Study Questions 8 Flashcards
- Define proximal-distal, anterior-posterior and dorsal-ventral limb axis.
● Proximal distal axis – 3 segments = stylopod (proximal), zeugopod and autopod (distal) that develop in sequence proximal to distal as cartilage first and then bone in the same order
● Anterior-Posterior axis – small fingers (pinky) are posterior (pp), thumbs are anterior
● Dorsal-ventral: palm is ventral, knuckles and nails are dorsal
Is the following statement true or false: Wnt proteins stimulate the degradation of Fgf10 in the lateral plate mesoderm, thereby initiating limb bud formation. Explain your reasoning.
False
● wnt – restrict fgf expression pattern to site of limb bud formation in lateral plate esoderm – act as inhibitor
○ result stabilizes fgf expression in hind and forelimb not degrades
● Fgf – secreted by mesenchyme cells of lateral plate mesoderm – inductive signal for limb formation
○ Evenly Expressed throughout lateral plate mesoderm until wnt expression
○ Induce AER in competent ectoderm between dorsal, ventral sides
○ Fgf8 from AER maintain mitosis and expression of Fgf10
- Describe the roles of Fgf10, Fgf8, Wnt2b, Wnt8c and Wnt3a in the initiation of the chicken limb bud formation. How the above molecules interact with each other to signal the formation of the limb bud?
Fgf10
· paracrine factor that act as inductive signal for limb formation
· Expressed evenly throughout lateral plate mesoderm
· induce formation of AER from ectoderm
· help with maintenance and function of AER · Regulate Fgf8 and Wnt3a
· Positive loop with Fgf8 (stimulate production of Fgf8)
• FGF10 from the mesenchyme induces AER formation and FGF8 secretion by the AER. FGF8 is a reciprocal signal back to mesenchyme that maintains mitosis and FGF10 expression. Positive feed back loop.
Fgf8
· express by AER
· maintain AER mitosis and Fgf10 expression · Positive loop with Fgf10 upregulate wnt3
Wnt2b assist forelimb bud formation Localize Fgf10 in forelimb area
Wnt8c assist hindlimb bud formation Localize Fgf10 in hindlimb area
Wnt3a assist in Fgf loop by inducing formation of fgf8 Influenced by Fgf10
Describe the roles of FGFs, Tbx5 and Tbx4 during limb bud formation in the chick. Outline the experiments that demonstrated the roles of the above proteins.
Fgfs induce formation of AER and help maintain mitosis of cells within it ● use of Fgf bead that was put into area b/w Tbx genes induced formation of chimeric limb
● half of limb became hind, half became fore depending on location (affected by Tbx) =show boundary b/w tbx5 & tbx4
Tbx5 Control forelimb specification
and Tbx4
Control hindlimb specification
● use of in situ hybridization – label complementary RNA strand that binds to target RNA (Tbx) which allows coloured location of protein expression
● Tbx5 was in forelimb bud area
● tbx4 was in hindlimb bud area
- What is AER (give the full name). What is PZ (give the full name)? How is the interaction between AER and PZ governing the limb growth?
AER = apical ectodermal ridge
● Major signalling centre for all 3 axis
● Controls mesenchyme cells of progress zone (PZ)
● Roles
○ Maintain PZ in state of proliferation to allow linear proximal-distal growth of limb
○ Maintain expression of molecules that generate AP axis
○ Interact w/ proteins that specify AP and DV axis to provide cells w/ differentiation info
● Control when cartilage forms from PZ cells
PZ = progress zone
● Area underneath AER
● Contains cells going through mitosis (proliferating distal bud mesenchyme)
○ Proliferation state maintain so limb bud grow and not form cartilage until right time
● Fgf8 from AER maintain state proliferation
AER, being signaling center, has three major roles:
1) Maintaining the underlying mesenchyme in a proliferating state so that there is linear proximal-distal growth of the limb
2) Maintaining expression of molecules that generate anterior-posterior axis
3) Interacting with proteins specifying dorsal-ventral axes
⇒each cell is given precise instruction on how to differentiate
- Outline the results of grafting experiments that helped defining the interaction between AER and PZ.
AER removal = limb development stop
- Extra AER graft onto existing limb bud = duplication = mirror image limb
- Leg mesenchyme placed below wing AER = beginning of limb is wing, end of limb is leg
- Limb mesenchyme replace by non-limb mesenchyme below AER = AER regress and limb development stop
- AER replace by Fgf bead = normal limb made b/c AER make Fgfs
a. Mesenchyme under AER determine what limb grow into
b. AER responsible for DP growth
Outline the experiments that showed that the specificity of the proximal-distal limb axis is determined by the limb mesoderm and not by the AER.
Experiment 1
● grafting only AER from younger to older embryo does not lead to duplication of structures
● graft only AER from older to younger does not lead to deletion of limb section
● remove AER early only stylopod form but if removed later, stylopod and zeugopod form
Experiment 2
● transplant early wing-bud PZ and AER onto late wing bud (w/ humerus, ulna, radius = zeugopods)
○ result in extra set of early and intermediate structures (another humerus, ulna, radius) forms
Experiment 3
● transplant late AER + PZ onto young embryo = region missing (only digits form)
● what is developed in PD axis is determined by PZ (limb mesoderm)
List and briefly explain four models for the mesodermal specification of the proximal-distal limb axis.
