Vertebrate limb development Flashcards
How do cells collectively organise themselves?
In order to acquire specific structure/identity
How do cells position themselves?
Along a 3D axis
What are the 3 axes of the vertebrate limb?
Anterior-Posterior:
- Anterior - thumb
- Posterior - little finger
Proximal-Distal:
- Proximal - shoulder
- Distal - tip of fingers
Ventral-Dorsal:
- Ventral - palm
- Dorsal - back of hand
What do the signals in the limb position?
Every cell that makes up:
- Nerves
- Tendons
- Blood vessels
- Bones
Of the limb
How do limb buds first appear?
As protrusions from the flank (side of the body) at PRECISE positions along the AP axis
Where do the limbs form from?
Limb fields at specific places along the AP axis
What determines where the limb fields are present?
INTRINSIC property of the LATERAL MESODERM
What happens if you graft a piece of mesoderm of the prospective limb into the flank which isn’t destined to form a limb?
What about if do this with an area of the flank –> area that forms a limb?
Forms a limb
No limb forms
What transcription factors specify limb bud IDENTITY?
Two T-box transcription factors
What T-box transcription factor is expressed in the forelimb?
Tbx5
What T-box transcription factor is expressed in the hindlimb?
Tbx4
What happens if force the expression of Tbx5 in the developing hindlimb?
BLOCK Tbx4 expression
In the chick, what happens if express Tbx4 in the forelimb and Tbx5 in the hindlimb?
In chick - reversal of activity
What happens if do loss-of-function of Tbx4 in the mouse?
Still from the hindlimb
Where is Pitx1 uniquely expressed?
What does Pitx1 do?
In the hindlimb - drives limb identity
What happens if KO Pitx1 in the hindlimb?
Loss of Tbx4 expression
Loss of hindlimb identity
What happens if force Pitx1 expression in the forelimb?
Express Tbx4
Hindlimb identity
How do specific limb fields form in specific areas along the AP axis?
Driven by Hox genes:
- Intrinsically expressed by the lateral mesoderm
- Drive specific Pitx and Tbx gene expression and positional identity
What is the boundary where the forelimb is going to form?
- At the boundary between Hox5 and Hox6
- The transition between the cervical and thoracic vertebrae in the axial skeleton
How is the limb bud/limb formation initiated?
How do we know this?
By FGFs (mainly FGF4 and FGF8)
If replace the limb field cells by beads soaked in FGF - limb field cells acquire limb bud identity
What is the suggested intermediate between Hox proteins and Tbx expression?
RA - produced by the paraxial and the lateral mesoderm
What is the model for the initiation of limb bud formation?
Hox proteins –> RA –> Tbx transcription factors –> Fgf10 –> FGF8
Describe the process of limb bud formation
1) Hox genes along the AP axis determine the position of the future LIMB FIELD
2) Depending on what Hox gene - determines forelimb or hindlimb (express different Tbx transcription factors)
3) Tbx transcription factors drive the expression of Fgf10 in the LATERAL MESODERM, which drives the expression of Fgf8 in the ECTODERM
What drives the expression of Fgf10 in the lateral mesoderm?
Fgf8 expression in the intermediate mesoderm
What restricts the expression pattern of Fgf10 and therefore Fgf8?
Wnt expression - restricts the size of the domains
What is the first thing to happen after the limb field is formed?
Thickening of the overlying ectoderm (apical ectodermal ridge)
What are the parts of the limb bud?
1) Apical ectodermal ridge (AER)
2) Progress zone
3) Zone of polarising activity (ZPA)
Where does the progress zone lie in the limb bud?
Directly behind the ectoderm (AER)
What cells make up the progress zone?
Loose mesenchymal cells
Where is the ZPA present in the limb bud?
At the posterior part of the limb bud
What does the ZPA express?
Shh signalling molecule
What happens when the AER of the chick embryo limb bud is removed at different stages in development?
What does this show?
Different effects at different stages of development:
3 days:
- Presence of proximal bone and SOME elements of the next part of the limb skeleton
- 5 days:
- All of proximal bone
- More LATERAL elements
4 days:
- Limb well formed but DIGITS missing
Shows:
- The longer you wait to remove the AER - the more distal structures will form
- The AER is NECESSARY for the outgrowth of the limb
What are the 2 models for proximo-distal patterning of the limb?
1) Progress zone model
2) Two signal model
Describe the progress zone model for the proximo-distal patterning of the limb
- AER drives the OUTGROWTH of the limb, by acting on the progress zone behind it (promotes proliferation of these cells)
- The MORE cells that divide in the progress zone - the more distal structure that will develop (more cell divisions = more distal stuctures)
Is the progress zone model for the proximo-distal patterning of the limb correct?
