Limb development Flashcards
LO
- Organogenesis
- Development of the Vertebrate Limb– or why is your leg different to your arm
What are the main players of limb development?
- Secreted signals
- Transcription factors
- Regions
Name some secreted signals in limb development
- FGFs
- SHH
- BMPs
- Wnts
Name some transcription factors in limb development
- Hox genes
- Tbx
Name some regions in limb development
- Limb field
- AER
- ZPA
- Dorsal limb ectoderm
What are some tools of limb development?
- Chick- manipulation and staining (protein/mRNA)
- Amphibian- manipulation
- Mouse-staining and genetic manipulation
- Null mutants
- Cre lox –stage specific deletion
- Overexpression
- Man-natural mutations
Tell me about the development of the pentadactyl limb
- What can be more curious than that the hand of a man, formed for grasping, that of a mole for digging, the leg of the horse, the paddle of the porpoise, and the wing of the bat, should all be constructed on the same pattern, and should include similar bones, in the same relative positions?
- 3 planes (stylopod, zeugopod and autopod (this changes the most between our species))
With a limb (in this examples a wing) tell me about the segmental structures
Stylopod: The upper part of the arm
Zeugopod: consists of radius (ra) and ulna (ul) in the forelimb, and tibia (ti) and fibula (fi) in the hindlimb
Autopod: comprises wrist and five fingers in the forelimb, and ankle and five toes in the hindlimb.
Tell me Edward Lewis’ role in limb development
- Homeotic mutations explained mechanisms of segment identity.
- Many homeotic genes are clustered in two chromosomal locations in Drosophila.
Whats the Homeobox?
What does it consist of and encode?
- 180bp conserved sequence in developmental regulator genes.
- encodes the homeodomain, 3 helices a DNA binding helix-turn-helix motif (helices 2-3).
- A homologous protein-coding sequence in Drosophila homeotic genes and its conservation in other metazoans. (Garber et al.,1984 Cell 3)
- 2 and 3 are important as they bind into the DNA
What are Hox genes and how do they act?
- Hox genes are a subgroup which regulate the body plan
- They may act as inhibitors (repressors) or enhancers of transcription and therefore regulate gene expression which is important in body plan regulation
Define the following:
Homeobox gene
Homeotic gene
Hox gene
- Homeobox gene- gene with a homeobox sequences (homeodomain) (not all Homeobox genes are Homeotic)- blue
- Homeotic gene- regulates a regions fate (almost all have homeobox motifs)- green
- Hox genes - are a subgroup group of homeotic genes – have homeoboxes (some homeotic genes are not Hox genes)- important in segmentalisation- red
- Some homeobox genes are homeotic genes and some homeotic genes are hox genes
Give an example of a Homeotic gene and what does it regulate
E.g., PAX genes-which are homeotic and regulate eye development bind DNA via a homeodomain (but are not Hox genes)
Tell me about the conserved hox gene cluster
- 4 sets of hox genes in mice where only 1 in drosophila
- Full set= 13 hox genes found in mouse on chromosome??
- Expression starts at 3’ end (towards head) and 5’ towards tail
- Regulated expression moving towards back end
- Expression is coming on in temporal manner (3’-5’)
What do the Hox expression domains set?
Transitions and the anterio-posterior axis in species
Limb development is about decisions, what are some things that need to be established before a limb can develop in a particular area?
The first decision is where the limb can form along the body axis.
The Limb field is where limb development will initiate
What are somites and what do they form?
somites are bilaterally paired blocks of mesoderm that form along the anterior-posterior axis of the developing embryo in segmented animals. In vertebrates, somite’s give rise to skeletal muscle, cartilage, tendons, endothelial cells, and dermis.
What is the Lateral plate mesoderm?
Its a type of mesoderm that is found at the periphery of the embryo
Limb fields
How do we define where limb development occurs?
limb development occurs at limb fields
The borders of Hox expression in the lateral plate mesoderm (LPM) decides where the limb field is
In chick and mouse Hoxc6, Hoxc8 and maybe Hoxc9 are expressed in the flank between the forelimb and hindlimb fields
These Hox genes repress limb formation in the flank and drive rib formation
How does the presence of Hoxc6/8/9 repress limb formation?
Where do the limbs start to grow now?
