Organogenesis

The vertebrate limb develops from a limb bud
The early limb bud has a core of mesenchymal mesoderm and an epithelial ectodermal layer.
Most of limb develop from the mesenchymal core but the muscle cells migrate into the bud from the somites.
The progress zone is at the tip of the bud and is composed of rapidly dividing and proliferating undifferentiated cells.
The apical ectodermal ridge (AER) lays directly above the progress zone.
Cells begin to differentiate after they leave the progress zone and cartilage is formed.
The proximal part of the limb begins to differentiate first and this ten proceeds distally as the limb extends.
Condensation of the cartilage, the packing of cells, occurs in a proximo-distal sequence (i.e. the humerus, the radius &ulna, the carpals and the three digits - 2,3 & 4).
The chick limb bud at 3 days is 1 mm wide by 1 mm long.
By day 10, the limb increases 10 X in size (mostly length).
This is followed by growth, bone replacement of cartilage and then innervation.

Patterning of the limb involves positional information
Limb bud is mostly a regulative developmental field but two organizing centres exist.
1)  apical ectodermal ridge (AER) at the limb bud tip and the
2)  zone of polarizing activity (ZPA) at the posterior mesenchyme.
Model of Pattern Formation based upon positional information approximates vertebrate limb development.
Structures appear to develop based upon their A/P and D/V position (P/D seems to based upon a temporal mechanism).

The apical ectodermal ridge induces the progress zone.
The apical ectodermal ridge (AER) consists of closely packed columnar cells giving the mechanical strength to keep the limb flat such that the length of the ridge directs the width of the bud.
The progress zone, rapidly proliferating mesenchyme just under the apical ridge, is where the limb bud grows from.
This is not a region of increased cell growth but one of continuing cell growth as the flanking cells decrease their rate of cell growth.
The apical ectodermal ridge is localized through the action of the gene radical fringe which is expressed on only one side of the D/V boundary.
The apical ridge is essential for outgrowth and Proximo-distal patterning of the limb and removal of it will result in loss of distal structures depending upon the time of surgery.
The AER signals to the underlying progress zone, as grafts of AER to dorsal surface of earlier limb bud will induce ectopic outgrowth (sometimes even digits).
The major signal is fibroblast growth factors, FGF-8 throughout ridge and FGF-4 in the posterior region.
FGF-4 (applied in a time-releasing bead) can functionally replace the AER to produce a normal limb.
The AER itself is maintained by signals from the underlying progress zone and the ZPA.

The polarizing region specifies position along the A/P axis.
The ZPA is similar to the Spemann Organizer in that a graft from the posterior ZPA to the anterior of another limb bud will induce a mirror image duplication (instead of digits 2-3-4, a catcherís mitt of digits 4-3-2-2-3-4 develops).
AER is lengthened and anterior cell division rate is increased.
The additional digits are composed of cells from the host bud (not the donor analogous to the Spemann experiment).
One theory is that different concentrations of a diffusible morphogen specifies A/P values (digit 4 at high and digit 2 at low).
 Sonic hedgehog (Shh) is normally expressed in limb polarizing region can induce mirror -image duplication of the digits (by transfection and beads) in this way.
Mouse mutant extra-toes gives additional anterior expression of Shh to result in polydactyly.
Shh "knock-out mice" develop proximal but not distal limb structures.
Ectopic FGF-4 induces FGF-8 in the ectoderm which induces Shh in the underlying mesoderm (progress zone) with in turn induces expression of embryoís FGF-4 to maintain an ectopic ridge and eventually an extra limb.

Position along the P/D axis may be specified by a timing mechanism
The Proximo-distal (P/D) axis is less understood but appears to be specified over time as the limb bud grows.
Possibly the counting of cell divisions gives a positional identity.
Removal of AER leaves distally truncated limb.
Killing cells or stopping their proliferation (by X-rays) at an early stage leads to the loss of proximal structures but as the progress zone recovers, distal structures are formed.