Why?
No
Using fate mapping:
- Early on in the developing limb field, there is progenitors that can contribute to ALL the skeletal elements present in the future limb
- If model was correct - there would only be cells that contribute to the most PROXIMAL skeletal elements
What is the 2 signal model for the proximo-distal patterning of the limb?
- Progenitors in the limb for ALL the possible skeletal elements
- BUT, these cells adopt a SPECIFIC FATE depending on the signals they receive
- Cells receive antagonistic signals from the distal and/or proximal of the limb as a gradient
- Depending on where the cells are along the gradient - determines if they will become a proximal or distal skeletal element
What molecule is released proximally in the limb bud?
Retinoic acid
What molecule is released distally in the limb bud? (in the AER)
Fgf
What does Fgf drive the expression of?
Hox genes
Which hox gene is expressed in the most distal element?
Hox 13
Which hox gene is expressed in the most proximal element?
Hox 11
What mesoderm releases RA?
Paraxial and PROXIMAL lateral mesoderm
What does RA drive the expression of?
Meis genes (Meis 1 and Meis 2)
What enzyme synthesises RA?
What happens if there is a KO of this enzyme
rhld2
KO:
- No RA
- No meis expression
- No proximal elements of the limb skeleton
How is the identity of the RA and Fgf translated downstream?
What does this specify?
By genes belonging to the hox transcription factor family
Specifies the proxmio distal axis
What is the proxim-distal patterning of the drosophila leg controlled by?
2 genes:
- Distalless (patterns distal elements)
- Homothorax (patterns proximal elements)
What is the homologue of Distalless in humans?
HoxD13
What is the homologue of Homothorax in humans?
Meis genes
When can the proximal distal axes of the limb bud be specified?
Following the initial formation of the limb bud
How is the proximal distal axes of the limb bud specified?
Through the activity of the antagonistic factors (RA and FGF) which are translated downstream by different genes belonging to the hox transcription factors
What structure is involved in patterning the AP axis of the limb bud?
The ZPA
What happens if the ZPA is ectopically transplanted from the posterior of the limb bud to the anterior?
What happens if do the same thing but with a shh soaked bead?
What does this show?
Results in a mirror image duplication of the digits
Also duplication of the ULNA
Similar outcome with soaked bead
Shows that the ZPA drives the patterning of the cells along the AP axis entirely because of the activity of shh
What lead to Wolpets french flag model?
The activity of shh from the ZPA which patterns the AP axis of the limb bud
Why when transplanting the ZPA anteriorly, is there a mirror image duplication?
Produces a morphogen gradient in the reverse direction
In the mouse, where does digit 1 form?
How are digits 2-5 formed?
Where there is NO shh signalling (most anterior)
Next digits are formed by progressively increasing shh gradient
What happens when there is shh KO?
- Complete loss of most DISTAL skeletal elements
- Truncation of the limb
- Loss of all the digits that require shh signalling but digit 1 remains (doesn’t require shh signalling)
- Also loss of the radius/ulna - single bone with no identity remains
When there is a shh KO, why is a defect in outgrowth along the proximal distal axis?
AP axis is coordinated with the PD elongation of the limb
How is the AP axis is coordinated with the PD elongation of the limb
What does this ensure?
Through reciprocal control of the events:
- Signals responsible for the AER that drive elongation of the limb are also required to MAINTAIN the expression of shh in the ZPA
- ZPA is required to MAINTAIN the expression of Fgf in the AER
Ensures that:
- As the limb grows, it is patterned along the AP axis
What is different/same between the ectoderm that covered the DORSAL part of the limb and the ectoderm that covers the VENTRAL part?
They are different:
- Wnt7a expressed dorsally
- Engraled1 (en1) expressed ventrally
Where is the transcription factor lmx1b expressed?
Domain of expression that divides the limb along the DV axis (expresse dorsally)
What does lmx1b control?
Dorsal fate
What is the drosophila wing homologue of lmx1b?
Apterous
What does KO of lmx1b cause?
What does this show?
Ventralisation of the limb (form palm instead of fur)
Shows that ventralisation of the limb is the default pathway
What is the proposed model for dorso-ventral patterning?
1) BMP signalling in the ventral part of the limb
2) This drives the expression of En1
3) En1 limits the extent of the expression of Wnt7a - is restricted to the dorsal extoderm
4) Wnt7a drives the expression of Lmx1b
