Limb Morphogenesis

Chick Limb Development

Limb buds form on either side of embryonic axis, from mesoderm.
Graft of limb bud mesoderm into ectopic site induces ectopic limb bud formation.
Graft of limb bud ectoderm has no effect.
 
Regulations of patterning and growth of the limb bud

Apical Ectodermal Ridge (AER) regulates proximo-distal patterning:
 
Limb bud mesoderm induces formation of ectodermal ridge at distal end, called the apical ectodermal ridge (AER).
Removal of AER stops further outgrowth of limb.  Distal parts fail to form.
Graft of AER to another limb bud induces secondary outgrowth from point of graft.  Recipient limb bud, with 2 AERs, thus  develops two sets of distal structures.
Maintenance of AER depends on factor from mesoderm.  Mesoderm from wingless mutant + wild type ectoderm with AER causes regression of AER, no limb.
AER maintenance factor appears to be concentrated in posterior zone of mesoderm.

Zone of Polarizing Activity (ZPA) regulates anterior-posterior polarity:

Rotation of entire tip of wing bud & regrafting to stump yields mirror-image duplicatiaon of distal parts.
Posterior of tip remains determined, produces posterior digits.  Anterior of tip, now relocated to posterior, changes fate and also produces posterior digits, with mirror image and opposite handedness.
Diffusible posterior determinant in stump mesoderm realigns A-P polarity in duplicated digits, but original dorso-ventral polarity maintained, thus produces opposite symmetry.

Grafting of tip to after 180 degree rotation to intact wing bud can produce 4 sets of digits.    Original posterior regions of host and graft produce posterior digits; anterior regions of both reorient to produce mirror-image duplications.

Posterior determinant located in posterior mesoderm:  Zone of Polarizing Activity (ZPA).
ZPA grafted onto apex of bud induces secondary digits in normal orientation; e.g., 2-3-4- 3-4.
ZPA grafted onto anteior of bud induces mirror image duplication:  e.g., 4-3-2-3-4.
ZPA grafted onto posterior has no effect.

Dorsal Ectoderm regulates dorso-ventral polarity:

Signals from the ectoderm control D-V positioning of distal limb structures.
Reversal of limb bud ectoderm in grafts onto limb bud mesoderm causes reversal of distal structures.
Removal of dorsal ectoderm, more than ventral ectoderm, decreases ZPA activity.

Signalling molecules

Retinoic acid

Retinoids have been known as teratogens.
Retinoic acid (RA) posteriorizes embryos.
Retinoic acid mimics ZPA activity in limb buds:
Implants of beads coated with RA produces mirror-image duplications like ZPA grafts.
RA is present in limb buds at 25 nM concentration; 20 nM RA applied exogenously is sufficient to produce digit duplications.
Concentration of RA is higher at posterior:  up to 50 nM.

Mammals have a plethora of receptors for retinoic acid:  RARalpha , RARbeta , RARgama, each with several isoforms, and RXR (retinoid X receptor), as well as a cellular retinoic acid binding protein (CRABP).
RARs and RXR all belong to the steroid receptor superfamily:
  receptor is present in the cytoplasm, complexed with chaperonin, hsp90;
  ligand (retinoic acid) diffuces across plasma membrane into cell and binds to the receptor; chaperonin dissociates;
  receptor:ligand complex translocates into nucleus, binds to specific promoter sequences in DNA to activate transcription of target genes.
RA may directly induce expression of hox genes.
But is RA really the morphogen produced by the ZPA, or does it induce formation of ZPA?
Implants of RA beads result in formation of ZPA, not around the bead, but distal to the bead, and only 16-24 hours after implantation.
RA beads induce expression of RAR , but ZPA implants do not.

Sonic Hedgehog (SHH)

Homologue of Drosophila hedgehog (hh) gene, expressed in imaginal discs.
Both HH and SHH are secreted proteins, autoproteolytically cleaved, with short- and long-range signalling capability.
Shh expressed in posterior mesoderm of limb bud, in region coinciding with ZPA, with same timing as ZPA activity.
Shh induced by ectopic RA, coincides with ZPA activity induced by RA.
Ectopic expression of shh in anterior of limb bud (mediated through chick fibroblasts infected with retroviral vector) results in mirror-image digit duplications.
SHH induces FGF-4 expression in AER.
SHH induces BMP2 (member of TGF  superfamily; homolog of Drosophila decapentaplegic).
Ectopic SHH activates Hoxd genes in same manner as ZPA implants.
Shh knockout mice have multiple defects in neural tube patterning, axial patterning, and loss of distal limb structures (Chiang et al. 1996).

