Chapter 19 Part 1: Tetrapod Limb Development

Question 1

Which type of mesoderm gives rise to the limb bud itself?

Answer 1

the lateral plate mesoderm

Question 2

the main direction of limb bud growth occurs along the proximal distal axis, aka from the ___ to the ___

Answer 2

from the somites to the mesoderm

Question 3

what three groups of cells make up the limb bud derivatices?

Answer 3

1) posterior lateral plate mersoderm 2) adjacent somites mesenchymal cells 3) bud’s overlying ectoderm

Question 4

3 main domains of a limb bud

Answer 4

1) proliferative zone: mesenchyme cells that actively proliferate to allow for limb bud growth 2) zone of polarizing activity: cells in the most posterior region or ZPA creates the anterior-posterior pattern of the limb 3) apical ectodermal ridge: thickening of ectodermal cells at the tip of the limb bud. Allows for distal extension and involved in specification

Question 5

this domain of the limb bud is critical for distal extension and proper specification into stylo/zeugo/autopod regions

Answer 5

the apical ectodermal ridge

Question 6

this domain of the limb bud creates the anterior-posterior pattern of the limb

Answer 6

zone of polarizing activity

Question 7

___- genes help determine specification of mesenchymal cells becoming the stylopod, zeugopod, or autopod

Answer 7

Hox genes

Question 8

How are hox genes broadly regulated?

Answer 8

regulated by signals from the flank (proximal) and AER (distal)

Question 9

In mice, which hox genes specify the stylo? the zeugo? the auto?

Answer 9

hox 9 and 10 = stylopod hox 11= zeugopod hox 12 and 13 = autopod.

Question 10

What is a paralogue? Provide an example

Answer 10

different transcripts that can be transcribed from the same gene. Hox 9 and 10 that specify the stylopod and receive input from the flank and aer are paralogues of the same hox gene.

Question 11

In humans, a homozygous mutation in the ____ results in polysyndactly, which is when fingers are fused together

Answer 11

mutation in the hoxd13 gene. (a stylopod specifier)

Question 12

LIMB DETERMINATION AND PLACEMENT (before limb bud growth): ____ mesoderm cells become forelimb buds when they express ____.

Answer 12

SOMITE mesoderm cells become forelimb buds when they express HOXC6. Somite mesoderm from non-limb area represses limb bud formation.

If there is hoxc6 overexpression in the thoracic vertebra/somite mesoderm, there will be larger limb buds.

Question 13

So, the limb is formed due to somite mesoderm expressing Hoxc6 in certain areas of the body, specific the thoracic vertebra area. But what induces actual limb bud formation in this area? There are 4 broad steps:

Answer 13

1) making mesoderm permissive for limb formation
2) specifying forelimb and hind limb
3) inducing epithelial-mesenchymal transition
4) establishing 2 positive feedback loops for limb bud FORMATION.

Question 14

step 1 of limb bud formation;

1) making mesoderm permissive to forelimb formation by ____ ____.

there is an antagonistic relationship of ___ ___ and _____ in somitogenesis. Explain it.

Answer 14

1) making mesoderm permissive to forelimb formation by RETINOIC ACID.

there is an antagonistic relationship of RETINOIC ACID and FGF8 in somitogenesis.

• fgf8 is expressed by the caudal (tail/posterior) progenitor zone located posterior to the forelimb field. RA is generated more anteriorly in somites and presomitic mesoderm of the forelimb field. Fgf8 is also expressed in more anterior regions above the forelimb field like the heart lateral plate mesoderm.
• this anterior and posterior expression of fgf8 besides in the actual forelimb, which contains an fgf8 inhibitor RA, inhibits forelimb bud initiation in other areas. Ie, fgf8 inhibits forelimb bud initiation, while RA permits limb bud formation.

Question 15

Limb bud initiation/formation:

RA is generated in the anterior portions of the somites and presomitic mesoderm adjacent to the forelimb field, whereas Fgf8 is expressed in the posterior flank regions as well as in the heart lateral mesoderm. RA ____ fgf8, which allows for:

Answer 15

RA inhibits fgf8, which allows for FORELIMB formation via the induction of tbx5 expression.

*if there is no fgf8, the lateral plate mesoderm is permissive for forelimb bud development.

