1
Q
Define Syndactyly
A
The fusion of the digits
2
Q
Define Polydactyly
A
Having more than 5 digits
3
Q
Define Preaxial polydactyly
A
before the thumb, an anterior defect
4
Q
Define Post-axial polydactyly
A
digits after the little toe or finger
5
Q
What is Cleft foot/lobster claw deformity?
A
- Cleft between 2nd & 4th metacarpals
* Third metacarpal/phalangeal bones absent
6
Q
Define Clubfoot
A
- Inward facing soles
* More common in males, sometimes hereditary
7
Q
What is Phocomelia?
A
- Seal like limbs that are seen in patients, typically seen after exposure to thalidomide
- The patterning of the limb is interrupted, the proximal elements are preserved and the intervening elements are missing such as the humorous, radius and ulna.
- The hands are formed almost normally
8
Q
Name the 3 major axis’ of the hand
A
On image
9
Q
Describe the structure of the limb bud
A
- The limb bud is a simple structure consisting of an ectodermal jacket surrounding a mesenchymal core
- At the distal tip of the limb bud is the apical ectodermal ridge which arises from the ectodermal cells. It runs from the anterior to posterior ridge. It is pseudostratified epithelium, which is formed by the physical movement of the epithelial cells along the distal tip of the limb bud.
- The ridge marks the boundary between the dorsal surface from the ventral surface on the left and right respectively.
10
Q
What does the ZPA stand for and pattern in the limb?
What happens if we graft the ZPA to the anterior region of the limb bud?
A
Zona polarising activity - found posteriorly - mesenchymal tissue - patterns limb on AP axis
- Anterior ZPA (zona polarising activity) graft
a. This experiment takes a section of the limb from the posterior margin of the limb bud (mesenchymal portion of the tissue). It is transplanted into the anterior margin of a host limb bud. This leads to a mirror duplication of the outgrowing limb. So we end up with two little fingers forming.
11
Q
What happens to the limb bud if we flip the ectodermal jackets at the dorsal and ventral poles?
A
- 180 degree rotation of the ectoderm
a. Here the ectodermal jacket is removed which is rotated 180 degrees so the ventral and dorsal surfaces have flipped. This causes a reversal of the dorsal-ventral axis of the limb. This tell us signals from the ectoderm pattern along the dorsal-ventral axis.
12
Q
What happens if we remove the apical-ectodermal ridge?
What does this tell us?
A
a. Pseudostratified epithelium is removed at the distal tip. This leads to the truncation of limb outgrowth and loss of distal structures. This tells us that the apical ectodermal ridge is required for the continued outgrowth and formation of the limb from the limb bud.
13
Q
Where is the skeleton derived from?
A
• Skeleton (including girdles) are derived from limb bud mesenchyme
14
Q
Where are the Tendons, ligaments, fascia, dermis derived from?
A
• Tendons, ligaments, fascia, dermis also derived from limb bud mesenchyme
15
Q
Where are the muscles derived from?
A
• Muscles are derived from myotome of somites
16
Q
Where are the nerves derived from?
A
• Nerves from spinal nerves (neural tube)
17
Q
What are the blood vessels derived from?
A
• Blood vessels from endogenous plexus and dorsal aorta
18
Q
What are the Pigment and Schwann derived from?
A
• Pigment and Schwann cells from neural crest
19
Q
Describe the origin of the limb bud musculature
A
- The myotome cells will migrate into the emerging limb buds.
- They will remain in an undifferentiated state because of the expression of a gene called pax3 which keeps them as a myoblast precursor
- Scatter factor causes the migration of the myoblasts into the developing limb bud.
- The cells split into a dorsal and ventral muscle mass that will give rise to extensors and flexors which will then be patterned depending on the limb environment itself.
- Migration of muscle precursors from the myotome of somites
- Splitting of dorsal and ventral muscle masses in the limb bud
- Signals (Scatter factor) from limb induce myoblast (c-Met RTK) migration
- Muscle patterning is dependent on the limb environment
- Pax3 mutation interferes with myoblast migration
20
Q
How do we know forelimb proceeds hindlimb development?
A
- The embryo on the LHS has 10 pairs of somites and the wing field has been specified (cell marking experiments have allowed us to see which tissue this will emerge from).
- A few hours later (5), the wing field has migrated upwards because the embryo has extended. In the chick it will always develop between somites 16 and 20.
- A few hours later the leg field has started to emerge, cell tracing experiments tell us this. The bulges in the flank of the chick show us the first morphological changes that give rise to the limbs. Forelimb development proceeds hindlimb development
- The embryo needs to decide where the limbs will actually grow out (along the dorsal ventral axis). Also, the forelimb has a different identity to the hindlimb, these differences needs to be specified.
