small block of mesoderm at the posterior junction of the young limb bud and the body wall
  • involved in establishment of anterior to posterior polarity
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18
Q

What does the ZPA being grafted anteriorly result in? And how might this be controlled?

A
  • results in a mirror image duplication of digits
  • additional digits come from the host not the donor ZPA
  • pattern of muscle and tendon also show mirror image duplication
  • potentially controlled via a diffusible concentration gradient of a morphogen to specify the position of cells along the AP axis (highest concentrations including digit 4 and lowest including digit 2)
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19
Q

What is the physiological process of formation of the limb by the AER and progress zone?

A
  • the progess zone is specified at the distal end of the limb bud by the AER
  • cells in the progress zone proliferate and aquire a positional value
  • when cells leave the progress zone, cartilidge elements differentiate and are laid down in a proximal-distal sequence
  • specification along the AP axis is a result of signals from the ZPA located in the posterior mesoderm
  • thought to be a morphogen concentration specifying posterior (digit 4) at high conc and anterior (digit 2) at low conc
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20
Q

What are the signalling centres in the limb bud?

A

ZPA

AER

21
Q

What is the function of the signalling centres in the limb bud?

A

give cells positional information along the AP (ZPA) and PD (AER)

22
Q

What is the involvement of FGF8 and FGF4 in the formation of the limb?

A

FGF8
-expressed in all of the AER
FGF4
-expressed in the posterior part of the AER

When the AER is removed, implantation of beads soaked with FGF can replace ridge signals and allow normal outgrowth
-FGFs allows AER to function in maintaining bud outgrowth

23
Q

What are the features of a limbless chick mutant?

A
  • fails to form an AER

- does not express FGF8 in the ridge

24
Q

What moelcules allow the AER to maintain bud outgrowth?

A

secreted FGFs by the AER allow outgrowth

25
Q

How is sonic hedgehog involved in limb development?

A
  • expressed in the ZPA
  • induced mirror image duplication when ectopically expressed in an anterior section of the developing limb bud
  • probably not acting as a morphogen gradient as the active N-terminal of sonic does not diffuse very far -> instead, activates the expression of BMP2 which is also expressed in the ZPA
  • BMPs expression induced by sonic may act as morphogen
26
Q

What happens when beads soaked in shh (or cells expressing shh) are grafted anteriorly in a developing limb bud?

A
  • act like a ZPA

- induce mirror image duplication

27
Q

What is the process of a positive feedback loop between FGF4 in the AER and sonic in the ZPA?

A
  • intermediate mesoderm expresses FGF10 and this induces FGF8 expression in the overlying component ectoderm
  • expression of hox genes establishes the AP boundary in the limb bud and therefore determines where sonic will be expressed
  • FGF8 in the ectoderm (AER) induces proliferation in the mesenchyme and sonic expression in the posterior mesenchyme
  • sonic then induces FGF4 expression in the posterior AER
  • sonic expression in the ZPA activates expression of BMP2 which maintains FGF4 in the ridge
  • sonic signalling also maintains the AER
28
Q

What triggers the positive feedback loop between FGF4 in the AER and sonic in the ZPA? And what experiment showed this?

A
  • a bead soaked in FGF protein and implanted into the presumptive flank of an early chick embryo induces an ectopic limb bud which expresses FGF8 in the AER, sonic in the ZPA and overlapping Hos D genes and develops into a complete ectopic limb
  • FGF protein
29
Q

What proteins are involved in a regenerating newt limb? And how is this different to morphologically equivilent developing limb buds?

A
  • shh is expressed in the posterior mesenchyme (shown by in situ hybridisation)
  • expresssion starts later and declines earlier than it does in morphologically equivilent developing limb buds
30
Q

What hox genes are involved in a regenerating newt limb?

A
  • 3’(HoxA9) and 5’ (HoxA13) genes are activated simaltaenously at the stump regardless of the level of amputation
  • coexpression of A9 and A13 specifies the distal most part of limb pattern -> consequently in the first part of regeneration the missing pattern to be specified is the most distal part
31
Q

What is involved in the growth and intercalculation of missing positional values in a regenerating newt limb?

A

the interaction of the stump with the newly specified distal structures (coexpression of A9 and A13)

32
Q

What is the molecular readout of the positional identity of cells in the progress zone?

A

expression of a certain set of Hox genes

  • vertebrate hox gene specify position along the AP axis of the body and provide positional values within the limb
  • the AbdB group of paralogues groups A and D (Hox D9-13 and HoxA9 -12) are expressed in the developing limb
  • Hox D9-13 are expressed as a concetntric overlapping set with centre at the ZPA (grafting of a ZPA to the anterior results in a mirrored expression of the hox genes)
33
Q

How are the AbdB group of vertebrate hox gene paralogues A and D (HoxD9-13 and HoxA9-13) expressed in the developin glimb and what is their prupose?