They function by preventing expression of the secreted signalling molecules Fibroblast growth factors, FGF10 and FGF8, in the flank
The presence of the Hox genes in the flank prevent the lateral plate mesoderm forming into the limb bud and therefore forming a limb. Therefore, meaning the limb field can be defined in the forelimb and hindlimb
Extended Hoxc6 and Hoxc8 expression domains in the python LPM inhibits fore limb development
As Hoxc6/8/9 are not expressed in the Fore- and hindlimb what does this mean for limb development. Tell me the next process that occurs/ feedback loops
- Hoxc6/8/9 stop the secretion of FGF10 and FGF8
- In the fore/hindlimb (the limbfields) FGF10 is expressed (FGF8 expression is only transient and soon lost from the LPM but acts to start FGF10)
- In The hindlimb, FGF8 expression –> Wnt8c –> FGF10
- In the forelimb, FGF8 –> Wnt2b –> FGF10
- Wnt has a positive feedback loop with FGF10 to stabilise its expression
What are FGF involved with?
involved in angiogenesis, wound healing, and embryonic development.
What are fibroblast growth factors, FGFs?
- In mammals 22 structurally related members of the FGF family FGF1 to 10 acts through one of 4 receptors FGFR1-4
- FGF10 have to interact with proteoglycans to work as go through a change in conformation allowing them to bind to their receptor
After FGF10 expression, what decides where limb buds form?
- FGF10 causes LPM (below) and somite cells (above) to rapidly divide and migrate laterally to form a bulge
What can induce the formation of an extra limb during development?
- Ectopic FGF10 (beads soaked in Wnt or FGF) in other areas of the flank induces the formation of an extra limb (red arrow)
Summary
- FGF10 first expressed throughout the LPM but expression stabilised to areas of Wnt2b(forelimb)/8c (hindlimb) expression (which itself is induced by the hox border)- it is to be noticed that fgf-8 is also first seen in the intermediate mesoderm
- A combinatorial Hox code (Hox4/ Hoxc6 and Hox8c) determines the segmental development of body
- The Hox expression boundary defines the limb field through expression of FGF10 in the LPM in this region which induces differential growth in LPM and somite
Limp position is now defined anterio-posterior but… how does limb morphology develop…?
- How does it define the proximal distal growth?
- How does it form segments? (stylopod, zeugopod, autopod)
- How does it form lateral/medial or dorsal/ ventral?
- How do the hind and forelimb specify?
Basic limb bud structures are conserved amoung vertebrates, tell me the embryonic day that the following appear in the mouse:
Forelimb bud
Hindlimb bud
Forelimb buds appear first (~E9 in mouse) followed by hindlimb buds (~ 0.5 -1day later).
What does each limb bud consist of?
Each limb bud consists of lateral plate mesoderm (LPM- expresses FGF10) and somite mesoderm (grows over LPM) outgrowths covered by ectoderm (grows over the top)
Tell me about Ross Harrisons manipulative experiments in Newts
- Showed that the main axes of the developing limb are determined independently and at different times, the anterior-posterior axis preceding that of the dorsal-ventral axis.
- However, his first experiments looked at Proximal/ Distal outgrowth
- Remove limb field mesoderm
- Transplant limb field ectoderm
- Remove limb field ectoderm
- Transplant limb field mesenchyme (all early stage in development)
- Remove limb bud ectoderm (later stage in development)
- he demonstrated that the main axes of the developing limb are determined independently and at slightly different times, determination of the anterior-posterior axis preceding that of the dorsal-ventral axis.
- Removal of mesenchyme (if sufficient) prevented limb formation- i.e., there are specific areas where they must form
What were Harrison’s manipulations?
- Remove limb field mesenchyme (mesoderm)
No limb formed- so this is the driver (mesoderm/mesenchyme)
- Transplant limb field mesenchyme
New limb formed (left mesenchyme to right side –second right limb formed)
- Splitting the limb field mesenchyme leads to 2 limbs
- Replace limb field ectoderm with other ectoderm (early stage)
Limb formed
- Remove limb field ectoderm (early stage)
Limb formed
- Remove limb bud ectoderm (ie later stage)
NO LIMB FORMED- ectoderm is significant later on
Summary II
- The initial outgrowth from the limb field is driven by mesenchyme signalling and expansion
- However continued limb-bud development is dependent on the overlying ectoderm
How does the limb grow out proximo-distally?
