limb development Flashcards
what tools do we have to inform us about limb development
• Chick- manipulation and staining (protein/mRNA)
• Amphibian- manipulation
• Mouse-staining -some physical manipulation- and genetic manipulation
Null mutants, Cre lox –stage specific deletion, Overexpression
• Natural mutations – help inform us
how do we study limb development
- Physically manipulate tissues
- Alter gene expression by KO or transgenic overexpression
- Add back some of the signalling molecules on beads etc
what are the main four secreted signals influencing limb development
- FGFs
- SHH
- BMPs
- Wnts
what are the three common segments of vertebrate limbs
Stylopod - humerus
Zeugopod - Ulna + radius
Autopod - Metacarpals + digits
who is considered the father of developmental genetics
Edward Lewis
how many sets of hox genes do mammals have
4
what are the two major transcription factors involved in limb development
hox genes and Tbx
what is the homeobox
180bp conserved sequence in developmental regulator genes.
- encodes the homeodomain, 3 alpha helices (and a non-structural loop).
- 3rd helix can bind into the major groove of specific sequences of DNA
seen in ALL hox genes and nearly all homeotic genes + some others
what is a homeotic gene
any of a group of genes that control the pattern of body formation during early embryonic development of organisms. - encode transcription factors that regulate a regions fate
(almost all have homeobox domains)
what are hox genes
a subgroup of homeotic genes - have homeoboxes
controlling the body plan along the cranio-caudal axis (aka anterior–posterior), and specify segment identity of tissues within the embryo
give an example of a gene that is homeotic with a homeodomain but not a hox gene
PAX genes-which are homeotic and regulate eye development bind DNA via a homeodomain but are not Hox genes
Hox genes are conserved in clusters, the polarity 3’-5’ encodes the axis in what positional and temporal colinearity
early and anterior to late and posterior
what can help camouflage hox gene mutations
a degree of overlap that can rescue mutations
what is the first decision made in limb development
where a limb can form along the body axis
in which two regions is the lateral plate mesoderm allowed to grow out
in regions where Hox genes c6 and c8 are NOT expressed
where are Hoxc-6 and Hoxc-8 expressed and what do they do there
expressed in the flank between the forelimb and hindlimb fields - together they repress limb formation in the flank and drive rib formation - by preventing expression of Tbx and preventing secretion of signalling molecules FGF10 and FGF8 in the flank
when does the fgf8 burst of expression occur and what does it induce
around e8 get small and short FGF8 expression in the lateral plate mesoderm causing Wnts to be expressed (this continues once FGF8 has disappeared) drive FGF10 to be expressed (prolonged) -
without what molecule do fibroblast growth factors not work
proteoglycans - help with interaction with (one of four of) their receptors
how do fibroblast growth factors work and what are there general roles
they bind to tyrosine kinase receptors, dimerase cross phosphorylate and turn on a signalling cascade
- Actin rearrangement cell shape changes, motion/migration
- Stop cells dying - antiapoptotic
- Cell growth and division
where is retinoic acid produced in the flank (before aer formation)
in the metanephros
what does fgf10 induce
FGF10 -Induces the lateral plate mesoderm (limb bones) and somite cells (muscle and dermis) to rapidly divide and migrate laterally to form a bulge
where is fgf8 first seen
in the intermediate mesoderm
what stabilises fgf10 expression in the lpm
areas of Wnt2b(forelimb)/Wnt8c (hindlimb) expression (which itself is induced by the hox border)
when do the forelimb and hindlimb buds appear in mice
Forelimb buds appear first (~E9 in mouse) followed by hindlimb buds (~ 0.5 -1day later).
what is each limb bud composed of
Each limb bud consists of lateral plate mesoderm (LPM) and somite mesoderm outgrowths covered by ectoderm.
what were Ross Harrisons manipulations of Newts
– Remove limb field mesenchyme - No limb formed- so this is the driver
– 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 – no difference
– Remove limb field ectoderm (early stage) - Limb formed - heals
- Remove limb bud ectoderm (ie later stage) - NO LIMB FORMED (or vvv reduced limb)
how does the limb grow proximo-distally
FGF10 from mesenchyme induces thickening of overlying ectoderm - the apical ectodermal ridge (AER) - the first organising centre for axis formation
what does the Apical Ectodermal Ridge do
- Maintains the proliferation of underlying mesenchyme the AER forms FGF8 which drives outgrowth of the limb by maintaining a cell dividing progress zone (PZ) and inhibits cartilage forming…………..an FGF-8/10 Feedback loop
(FGF-8 also helps induce formation of the zone of polarising activity(ZPA) *in the posterior mesenchyme )
what is the FGF8/10 feedback loop that occurs in the AER
Underlying mesenchyme produces FGF10 which binds to its receptor FGFR2b on the surface ectoderm which responds to binding by growing larger and producing FGF8.