. Progress Zone Model – amount of time mesodermal cell in mitosis in PZ determine how distal its specification is (More time = more mitosis = more distal
o Proof = if AER removed, cells cannot divide and only stylopod formed
2. Early Allocation and Progenitor Expansion Model – early cells are specified and division increases discrete cell population (proximal cells proliferate and expand)
o Proof = if AER removed ,apoptosis destroy distal precursor = only proximal structure form
3. Reaction-Diffusion (Activator-inhibitor) model – sequential patterning in limb bud = 3 areas of cells respond to Paracrine factors as stabilize mechanism
o Apical Zone (PZ) – Fgf signal prevent expression fibronectin so no aggregation of mesenchyme cells, mitosis occur
o Active Zone – no mitosis, fibronectin present = aggregation
o Frozen zone – nodules differentiate into cartilage (high [fibronectin])
4. Two signal gradient model – Mixing of Proximal-distal RA and Distal-proximal AER-FGF gradient give cell position value by inducing genes of proximal/distal limb axis
o Two gradients of diff conc. = diff production of Transcription factors
What is the role of Hox genes in the specification of the proximal-distal limb axis?
● Hox genes controlled by Retinoic acid and Fgf gradients
● Influence where limbs form and specify fate of mesenchymal cell into stylopod, zeugopod, autopod
● E.g. 9-13 determine forelimb, 10-13 determine hindlimb
● Forelimb initiation:- anterior-most point of Hoxc6 expression
● Hox genes (controlled by RA and FGF gradients) not only play a role in where the limbs will form, but also in specifying whether a mesenchymal cell will become part of the stylopod, zeugopod or autopod.
● The 9-13th paralogue groups appear to be active in limbs of mice.
Define the zone of polarizing activity (use diagram).
Zone of polarizing activity = small block of mesodermal tissue that specifies anteriorposterior axis. It can induce formation of mirror wing limb in chicken if grafted.
- What is the role of Shh in anterior-posterior limb axis determination? How is Shh activated in the ZPA (feedback between AER and ZPA)? What is involved in determination/formation of each of the digits?
● Protein involved with posterior parts of limb development, expressed in Zone of Polarizing Activity > greater Shh concentration = more posterior
● Shh activated by Fgf8 and dHand in posterior area of limb
○ AER secrete fgf that make mesenchyme cells with expression of Gli3 and dHAND (before Shh expression) and hoxb-8 to induce Shh expression only in posterior = form ZPA which specifies AP axis
● Shh maintain Fgfs and Fgfs maintain Shh
● Determination/Formation of digits
○ Det. By gradient and time exposure to Shh
○ Digit I = Shh independent
○ Digit II = depend on [Shh] maybe (have Shh responding cells)
○ Digit III = depend on [Shh] and amount of time exposed to Shh
○ Digit IV, V = depend on duration of high level Shh signal
○ Shh release graded positional value across AP axis
What is the role of BMPs in digit formation?
BMP influence digit identity by establishing a gradient which affects apoptosis of interdigital regions by conveying digital and ID fates via:
· Repression of AER
· Cause Apoptosis of ID regions and AER
· Control phalanx-forming region – nail area
Gremlin creates apoptosis through BMPs
Interdigital (ID) region specifies the digit which is anterior to it.
Removal of an ID region before its “scheduled” apoptosis, or change in BMP concentration by adding BMP inhibitors (e.g. Noggin or Gremlin soaked beads) has similar effect.
removal of ID region 2 between digit primordia 2 and 3 will change the digit 2 structure into (anterior) digit 1 structure (B)
Shh and HOX transcription factors create a posterior-to-anterior gradient (purple to peach) that establishes interdigital (ID) signaling centers through a BMP gradient (brown to yellow).
The BMP gradient participates in conveying digital and ID fates through
the region which is “capping” digits - the phalanx-forming region = nails forming region - mesenchyme directly under AER in digit primordium region:
• repression of the AER in the ID regions
• apoptosis of ID regions
• Apoptosis in the ID region happens together with the regression of the associated AER (over stippled region).
• The AERs in the area overlying the digits persist, and digit growth continues
• The nail-forming region regulates digit construction at this point.
Explain why inserting a bead containing Shh into the anterior region of an early limb bud will result in a mirror image duplication of the limb.
● Shh is responsible for posterior development of limbs therefore inserting a shh bead into the anterior region will cause development of posterior parts of a limb which will mirror the development in the posterior region of an early limb bud
● Shh comes from Zone of Polarizing Activity = similar result as grafting ZPA into a recipient
. What is the signaling pathway responsible for dorsal-ventral limb axis formation? Which germ later is initiating signal transduction events leading to dorsal-ventral limb axis formation?
● Notch signalling pathway responsible for dorsal-ventral limb axis formation
● The ectoderm initiates the signal transduction events leading to dorsal ventral limb axis formation? – causes expression of Wnt7a which is expressed in dorsal but not ventral area = needed for dorsal patterning
● Determined by the whole ectoderm enveloping the limb bud.
● Wnt-7a expressed in dorsal ectoderm induces Lmx-1 in the dorsal mesoderm.
● Radical fringe is also expressed in the dorsal ectoderm.
● Engrailed represses radical fringe and Wnt-7a in the ventral ectoderm.
● AER forms at the junction between the dorsal and ventral sides.
● The Notch signaling pathway appears to be important as well in relaying the dorsal ventral signal into the cells that will form the AER.
- What type of morphogene is Lmx1? What happens if there is a mutation in Lmx1 gene in humans (wt / Lmx1 mutant heterozygotes)
● Lmx1 is a homeodomain transcription factor morphogen (and dorsal ventral gene)
● Mutation causes decrease in communication w/ other proteins
○ Heterozygote mutant result in nail patella syndrome (induction of dorsal fate when expressed in ventral cells) = poorly developed nails and dorsal structures
○ Lim protein specify dorsal cell fates
● Lmx1 is a LIM-homeodomain transcription factor; LIM is transcription activation domain - responsible for protein-protein interactions; different mutations in LIM region are possible and they lead to different mutated phenotypes (affects nails, skeletal system, kidneys, eyes…) – Nail patella syndrom (NPS; patella – knee cups)