FGF10 from the mesenchyme induces thickening of overlying ectoderm forming the Apical Ectodermal Ridge. (AER) the first organising centre for axis formation
The AER is the major signalling centre for continued outgrowth
How does the Apical ectodermal ridge (AER) help with outgrowth during limb development?
- Maintains the proliferation of underlying mesenchyme the AER forms FGF8 which drives outgrowth of the limb by maintaining a proliferating progress zone (PZ) and inhibits cartilage forming…an FGF-8/10 Feedback loop (+ve feedback loop)
- FGF-8 also helps induce formation of the zone of polarising activity (ZPA) *in the posterior mesenchyme
FGF8 in AER and FGF10 in mesoderm +ve feedback loop. Causes cells to grow more rapidly. These cells are known as the progress zone cells (PZ)
Once AER destroyed the limb will stop growing out. FGF8 part of feedback loop done in experiment where AER was removed but FGF8 was put in its place
FGF8 expression in the AER
Further removing the AER at time points in development limits growth in a proximo-distal manner, what does this suggest?
Name a disorder that can come about from the failure of the middle of the AER late in limb development?
Ectrodactaly
Roles of the growth factors- FGF10 in limb bud
- However, loss of FGF-8 from the AER is not so severe as loss of FGF-10
- suggesting other AER factors are important
- removal of FGF10 removes limb altogether, if FGF8 removed the limbs still grow which suggests that theres something else in the AER that contributes to limb development
- mesoderm defines what limb forms in what place
- FGF10 is crucial
- FGF8 is only part of the story
Where does the specification of the medial/lateral axis of the limb (which way round the thumb and fifth finger or radius and ulner) start?
starts in the early thickening of the limb fields as anterior /posterior changes
In the pre limb bud what is there a graded expression of anterio-posterior of what 2 transcription factors?
dHAND (Hand2) and Gli3
dHAND is found posteriorly
Gli3 is found anteriorly
What does transplanting ZPA induce?
Mirror image formation about the proximo-distal axis
What is the morphogen producing gradient?
TF Gli3 and dHAND are expressed in an asymmetric manner
dHand is a positive effector/ activator (says yes to ZPA formation)
Gli3 is a transcriptional repressor (says no to ZPA formation)
What does Gli3 and dHAND help to form?
The Zone of polarising activity (ZPA)
What is the ZPA and what does it define?
Its an area of Distal limb bud mesoderm and it defines the AP axis a second organising centre for axis formation
During early expression, what is expressed to induce the formation of ZPA?
The ZPA is induced to form through the early expression of Hoxd8, Hoxb8 and dHAND
What does the ZPA form?
signalling factors like SHH (actually starts to form in very early limb bud development)
How does the TF Gli3 affect ZPA formation?
Gli3 is believed to prevent its development too anteriorly
What in the AER maintains the ZPA?
FGF8
What is SHH important for and what would a point mutation in this protein mean?
- SHH (sonic hedgehog)- is important for the A/P axis of the limb dub
Image shows the Hx mutation from G to A in the SHH gene
- Gli3 binding site on SHH promotor (Hx mutation prevents this Gli3 binding)
- Extra digit on medial surface in man
- Gli3 inactivate gives duplication of digits as shown by red * in photo above
- Too much dHand= too much Shh which presents mirror image of lateral side of limb onto the medial side of the limb as shown above. Also zeugopod= 2 ulnars
What does the ZPA produce?
Zone of Polarizing Activity (ZPA) produces Sonic hedgehog (SHH) a secreted morphogen.
How is SHH maintained?
SHH is maintained though a feedback loop between the ZPA and the AER- It induces the AER over this region to produce FGF8 and FGF4 which maintains the expression of SHH in the ZPA
What does SHH induce?
SHH induces the organised expression of Hox genes in the leg (SHH beads recapitulates ZPA replacement experiment)
Tell me what the anterior and posterior ends of the AER drives?
In the anterior end FGF8 drives PZ and FGF10 and at the posterior end, FGF4 and 8 drives ZPA and Shh
Full progress zone outgrowth is driven by what?
Both FGF8 and FGF4 from the AER
What does the loss of both FGF4 and FGF8 from the early E9 AER give?
Severe limb failure
Hox genes define the segments but…How does the limb pattern occur proximal distal in a regulated manner?