21
Q
Once the limb bud has formed what now needs to happen to the axis’
A
Once the limb bud has emerged it needs to be patterned along the three major axis: the proximal-distal axis, dorsal-ventral axis and what determines anterior-posterior identity
22
Q
Label the major limb elements
A
On image
23
Q
What is variation in tetrapod’s achieved by?
A
• Variation in tetrapod forelimb morphology is achieved (largely) by modification of the distal elements (autopod)
Most variation arises from the autopod region
24
Q
What happens when we knockout the prx1 gene in the mouse?
What happens when we expresses prx1
What does this gene do?
A
- In the knock out the proximal region is not greatly affected, the zeugopod might be shorter and the autopod are shorter.
- In the prx1 enhanced gene, we can see an increase in length of the zeugopod and autopod elements.
- Prx1 is important for the proliferation of the early mesenchyme of the limb. The bat enhancer is changing the duration of the prx1 gene expression.
25
Bat wing development
Bat wing development
26
How many phases are there of limb patterning?
There are 3 phases of limb patterning
27
Describe the 3 stages of limb bud patterning?
* There are 3 phases of limb patterning
* There are the early events that occur in the lateral plate mesoderm (along the body axis of the embryo). These early events will pattern the limb along the proximal-distal axis, anterior-posterior axis and dorsa-ventral axis.
* This early initiation is followed by the developing limb bud stage. There is pattering in the same 3 primary axis
* Finally there is digit patterning occurs in the 3 axis again.
* The slide summaries the main growth factors that are involved in each process.
28
What GFs are expressed in each of the 3 stages of limb bud patterning?
On image
29
What is limb territory specified in?
• Limb territory is specified in the lateral mesoderm
30
What genes are expressed along the cranial to caudal end that marks forelimb and hindlimb territory?
The same goes for dorsal - ventral patterning
• Limb buds form at the intersection of gene expression domains, governed by:
o Rostro-caudal - Hox b8 & c6 (marks the territory for the forelimb – first thoracic), hox c9 (hind)
o Dorso-ventral – Wnt-7a, en-1 (a repressor of gene expression)
• Requires somites and retinoic acid
o RA early for proximal elements (inducing of the Meis genes)
o RA later for AER expansion & activation of Hand2 for ZPA
31
What does fgf10 do in the AER?
• Fgf10 induces Fgf8 in AER (wnt mediated positive feedback). FGF8 then maintains fgf10
32
What does Retinoic acid and Raldh2 do in the developing limb?
What does the expression of RA then activate?
* This is a key signal that initiates limb bud initiation
* Raldh2 is involved with the final steps in the formation of retinoic acid. If we knockout raldh2 in mice the embryos have no forelimbs.
* The retinoic acid expression is required for the expression of other transcription factors downstream of it such as hand2, tbx5 in the forelimb and Meis expression
33
What happens if you cut the limb bud and implant with a bead soaked in RA?
* If you cut the limb of a developing chick embryo and implant a glass or plastic bead at the stump that has been soaked in RA (which serves as a localised source of RA). This causes the cut stump to regenerate the remainder of the limb. The limb that regenerates is along the entire proximal distal axis. You get all the pods forming from the bead.
* This can also be done in the newt. If you cut the tail of the newt, then the cut tail will regenerate. If we apply RA to the cut stump we will regenerate additional limbs from stump. This includes all the pods.
34
Describe how the forelimbs form in the chick
* The limb territory will form at the boundary of Hoxb-8 expression.
* Signals are released from the somites, primarily RA that induce the expression of hand2. This expression will drive the expression of fgf10 in the mesenchyme.
* This fgf10 then signals to the overlying ectoderm to induce the expression of fgf8 which starts to cause the differentiation of apical-ectodermal ridge.
* The fgf8 signals back to the underlying mesenchyme to maintain the expression of fgf10. This maintenance of the fgfs requires the formation of the ZPA. Shh is induce by fgf8.
* The maintenance of fgf8 in the overlying ectoderm and fgf10 in the mesenchyme leads to the outgrowth of the limb bud.
35
What is the role of tbx5,4 and pitx1 in limb development?
• Tbx5 in forelimb bud mesenchyme
o Tbx5 KO no forelimb outgrowth or AER, no bone elements, including shoulder
• Tbx4, Pitx1 in hindlimb bud mesenchyme
• Tbx4/5 do NOT establish limb identity
• Tbx4 rescues forelimb outgrowth in Tbx5 KO
• Pitx1 confers some hindlimb characteristics on this rescued forelimb
• Tbx4/5 interchangeable, function to induce early fgf10 expression
• Minguillon et al 2005 Dev Cell 8, 75-84
• Pitx1 required for hindlimb initiation
36
From an evolutionary point of view describe how Tbx5 and 4 has evolved
Tbx4/5 has diverged from a common ancestral form with the acquisition of distinct forelimbs and hindlimbs seen in cartilaginous fish
In a primitive organism they have a single fin and don’t have distinct tbx4/5 genes. They have a gene that represents both.