The D/V axis is controlled by the ectoderm
In the chick wing, large feathers are only on the dorsal surface and the muscles and tendons have complex D/V pattern.
Examined by transplanting composite limb buds with left ectoderm and right mesoderm or vice versa then grafted to host flank- just D/V of ectoderm inverted.
In general, proximal regions develop as mesoderm dictates but the distal (hand) region is reversed.
Therefore, the ectoderm can specify D/V limb patterning.
In Wnt7a mutants, double ventral limbs.
Since Wnt7a is normally expressed in the dorsal ectoderm, ventral pattern must be the ground state.
When mutant for the engrailed gene (normally in the ventral ectoderm), results in ventral expression of Wnt7a and results in double dorsal limb fates.
When mutant for engrailed (normally in the ventral ectoderm) double ventral fates are assumed.
Wnt7a induces Lmx-1, a LIM homeobox gene, in the underlying ectoderm which induces dorsal fate.
Wnt7a mutant mice are often missing posterior digits which suggests that there is an integration of signaling along the different axes.

Different interpretations of the same positional signals give different limbs
Positional signals are the same in fore and hind limb but are interpreted differently.
Graft of polarizing region of the wing bud to the anterior leg bud induces extra toes.
The "extra digit" signal is received but development follows the leg program.
Fore and hind limbs differ in developmental history along the A/P axis of the body.
Grafting proximal leg (presumptive thigh) cells to the tip of the wing gives rise to toes with claws on the wing tip.
The tissue acquires a more distal positional value but still interprets it according to the leg program.

Homeobox genes are involved in patterning the limbs and specifying their position
23 Hox genes are expressed in the developing chick limb.
Hoxa and Hoxd clusters are expressed in both fore and hind limb buds.
Hoxd9-13 are expressed sequently nested pattern such that Hoxd9 is expressed throughout but all Hoxd9-13 are expressed in the small posterior region.
Hoxd seem to control A/P (i.e. finger) identity.
Hoxa9-13 are expressed in a nested proximo-distal pattern.
Ie Hoxa9 only in the presumptive upper limb, Hoxa9-11 in the lower limb region and Hoxa9-13 region give rise to wrist & digits.
Analysis of knock-outs of Hox paralogous groups (to account for redundancy) clearly show regions of Hox influence.

Self-organization may be involved in pattern formation in the limb bud.
Limb bud mesenchyme that has been mixed, reaggregated and placed in ectodermal jackets develop as limbs without a polarizing region.
Proximal regions are abnormal but distal elements are (digits) are formed.
A reaction-diffusion mechanism, where a proximal single peak in a morphogen could pattern the humerus but more distally, several peaks could generate the digits.
Reaction-diffusion mechanisms are responsible for pigmentation patterns in both mammals and fish.

Limb muscle is patterned by the connective tissue
The limb muscles migrate from the somites, form a dorsal and a ventral block of presumptive muscle tissue which eventually give the final muscle mass.
Grafts of neck somites to the limb region which contribute limb muscle demonstrate that the connective tissue laid down by the progress zone.
The initial development of cartilage, muscles and tendons is autonomous but the connection between them appear to be non-specific.
Separation of the digits is the result of programmed cell death.

Drosophila appendages develop from imaginal discs.

Signals from the A/P compartment boundary pattern wing imaginal discs.
In wing discs, cells at the A/P boundary form a signaling region.
engrailed is expressed in the posterior compartment and reflects expression of its embryonic parasegment.
engrailed expressing cells also express the segment polarity gene hedgehog (hh) which encodes a secreted protein.
hh causes adjacent anterior compartment cells to express decapentaplegic (dpp) by inhibiting proteins that repress dpp.
dpp (a TGF-beta family member) is then secreted at the A/P boundary and probably directs patterning of both the anterior and posterior compartments along the A/P axis.
In one instance, expression of spalt (a wider band superimposed over dpp) depends upon the presence of a "threshold level" of dpp.
Misexpression of hh in the posterior has little effect but in the anterior compartment an ectopic mirror-image duplication results.
Normally, wing veins can be numbered 1-5 (A to P) such that the compartment boundary is between veins 3 & 4.
Ectopic anterior expression of hh could give the pattern of 12332112345.
hh sets up new dpp expression which seems to direct this pattern.

