Intermediate Range Signalling

when do most structures form in the embryo

by about 8 weeks

what type of signalling is able to work across precursor structures to regulate their formation

paracrine

how many cell diameters does paracrine signalling operate at

1-100 cell diameters

what organism was used in much of the work to understand the role of paracrine signalling in limb development

chick embryos

why are chick embryos used to help understand the role of paracrine signalling in limb development

the chick develops inside an egg so it is accessible to surgical manipulation by removing the shell to access the embryo

when does limb formation in the chick start

around 3 days after fertilisation

what do the mature limbs develop from in chicks

tiny limb buds

what is the proximal-distal axis

base to tip, proximal is close to body and distal is distant from body

what is the anterio-posterior axis in the hand

along the body axis, the digits are numbered 1-5 with 1 being the thumb and 5 being the little finger

how many digits does the wing of a chick have

3 digits

in simple terms, how do cells adopt different fates along each of the three axes

paracrine signalling plays a central role, patterning occurs when the limb is small, there is subsequent growth to reach final size, this is mediated by cells signalling over a 0-1mm range

what is the name of the structure at the tip of the limb bud

the apical ectodermal ridge (AER)

what does the position of the AER suggest

the hypothesis that it is important for limb formation along the proximal-distal axis

how can the hypothesis that the AER is important for limb formation be tested

by manipulating the AER, if limb formation is affected, then this supports the hypothesis, conversely, if manipulation has no effect on limb formation, this falsifies the hypothesis

give an outline of the process of the AER removal experiment

the AER was surgically removed from the wing bud at different stages over the developmental period of 3-4 days, development was allowed to proceed

what were the results and interpretation of the AER surgery results

the later the AER was removed, the more distal limb structures form, therefore, manipulating the AER disrupts wing development along the proximal-distal axis, supporting the hypothesis, the earlier the AER removed, the less wing forms along the proximodistal axis, this supports an additional hypothesis that the AER continues to be important over a developmental period as opposed to just being important early on and then becoming dispensable

what are the hypothesis and prediction for the molecular mechanism of the AER

AER produces secreted signalling molecules which instructs limb formation along the proximo-distal axis, this signalling molecule will be able to replace the function of the AER and instruct limb formation along the proximodistal axis

what is the name of a secreted protein in the AER

FGF4 (fibroblast growth factor 4)

can a source of FGF4 rescue limb truncation caused by AER removal

yes, an FGF4 soaked bead can replace function of surgically removed AER

what does showing that the function of th AER can be replaced by an FGF4 soaked bead demonstrate

that the FGF4 signal alone is sufficient to instruct limb development

what is the sausage dogs short leg phenotype due to

ectopic expression of FGF4 in developing limbs

what is a morphogen

a substance active in pattern formation whose spatial concentration varies and to which cells respond differently at different threshold concentrations

what does the french flag model describe

how morphogen concentration gradients can be translated into a pattern

give an outline of the key ideas behind the french flag model

each cell has the potential to develop as blue, white, or red, position of each cell is defined by the concentrations of morphogen, positional value is interpreted by the cells which differentiate to form a pattern

for the french flag method to work on the anterior-posterior axis of the hand, what would there need to be

a source of morphogen on one edge of the developing limb anterior-posterior axis, a resulting anterior-posterior concentration gradient, a specific range of morphogen concentration would specify each digit along the anterior-posterior axis

what is the zone of polarising activity (ZPA) in a chick limb

a region at the posterior edge of the limb bud

what is a plausible hypothesis involving the ZPA

the ZPA secretes morphogen molecules that specify digits along the anterior-posterior axis according to the french flag model

give an overview of the ZPA grafting experiment

the ZPA from a donor embryo is grafted onto the anterior edge of the limb bud of a host embryo, the result is a mirror image wing, these results make sense if the donor ZPA creates an anterior to posterior morphogen concentration gradient mirroring that created by the host ZPA

what protein is a strong candidate for the ZPA signal

sonic hedgehog

what does carrying out the grafting experiment using sonic hedgehog in place of a ZPA graft cause

the development of a mirror image wing, sonic hedgehog is therefore sufficient to provide the ZPA morphogen activity

what is an organiser

a signalling centre that directs the development of the whole embryo or part of the embryo

why is the ZPA an example of an organiser

because it signals to the developing limb and directs differentiation between digits along the anterior-posterior axis