Shh in posterior region of limb
37
Describe the expression of hox C6 and C8 in the required for forelimb formation
* If we look at hox genes within the flank, hoxb5 is expressed throughout the rostral-caudal axis of the chick
* Hoxc8 and c6 are restricted to just the thoracic region
* The overlap marks the territory for the forelimb
* In the python there is expansion of hoxc6 and c8 through the proximal distal region, so they don’t form a forelimb, but the caudal boundary marks the position where a hindlimb can form.
38
How can a limb bud be initiated to form in the snake?
* Snake hindlimb bud has no AER or AER genes
* Snake limb bud can be elongated by FGF bead, no AER means no fgf expression
* AER is missing from the python limb bud
* Downstream transcription factors don’t respond in python
* But we can get outgrowth of limb bud in the python if we add fgf ectopically.
39
Feedback loops between signalling centres
* Signals from the dorsal ectoderm such as Wnt7a which is necessary for the induction of Shh in the posterior mesenchyme of the limb bud
* Shh is required for the induction of fgf8 in the apical ectodermal ridge (AER) which is in turn needed for fgf10 expression
40
What determines dorsal ventral patterning of the limb in the embryo?
• It is the Ectoderm that determines DV pattern
• Wnt7a signals in dorsal side are responsible for DV patterning (mutants have sole pads on both sides, double ventral, loss of Wnt7a prevents the formation of those dorsal structures)
Al-Qattan Palmar duplication syndrome
• This child has a mutation in Wnt7a, they have a palm on both sides but don’t have nails – a double ventral phenotype
41
What does Wnt7a do in the dorsal mesenchyme?
* Wnt7a induces the transcription factor Lmx1 in dorsal mesenchyme (LMX1 (humans) mutations – nail patella syndrome, loss of dorsal structures, patients lack nails and patella)
* Wnt7a in the ectoderm is required for Shh in Zona polarizing activity, this induction is driven by the action of RA to induce HAND2.
* Ectopic Wnt7a>ectopic Lmx1>double dorsal phenotype
Wnt7a (dorsal ectoderm) -> Lmx1 (dorsal mesenchyme) -> dorsal phenotype
42
How is the DV axis initiated?
Mesoderm signals to the overlying dorsal and ventral ectoderm and the AER
43
How does the AER become restricted to the tip of the limb bud?
* Wnt7a is expressed in dorsal ectoderm
* En1 is expressed in the ventral ectoderm
* The AER epithelium is expressing FGF8 induced by the underlying fgf10 of the limb bud.
* En1 is expressed in the ventral territory, there is an overlap with fgf8
* As the limb bud starts to grow outwards it will undergo a morphological change. The epithelium is changing of the AER. The cell become pseudostratified. So the AER is restricted to the tip where fgf8 is restricted to.
44
How is the boundary between the dorsal ectoderm and ventral ectoderm established?
* Wnt7a in the dorsal ectoderm induces lmx1 in the dorsal mesoderm.
* This dorsal mesoderm signals back to the ectoderm to modify the expression of radical Fridge (modifier of the Notch signalling pathway)
* This set the boundaries and formation of the AER.
* In the ventral mesoderm En1 is induced and will supress Wnt7a and radical Fringe, to confine these to the dorsal territories.
* Dorsal ectoderm expresses Wnt7a and radical fringe
* Ventral ectoderm expresses En-1
* Radical fringe becomes restricted to dorso-ventral boundary, where the AER will form.
* Radical fringe is a GlcNAc transferase that glycosylates Notch receptors and alters its binding specificity for DSLs (notch ligands)
* Fringe inhibits Serrate but potentiates Delta activation of Notch?
* FGF10 from mesoderm required
* FGF8 then secreted by AER region maintains mesoderm as proliferative mesoderm and production of fgf10
* Ectopic radical fringe induces an ectopic AER
Radical fringe is expressed throughout the dorsal ectoderm, early on. Along with Wnt7a
En1 will repress Wnt7a and radical fringe.
45
Have a look at what genes form the axis' in the wing imaginal disk
On image
46
How can we determine that limb structures are forming proximally to distally?