Tell me about the three models debated and why may the Pz model be limited
- Three models debated
- Spots- not committed
- Block colour- committed
- Here as the tissue leaves the progress zone it differentiates in a defined sequence
- So, limb develops in an order – proximal regions first distal last
- Hence the regions are not predefined
- different regions perhaps caused by length of time close to the AER and numbers of cell divisions?
- Pz model limited because FGF4/8 ko has a proximal limb development, further marking a single PZ cells with dye shows its progeny end up in a similar region…hence specification model
Evidence for progress zone specification
Evidence against progress zone specification
- KO of both FGF4 and FGF8 (from the late limb bud) from the late E10.5 AER
- theoretically you should have no autopod
- But you actually do get an abnormal hand forming
Tell me about the early specification model
- The different parts of the limb are defined early after limb field formation
- growth merely expands these regions
Tell me the evidence for early specification
What happens during late FGF4 and FGF8 KO (knockout)?
Removal of the AER this causes death of cells up to ~200µm behind the AER-simple size of limb bud as early time points may explain this
3-self regulated turning model- general pattern formation
Which model is the best?
- I don’t know
- Possibly all three
- Turing excludes needs for the Hox genes in limb segmentation – but they are important
What is the AER needed for?
What do the HOX genes a/c/d define?
The AER is needed for outgrowth, Hoxa, Hoxc and Hoxd genes define the proximal- distal segments (they are needed for limb segmentation)
Additive Hox gene expression in the limb, are hox genes only present in one section or at various parts of the gene?
Additive hox gene expression
here is an additive effect as growth continues, however loss of the latest added Hox gene alters tissues being formed at the developmental point it its added
Where are Hox11 group genes required?
Hox11 group genes are required in the forelimb zeugopod (but individually redundant)
Summary III
- Continued outgrowth of the limb is induced by formation of the AER caused by expression of FGF 10 in the underlying mesoderm
- The AER drives growth, caused by FGF8 and FGF 4 induces growth of the progress zone (these are maintained by SHH from the ZPA)
- The mesoderm defines limb form (ie fore and hind and different segments)
- ZPA defines the A/P (which is later the latero medial) axis
- As the limb bud extends sequential Hox gene expression drives segmentation effects
How many forms of the sonic hedgehog protein are there?
There are three forms and SHH is just one of three
What are the hedgegog proteins?
secreted signalling molecules induces a gradient of activity and transcription factor expression
Tell me about the range of effect for the SHH protein
SHH has short range effects - does not diffuse far beyond the ZPA
Whats the current hypothesis for SHH proteins?
- Length of time precursor cells in contact/producing SHH and concentration of SHH defines their fate (cells near ZPA in contact longer and at higher concentrations)
- SHH activates secretion and a gradient of BMP2 /BMP7 from the ZPA
Together these regulate expression of HoxD genes
Tell me about SHH and BMP secretion in the digits
How do their concentrations differ among the digits?
What does SHH induce the expression of?
SHH induced BMP expression- these diffuse over limb as a gradient from the ZPA
What is BMP?
What family is it part of?
What does it bind to and this induce?
- BMP- Bone Morphogenic Proteins
- part of TGFβ cytokine family
- Bind to receptors which induce the serine/threonine kinase receptors
What do BMPs signal via?
signal via SMADs which act as transcription regulators
Tell me about BMP regulation and what are their receptors inhibited/ blocked by?
- They are highly regulated and blocked by binding to extracellular inhibitors (noggin, chordin, gremlin and follistatin)
What sort of things does BMP signalling have a role with?
- mesenchyme to cartilage /bone transformation (BMPs 2-8)
- Roles in the later limb pattern formation
BMP signalling
What are the digits specified by?
Digits are specified by a gradient induced by SHH in cells next to ZPA and diffusing BMPs-2 and -7 (Morphogens) for cells further away
causes a graduated differentiation
What causes the differentiation of the mesenchyme to cartilage?
- Digits are specified by a gradient induced by SHH in cells next to ZPA and diffusing BMPs-2 and -7 (Morphogens) for cells further away
- causes a graduated differentiation
- These regulate the transcription of 5’ (late) HoxD genes (e.g., Hoxd-13) and cause differentiation of the mesenchyme to cartilage
What leads to altered digit specification?
How was this tested?
Removal of specific interdigital areas alters gradient of morphogen and alters digit specification
Placing beads of noggin (BMP inhibitor) in the interdigital area- causes induction of lower digit to one below i.e., place in 3-4 induces 3 to become 2
What does BMP2 and BMP7 do?