FGF8 travels bakc into the mesenchyme and binds to its receptor FGFR2c causing the mesenchymal cells to keep dividing and producing more FGF10
- positive feedback loop forming the progress zone where the limb eventually is
how does the FGF8/10 feedback loop result in limb formation and differentiation
Cells driven to continue dividing by continued expression of FGF8 (mesenchyme) and FGF10 (AER) – called progress zone. Where the limb eventually is. The cells further away get less fgf8 and start to differentiate
removing AER ________, but replacing it with _____ restarts it
Removing AER stops growth, replacing it with an FGF8 bead restarts
what does the LPM do
initiates limb bud formation and specifies product
the later you remove the AER, the ___________________
The later you remove the AER the more of the limb is able to develop
what is ectrodactyly
a condition characterized by absence or malformation of one or more of the fingers or toes - caused by premature apoptosis of the AER in late limb development causing defect sin the autopod
what happens if you knock out FGF10 in the limb bud
there is no morphological appearance of an AER so no limbs really forms
what happens when you knock out FGF8 in the limb bud
you get a much smaller limb form
suggesting other AER factors are important
when does the specification of the medial/lateral axis start
in the early thickening of the limb field as the anterior/ posterior changes (in the pre limb)
in the prelimb bud there is graduated expression anterior-posteriorly of what two transcription factors
the transcription activator dHAND is present posteriorly and transcription repressor Gli3 anteriorly
what is the Zone of Polarising Activity
– an area of Distal Limb Bud mesoderm defines the AP axis (by producing morphogens)
a second organising centre for axis formation
what induces the zone of polarising action to form
The ZPA is induced to form through the early expression of Hoxd8, Hoxb8 and dHAND
(Gli3 prevents its development too anteriorly)
what signalling factor is produced by the ZPA and is important for the A/P axis formation of the limb bud
Shh
transplantation of the zone of polarising action causes what defect
Transplanted the region in to a new animal causes repetition of the area along the symmetrical axis/point
what happens when you get a mutation in Gli3 that inactivates it
Gli3 cant bind to the inhibitor stopping production of Shh so you get a wider feild of expression and another 5th digit
what happens when you get a mutation causing overexpression of dhand
wider expression of Shh and repetition of digits in a mirror image
how is Shh expression maintained
a feedback loop between the ZPA and the AER-
SHH induces the AER over this region to produce FGF8 and FGF4 which maintains SHH expression in the ZPA
beads of ____ can recapitulate the ZPA
Shh
what is the full outgrowth of the progress zone driven by
FGF8 and FGF4 from the AER
what three genes define the proximal-distal segments (styolopod, zeugopod ot autopod)
Hoxa, Hoxc and Hoxd genes
which hox genes are expressed in limb buds and result in definition of each part of the limb
Hox 9-13 are expressed in limb buds
– positional and temporally controlled manner
which set of the hox genes isnt expressed in the forelimb
hoxc
if you only knockout out one set of hox genes what happens
limb still forms but you get deformity
what sets of hox genes are expressed in the forelimb
only a and d
what sets of hox genes are expressed in the hindlimb
a, c and d
hox11 codes for which part of the limb
the zeugopod
what is the current hypothesis as to how the ZPA - Shh directs limb medial/ lateral polarity
– 1. Length of time precursor cells in contact and concentration of SHH defines their fate (cells near ZPA in contact longer and at higher concentrations)
– 2. SHH activates secretion and a gradient of BMP2 /BMP7 from the ZPA (BMP can diffuse out and have a wider effect)
Together these regulate expression of Hox genes
highest Shh in pinky (lateral)
what are the concentrations of Shh in each of the digits
digit 1 (thumb, medial) - Shh independent
digit 2 - low shh conc
digit 3 - brief shh expression, moderate shh conc
digit 4 - moderate shh expression extended length of time
digit 5 (pinky, most lateral) - extended shh expression
why does shh have short-range effects
because it binds to the matrix and doesn’t diffuse beyond ZPA
what do BMPs do in limb development
1/ Cause mesenchyme to cartilage /bone transformation (BMPs 2-8)
2/ Roles in the later limb pattern formation
how does BMP signalling work
Need two bmps to interact with their receptors, cross phosphorylate and smad intermediate signaling molecule comes, is phosphorylated, bind with co-smad, and is able to enter nucleus
how are the digits 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)Hox genes (eg Hoxd-13) and cause differentiation of the mesenchyme to cartilage
give 3 examples of BMP inhibitors
noggin, chordin and gremlin
what happens when you place beads of BMP inhibitors in the interdigital area
causes induction of lower digit to the one below
- eg inbetween 3 and 4 the digit 3 becomes a 2
what causes digit septation
BMP2, and BMP7 induce cell death in interdigital mesenchyme, by apoptosis
if BMPs are allowed to full signal what happens
will induce cells death - we only want this inbetween digits
what allows formation of digits
BMP inhibitors expressed in digits prevent cell death by BMP ovverexpression
what allows ducks to have webbed feet but chicks not to
in ducks: Gremlin stops bmp being expressed in between digits so no cell death and webs.
Noggin in chick expressed in digits stopping cell death so digits can form
what induces the dorsal/ ventral axis to form
the overlying ectoderm
- wnt7a expression only in overlying ectoderm dorsalises the limb. Wnt 7a causes Lmx-1 expression in the underlying mesoderm (ventralising))
what defines whether a forelimb or hindlimb forms
mesenchymal expression of Tbx genes
Tbx5- expressed in the presumptive fore limb (wing) area mesoderm ,
Tbx4 -in the hindlimb mesoderm
- both initially induced by mesenchymal FGF-8.
what are Tbx genes
form T-box proteins, transcription factors (no homeobox!) control which limb forms, (homeotic genes that don’t contain a homeodomain and aren’t hox genes)
what gives full hindlimb formation
Pitx WITH Tbx4
what happens when you insert a bead of FGF8 close to the forelimb, close to the hindlimb o in the middle
fgf8 close to forelimb region. More induction of tbx 5 get another wing forming
But if inserted closer to the bottom get another leg
If inserted in the middle you get a weg basically a mixture of both (because induction of both tbx genes
what combination hox code determines the segmental development of the body
Hox4/ HOx6c and Hox8c
what defines the limb feild
the hox expression boundary through stable expression of FGF0 (FGF8 initiator wnt maintenance) in the lateral plat mesoderm in this mesoderm induce growth