Question 16

Okay, so the mesoderm is permissive to forelimb bud formation by retinoic acid because it suppresses limbbud formation-repressor fgf8. which gene expression determines whether the limb is fore limb or hind limb?

Answer 16

for the forelimb, RA induces Tbx5 expression, while genes encoding for islet 1, tbx4, and pitx1 are expressed in presumptive hindlimbs.

this is for limb bud formation, this all happens before the limb bud is actually seen in the embryo.

Question 17

for the forelimb formation, RA induces ____ expression, while genes encoding for ___, ____, and ____ are expressed in presumptive hindlimbs.

Answer 17

for the forelimb, RA induces Tbx5 expression, while genes encoding for islet 1, tbx4, and pitx1 are expressed in presumptive hindlimbs.

Question 18

In addition to tbx 4 in hindlimb and tbx5 expression in forelimb necessary for the limb bud formation, what other factor must be present?

Answer 18

fgf10 is the primary inducer for limb bud growth and proliferation.

prior to actual limb bud formation, there are normally regions of lateral mesoderm where tbx4 is expressed (because of posterior RA) and tbx5 is expressed (because of anterior RA). Adding fgf10 to these regions promotes the limb growth that tthe tbx genes specified.

therefore, fgf10 + tbx5 = forelimb. and fgf1- + tbx4 = hindlimb.

Question 19

___ initiates the growth of limb that is specified by tbx 5 or tbx4 depending on location, and is present in both the forelimb and hindlimb. In what germ layer is this paracrine factor found in?

Answer 19

FGF10 initiates the growth of limb that is specified by tbx 5 or tbx4 depending on location, and is present in both the forelimb and hindlimb. FGF10 is found in the lateral plate mesoderm.

Question 20

Outline the role of Pitx1 in limb bud initiation

Answer 20

pitx1 is a TF invovled in the initiation of the HIND LIMB. promotes the development of muscles, bones and tendons of the hind limb. it also helps to activate tbx4 as well

a hind limb will still form wihtout pitx1, but it forms funny. therefore it is suggested that pitx1 expression is important for hind limb patterning instead.

Question 21

Clarification: RA can induce Tbx5 expression for proper forelimb development. Tbx5 upregulates fgf10 for further proliferation. Fgf10 can then induce more tbx 5 (like a positive loop).

This is also seen in hind limb development for tbx4. However, it is unknown what actually induces tbx4 to begin with, because if you were to antagonize RA, the hindlimb bud still grows, so it probably isn’t as dependent on RA as forelimb growth is.

Question 22

outline the role of islet1 in limb bud induction/initial formation

Answer 22

islet 1 is important for hindlimb formation. It is seen to be transientyl expressed in the hindlimb before fgf10.

• when islet1 is inactivated in the lateral plate mesoderm, no hind limb forms. Therefore it has a role in hindlimb initiation.

In mice, islet1 seems to replace the role of tbx4 in terms of hind limb initiation.

Question 23

Early limb bud induction requires epithelial-to mesenchymal transition of cells from epithelium to form the somitic mesoderm. Regions where limb fields form actually undergo EMT before other flank regions. What regulates this process of EMT?

Answer 23

Tbx5 expression is a major regulator of EMT in the forelimb field

Recall in chapter 13: Neural ectoderm cells that will form the posterior neural tube expresses Sox2, where as somitic mesoderm will express Tbx genes to undergo EMT into the embryo and form somites. When inside the embryo, the somitic mesoderm cells are no longer being affected by BMP so they don’t need to express any sox2 to prevent epithelial tissue formation. When mesoderm cells ingress (gastrulation or EMT), Tbx is expressed to form somites, suppressing neural induction by sox2.

Question 24

Through the upregulation of tbx5 in the forelimb, and islet1 (or tbx4 for chicks) in the hindlimb, the mesenchyme commit otward limb development and secrete the paracrine factor ____. What does this do?

Answer 24

Through the upregulation of tbx5 in the forelimb, and islet1 (or tbx4 for chicks) in the hindlimb, the mesenchyme commit otward limb development and secrete the paracrine factor FGF10. Fgf10 signals and initiates limb bud formation by inducing the GROWTH and proliferation of limb buds.