BMP2, and BMP7 induce cell death in the interdigital mesenchyme, by apoptosis NOT necrosis
Why does BMPs cause death and differentiation?
- BMPs presence is widespread, but gradients and regulated presence of the BMP inhibitors noggin and gremlin limit BMP activity and spare groups of cells – (cause digit and joint formation)
- Not a necrotic zone by an apoptotic zone (wrong name given by early development biologists)
- When gremlin or noggin inhibit BMP then it means apoptosis doesn’t occur between digits and webbing is retained
- Necrosis= pathological event and apoptosis is a regulated/ driven event so they both cause cell death but are different
Summary IV
- A/P (later Medio –Lateral) specification depends upon ZPA
- Its formation depends upon dHAND and Gli3 gradient expression in mesoderm
- ZPA produces SHH and is in a feedback with the posterior AER Fgf4 and 8 (this is a mutual feedback)- this also gives further limb elongation P/D
- SHH induces BMP expression
- The BMP gradient and SHH concentration / length of expression induce A/P specification (later Medio –Lateral)
- Digit separation (and zeugopod bones) driven by BMP and its inhibitors
The third acis of limb formation is the dorsal-ventral axis. What does this axis control the development of?
This axis is the knuckles, patella and fabellas, nails and pads on separate surfaces,
What is the dorsal-ventral axis induced by?
This is induced by the overlying ectoderm- a third signalling “centre”, rotating only this (not underlying mesoderm) causes transposition of the dorsal ventral axis
What is expressed only in the overlying ectoderm and what does this have a role in?
Wnt7a expressed only in the overlying ectoderm, “dorsalizes” the limb.
Wnt-7a causes Lmx-1, (a Lim gene homeobox transcription factor), to be expressed.
The mice lacking Wnt-7a or Lmx-1 produced ventral tissues on both sides of the paw.
What do human mutations of Lmx-1 lead to?
mutations of Lmx1- leads to a lack of patella, and nails are atrophic (don’t get the proper formation of the upper surface)
Summary V
- D/V (dorsal-ventral) specification depends upon overlying ectoderm
- Ectoderm produces Wnt7a. Its formation depends upon Wnt7a in the ectoderm driving dorsal mesoderm to form the TF and homeotic gene Lmx-1
What do Hox genes specify position on?
Flank
What does the mesenchyme define?
Mesenchyme defines whether leg or arm (proven by replacement experiments). So, what’s different between fore and hind mesenchyme?
What do Tbx genes form?
Tbx genes –form T-box proteins, transcription factors (not homeobox!) control which limb forms, induced by combinatorial Hox expression and early short burst FGF-8 in LPM key
how they are regulated is not yet known but early short burst of FGF-8 in LPM key. (see earlier)
Name 2 T-box genes and where they are expressed/ induced
Tbx5- expressed in the presumptive fore limb (wing) area mesenchyme,
- *Tbx4 -**in the hindlimb
- *both are initially induced by mesenchymal FGF-8.**
Why is your foot different to your hand?
When FGF8 is placed in the 23rd somite position, what does it induce?
both Tbx genes (producing an intermediate limb/wing)
But Tbx4 and 5 and expressed in specific limb fields and induce the same down stream signal. So why are there different outcomes?
A second transcription factor Pitx 1 (has a homeobox!) with Tbx4 gives full hindlimb formation
Final summary
- Position of limb field depends on Hoxc 6/8 expression boundary inducing FGF-8 initially
- These in the LPM mesoderm
(1) causes Tbx 5 and Tbx4 expression, these are specific for fore and hind limbs.
(2) induces FGF10 in the LPM, thought to be maintained by Wnt signals
- Fore/ Hind limb specification
- Tbx default path is a forelimb - Tbx4 with Pitx1 produces the hind limb pattern. They drive the Wnt signalling needed for continued FGF10 and indirectly specific Hox genes
- Axis Organisers- AER (FGF-8/4) - grow out the limb and drives the ZPA (SHH) in a positive feedback SHH induces BMPs and regulates limb Hox gene expression and define mediolateral limb axis and limb shape
- BMPs cause digit specification and also cause death of “non protected” cells
- allowing separation of digits and formation of joints as well as cartilage development
- Axis Organiser- Wnt signals from dorsal ectoderm defines the dorsal ventral surfaces