* in the chick wing if we remove the apical ectodermal ridge, via surgery and discarding this tissue
* Embryos can be stained to look at the cartilage and bone that have started to form in the limb
* At 3 days is a partial limb, that just contains the stylopod element, and remnants of the zeugopod
* If we remove the AER at 3.5 days you have the stylopod and zeugopod
* At 4 days we see a complete stylopod, zeugopod and begins of the autopod forming
* This tells us that structures within the limb are actually forming in proximal to distal order.
47
Identify the progress zone
* Structures form in proximal to distal order
* The yellow is a region of cells called the progress zone (cells within the mesenchyme that are rapidly proliferating). The progress zone remains constant as the limb bud grows outwards.
48
What FGFs following expression of FGF 8 and 10 are then expressed by the AER?
What happens if we knock out FGF8?
What happens when we knock out FGF 4,9 and 17?
What happens if we knock out FGF 4 and 8?
* Fgfs such as 4, 9 and 17 expressed at later stages in the AER are later expressed (posterior regions of the AER - this region will form the future digits)
* Fgf8 KO - distal deletions
* Fgf4, 9 or 17 KO alone are normal
* Fgf4/8 KO - no limb
49
What does the progress zone model state?
1. The progress zone model – the yellow territory of mesenchymal cells remain undifferentiated because of signals the eminent from the AER. As the limb bud grows outwards the progress zone is moving further away from the first groups of cells that are laid down. Cells will leave as the bud grows.
* The progress zone is maintained as proliferative by FGF8 from the AER
* As cells leave the progress zone they have a proximo-distal identity
* The first cells to leave are proximal
* Removal of AER results in distal truncation
* Specification of tissue within the proximal – distal fate is because cells are counting the number of divisions they have undergone in the progress zone
50
What does the prespecification model suggest?
* This model says there are already territories laid down in the early limb bud that mark different regions within the limb. Blue for proximal elements (stylapod), white for zeugopod and red for distal formations (autopod).
* During development the territories are expanding after being prespecified.
* When the AER is removed surgically, you see cells death in the progress zone. This model suggests that if you remove the AER early enough, programme cell death will kill the white and red territories before they have expanded. If the AER is removed later then the cell death will only affect the red tissue, because the white tissue has already expanded and therefore lose the autopod.
51
What does the two signal model state?
* RA is produced in the flank and FGFs in the AER.
* RA acts as a proximalising signal and FGFs act as distalising signals.
* They turn on different transcription factors
* Meis 1 specifies the stylapod element
* Hoxa11 specifies the zeugopod
* Hoxa13 specifies the autopod
52
What is the evidence for the two signal model state?
* If we take a late limb bud and graft this tissue which will form the distal elements of the limb bud: the zeugopod and autopod.
* If we take this same graft and transplant it into a host embryo but treat it with RA, you get a limb that has got an additional element: all pods present
* If you take an early limb bud and take the distal tissue from the limb bud and transplant it into an early host, the limb that grows out from that is a complete limb bud. It has all pods present. If we transplant this tissue into a late host there won’t be a proximal element, not stylapod will be present.
* The tissue is capable of forming all the elements, but grafting it into a different environment causes it to change via environmental signals so the proximal elements have informed.
* If we grow this same tissue graft in culture in Wnt and FGF it will induce Hoxa11 and Hoxa13 transcription factors
* If we grow this same tissue graft in culture in Wnt and RA it will induce Meis 1 transcription factor
53
How are the RA and FGF molecules interacting in more detail
* In the early limb bud there are high levels of RA in the flank, this expression drives the expression of the Meis1 and 2 genes throughout the early limb bud.
* High levels of RA inhibit Hox genes in the distal region such as Hoxa13.
* As the limb bud grows there is induction of fgf10 in the mesenchyme and fgf8 in the ectoderm as the bud grows outwards. The two opposing signals of RA and FGF8 are balancing each other out.
* Low levels of RA cannot then inhibit the distal hox genes. The degradative enzyme of RA is also turned on my FGF. So RA levels decrease, which increases FGF levels rise.
* Histone deactelase activity changes that allows the expression of Hoxa13 which marks the autopod 13 which is inhibitory for hoxa11.
54
Hox gene expression defines late limb identities
On image
Development and Disease
flashcards
Decks in class (19)
# Cards
-
The concepts and language of development54
-
Animal Models in Development19
-
Methods in Development35
-
Signalling Pathways42
-
Primary Cilla, signalling and development21
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Genome editing and sophisticated transgenesis59
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Neurulation42
-
Boundary Formation41
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Somitogenesis34
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Organoids and stem cells30
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Eye development39
-
Ear development35
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Neural Crest Part 126
-
Neural crest part 217
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Heart development46
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Heart Development Part 223
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Limb Development54
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Limb Development Part 210
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Cortical Development53