Question 25

Through the upregulation of tbx5 in the forelimb, and islet1 (or tbx4 for chicks) in the hindlimb, the mesenchyme commit otward limb development and secrete the paracrine factor FGF10. Fgf10 signals and initiates limb bud formation by inducing the GROWTH and proliferation of limb buds.

How is the fgf10 signal maintained to continue inducing the growth of the limb bud?

Answer 25

Fgf10 signal is maintained by the positive feedback loop instilled between it and tbx5 (forelimg) or islet 1(mammal hindlimb).

Tbx5/tbx4/islet 1 induces fgf10 expression, which induces proliferation, but also exerts positive effects back onto tbx5/4 and islet1, AS WELL AS WNT+BETACATENIN PATHWAYS.

Therefore, in the forelimb field for example, Tbx5 that was induced by RA in forelimb field somitic mesoderm induces Wnt2b, which upregulates fgf10, which in turn positively feedbacks to maintain activation of both wnt2b and tbx5, while also induced proliferation (mitosis on diagram)

Question 26

Fgf10 is originally upregulated by tbx4/5/islet1, which induces a positive feedback loop for sustained limb bud proliferation in mesenchyme. What does fgf10 also do to the overlying ectoderm?

Answer 26

Fgf10 secreted by the limbfield mesenchyme induces the overlying ectoderm to form the apical ectodermal ridge (AER).

Question 27

Fgf10 secreted by the limbfield mesenchyme induces the overlying ectoderm to form the ___ ___ ___

Answer 27

Fgf10 secreted by the limbfield mesenchyme induces the overlying ectoderm to form the apical ectodermal ridge (AER)

Question 28

How does fgf10 in the lateral mesenchyme limb bud tissue induce the apical ectodermal ridge?

Answer 28

fgf10 in the lateral mesoderm induces the cells above it in the ectoderm to form a distal apical ectodermal ridge. This is done because fgf10 stimulates Wnt3 in the prospective limb bud surface ectoderm. Wnt protein acts via canonical beta catenin pathway to induce fgf8 expression in the ectoderm. Once fgf8 is made in the surface ectoderm, the surface ectoderm elongates to physically become the AER.

Question 29

fgf10 in the lateral mesoderm induces the cells above it in the ectoderm to form a distal apical ectodermal ridge. This is done because fgf10 stimulates ____ in the prospective limb bud surface ectoderm. ___ protein acts via canonical ____ pathway to induce ___ expression in the ectoderm. Once ____ is made in the surface ectoderm, the surface ectoderm elongates to physically become the AER.

Answer 29

fgf10 in the lateral mesoderm induces the cells above it in the ectoderm to form a distal AER. This is done because fgf10 stimulates Wnt3 in the prospective limb bud surface ectoderm. Wnt protein acts via canonical betacatenin pathway to induce fgf8 expression in the ectoderm. Once fgf8 is made in the surface ectoderm, the surface ectoderm elongates to physically become the AER.

Question 30

there are two positive feedback loops formed during limb bud induction. the first one is when fgf10 is upregulated by tbx5/4/islet1, which promotes the formation of wnt2, as well as tbx5/4/islet1 (+ feedback). What is the second?

Answer 30

In addition to ensure the mitotic proliferation of limb bud mesenchyme via tbx5/4/islet1 and wnt2 positive feedback loop, another feedback loop is through AER formation.

fgf10 upregulates ectodermal wnt3, which activates fgf8. Fgf8 in the ectoderm (AER) can induce fgf10 even more, making this the second feedback loop.

Mesodermal fgf10 tells the surface ectoderm to continue to make fgf8, which induces AER formation and makes the underlying mesoderm to make fgf10. Each fgf thus activates the synthesis of the other. This continued expression of fgfs maintains mitosis in the mesenchyme beneath the AER, which fuesl the outgrowth of the limb.

Question 31

Important note:

Mesodermal fgf10 tells the surface ectoderm to continue to make fgf8, which induces AER formation and makes the underlying mesoderm to make fgf10. Each fgf thus activates the synthesis of the other. This continued expression of fgfs maintains mitosis in the mesenchyme beneath the AER, which fuesl the outgrowth of the limb.