A

The AbdB group of vertebrate hox gene paralogues A and D (HoxD9-13 and HoxA9-13) postion along the AP axis of the body and provide positional information within the limb
-HoxD9-13 are expressed a a concentric overlapping set with centre at the ZPA

34
Q

What does grafting a ZPA to the anterior result in~?

A
  • a mirror expression pattern of the hox genes

- ZPA control the hox gene expression

35
Q

What protein/genes are involved in the establishment of dorsal to ventral polarity?

A

Wnt7a

-involved in maintaining the activity of Shh and FGF4

36
Q

What have experiments that remove the dorsal ectoderm and in Wnt7a deficient mice show?

A

Remove the doral ectoderm
-results in loss of the dorsal and posterior elements of a developing limb
Wnt7a deficient mice
-ventral pads forms both dorsally and ventrally (dorsal axis not specified)
-no posterior digits form
-no expression of Shh and FGF4

Shows: the establishment of all three axis is tied in together

37
Q

How is the establishment of all three axis in a developing limb bud tied in together?

A

Coordinated regulation of pattern formation in the developing limb

  • AER signals maintain the ZPA
  • ZPA maintains the AER
  • dorsal ectoderm (Wnt7a) is needed for FGF4 and Shh expression
38
Q

What are the features of coordinated regulation of pattern formation in the developing limb?

A

The establishment of all three axis is tied in together

  • AER signals maintain the ZPA
  • ZPA maintains the AER
  • dorsal ectoderm (Wnt7a) is needed for FGF4 and Shh expression
39
Q

What type of regeneration is necessary for regeneration of the uroldele limb?

A

Epimorphosis

-occurs by proliferation of new tissue from the wound site and progressive replacement of the missing parts

40
Q

What is a special feature of a newt limb?

A
  • when amputated it regenerates perfectly
  • remaining cells are able to reconstruct a complete limb with all its differentiated cell types and with the correct pattern along all three axis
41
Q

What are the features of newt limb regeneration?

A
  • remaining cells can reconstruct a complete limb with all differentiated cell types
  • with the correct pattern along all three axis
  • positional information is retained within the adult tissues (amputation at differentiation results in regeration in only the missing parts)
  • can take from 3 weeks to several months
42
Q

What is the morphogenic process of epimorphosis?

A
  1. Wound closure: the epithelium spreads over the wound by cell movement, epidermis will form the apical ectodermal cap
  2. Formation of the regeneration blastema: the epidermis bulges out, becoming conical in shape
    - mass of cells accumulate under the epidermis which are actively proliferating and for the regeneration blastema
43
Q

What are some observations about regenerating limbs?

A
  • never contain duplicated or missing tissue - all the normal limb elements are laid down to form in the correct order
  • even when cultured in isolation a blastema will forma limb appropriate to where it was amputated
  • regeneration always occurs in a direction distal to the cut surface (everything distal to the cut is regenerated)
  • there is memory in the adult tissue so that the regenerating limb is able to read positional value at the site of amputation and can then regenerate everything distally
44
Q

What experiment was done to show that regeneration always occurs in a direction distal to the cut surface?

A
  • Newt limb was amputated
  • limb was then inserted into the belly, vasocular connections were established then the humerus is cut
  • regeneration starts from the proximal and digital surfaces of the humerus
  • both proximal and distal surfaces regenerate distal structures

Therefore:
-there is memory in the adult tissue so that the regenerating limb is able to read positional value at the site of amputation and can then regenerate everything distally

45
Q

What process must cells in the amputated region (including connective tissues, bone and cartilidge cells and skeletal muscle cells) participate in during regeneration?

A

De-differentiation to give rise to embryonic like cells in the blastema

46
Q

What experiment was done to show that dedifferentiation can happen in newt but not in mammals and avians?

A

-in mammals and avians, after fusion, muscle cells are refractory to any serum growth factors and will not re-enter the cell cycle
-normally muscle repair is done by mobilising mononucleate satellite cells that act as reserves
HOWEVER
-Newt myotubes respond to high serum by re-entering the cell cycle
-transfer of cultured labelled mytotubes (multinucleate) into the blastema will give rise to a significant number of mononucleate, proliferating cells
-cells will go on to form muscle and some cartilidge
-indicates that the local environment of the blastema promtotes a revelsal of the mono to nucleate cells tha toccurs during myogenesis

47
Q

When do the limb buds appear on the developing chick embryo?

A

Form on the third day of development

consist of loose mesoderm surrounded by an ectodermal jacket

48
Q

What is the function of the mesenchyme in limb outgrowth?

A
  • induces competent ectoderm to form the AER at the DV margin
  • determines the type of limb that will form
  • signals to maintain the AER

Developmental Biology (14 decks)

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