The D/V boundary of the wing acts as a pattern-organizing centre.
D & V compartments form after the A/P.
The homeotic selector gene apterous (dorsal compartment only) activates expression of fringe and Serrate.
Dorsal cells expressing Serrate interact with ventral cells via Notch receptor protein to form the wing margin.
Ectopic fringe (the ventral compartment) results in extra wing margins.
Clones of apterous minus cells in the dorsal compartment will adopt a ventral fate leading to ectopic margin formation.
Like the A/P boundary, the D/V boundary acts as an organizing centre and expresses the secreted Wnt protein, wingless (wg) which acts in a manner similar to dpp but along the D/V axis.
The wing veins and inter-vein areas are specified by a pattern of signals.

The leg disc is patterned the same as the wing disc, except for the P/D axis.
An imaginal leg disc is a flat cone of concentric rings of epithelial cells.
At metamorphosis, the disc extends via cell shape changes such that the outer ring becomes the base and the centre becomes the distal tip.
First step is similar to the wing: engrailed is expressed in the posterior which induces hh and dpp is induced in the dorsal region of the leg disc.
However, hh induces wg in the ventral region such that wg and dpp mutually inhibit each other and (where they meet) induce Distal-less (Dll) expression at the tip.
Dll controls P/D axis & aristaless (same expression), specifies distal structures.
Surprizingly, the wing patterns of butterfles utilize these genes to generate the circular rings that lead to spot formation.

The segmental identity of imaginal discs is determined by the homeotic selector genes.
The wing and leg discs interpret their positional differently due to the Hox genes.
Legs and antennae are similar structures, but when Antennapedia is expressed in the head, the antenna become legs.
As in the vertebrate limb, positional information is similar in appendages (i.e. wings and legs) but the interpretation differs.
Segment-specificity (interpretation) is determined by the Hox genes (legs only on thorax, not abdomen; wings only on 2nd thoracic segment) as demonstrated in the postbithorax mutant.
Genes required for leg and wing discs are suppressed in the abdomen.
Hox genes specify leg identity
(Antp->2nd pair of legs; Ubx->3rd pair).
Leg imaginal discs arise from ~25 ectodermal cells in parasegments 3-6 during blastoderm growth at the parasegmental boundary.
Posterior and anterior compartments of adjacent parasegments contribute to each disc.
In the future 2nd & 3rd thoracic segments, the leg discs split off a second disc (wing in the 2nd and haltere in the third).
The discs increase during larval growth (the wing discs ~1000 fold).

The Insect Compound eye

Signals maintain progress of the morphogenetic furrow and the ommatidia are spaced by lateral inhibition.
Mutations in genes that block the movement of the morphgenetic furrow across the eye disc block ommatidia differentiation and produce flies with small eyes.
Although there is n A/P axis in the eye, cells behind the passing furrow resemble posterior cell because they secrete hh to trigger ddp in the furrow which initiates R8 differentiation.
As the furrow moves forward, dpp is turned off and hh is expressed which drives dpp expression ahead.
wg expression prevents furrow formation in the lateral parts of the eye.
Spacing of R8 is based on lateral inhibition.
The cohort of cell begin to differentiate as R8 cells but one cell inhibits its neighbours to become the only R8 cell probably by secretion of a protein, perhaps scabrous (a fibrinogen).
Notch may also play a role in lateral inhibition.
The patterning of the cells in the ommatidium depends on intercellular interactions through a series of inductive steps involving the Drosophila epidermal growth factor receptor (DER).

The development of the R7 depends on a signal from R8
sevenless and bride of sevenless (boss) mutants do not develop the R7 cell.
sevenless encodes a transmembrane receptor tyrosine kinase is expressed in a number of cells.
boss (encodes an integral membrane protein) present on the apical surface of the R8 cell where it makes contact with the R7 cell.
Once boss and sevenless protein bind together, boss is brought into the R7 cell and a signal transduction pathway causes gene expression to change.
eyeless (Pax6 homologue) is the eye selector gene and activation it can initiate ~2000 genes & ectopic eye development.