Fibroblast Growth Factor (FGF)

 Various members of FGF family (FGF-2, FGF-4, FGF-8) expressed in AER.
 Can replace AER to direct outgrowth and patterning of limb.
 FGF-4, in particular, expressed in posterior of AER.
 FGF-8 expressed uniformly throughout AER.
 FGF-4 expression increases in anterior AER in response to ZPA graft.
 FGF-4 implant in proximal region induces SHH & ZPA activity.
 FGF-4 and PZ cells induce ectopic limb

Transplants of latex beads soaked in FGF-1, FGF-2, or FGF-4 into lateral interlimb mesoderm can induce ectopic limb; FGF-2 induces most complete limbs with digits (Cohn et al. 1995).  However, none of these FGFs are expressed in lateral mesoderm prior to limb bud initiation.
FGF-8 implants can also induce ectopic limb formation, and is expressed in lateral interlimb mesoderm at time of limb bud initiation (Crossley et al., 1996).

Wnt7a

 Member of Wnt family of secreted signalling proteins.
 Expressed in dorsal ectoderm of limb buds.
 Drosophila wingless gene induces expression of hedgehog at D/V boundary
 Wnt7a knockout mice show lack of dorsal structures on limbs (Parr & McMahon, 1995)..

Interactions among signalling molecules and axes

 FGF-8 induces formation of limb bud ectoderm (AER), and expression of FGFs in AER.
 FGF-4, FGF-8 from AER induces Shh in posterior mesoderm (ZPA) & regulates cell proliferation in progress zone.
 SHH from ZPA induces FGF-4 in posterior of AER and polarizes limb mesoderm.
 RA induces both Shh and FGF-4 - affects, D-V, A-P, and P-D axes.
 Wnt7a from dorsal ectoderm is required for Shh expression and maintenance, establishes dorsal compartment.  En establishes ventral compartment.
 Boundary of D/V compartments, proximity to AER establish location of ZPA.
 Cells in progress zone receive all 3 signals to establish pattern in developing limb bud.

Establishment of the AER at D/V boundary

Correct positioning of AER depends on boundary of expression of Radical Fringe (homolog of Drosophila fringe) (Rodriguez-Esteban et al., 1997; Laufer et al., 1997).
Radical Fringe expressed in dorsal ectoderm prior to formation of AER, and repressed in ventral ectoderm by Engrailed-1.
Misexpression of Radical Fringe or Engrailed-1 in ventral ectoderm disrupts AER or causes division of AER or ectopic AER.

Homeotic genes

Hox genes expressed in limb buds along proximal-distal axis, with 3' gene expressed first (most proximally) and 5' genes expressed last (most distally).
Targeted disruption of hoxd-11 and hoxa-11 simultaneously causes missing radius and ulna in mouse forelimbs (Davis et al., 1995).

Induction of ectopic limbs causes reprogramming of Hox (Hox b9, c9, d9) gene expression patterns in lateral plate mesoderm (Cohn et al., 1997).

References

Chiang, C., Y. Litingtung, E. Lee, K.E. Young, J.L. Corden, H. Westphal and P. Beachy, 1996.  Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function, Nature 383:407-413.

Cohn, M.J., K. Patel, R. Krumlauf, D.G. Wilkinson, J.D.W. Clarke and C. Tickle, 1997.  Hox9 genes and vertebrate limb specification, Nature 387:97-101.

Cohn et al., 1995.  Fibroblast growth factors induce additional limb development from the flank of chick embryos, Cell 80:739-746.

Crossley et al., 1996.  Roles for FGF8 in the induction, initiation and maintenance of chick limb development, Cell 84:127-136.

Davis et al., 1995.  Absence of radius and ulna in mice lacking hoxa-11 and hoxd-11, Nature 375:791-795.

Fallon et al., 1994.  FGF-2:  apical ectodermal ridge growth signal for chick limb development, Science 264:104-107.

Feldman et al., 1995.  Requirement of FGF-4 for postimplantation mouse development, Science 267:246-249.

Isaac, A., M.G. Sargent and J. Cooke, 1997.  Control of vertebrate left-right asymmetry by a Snail-related zinc finger gene, Science 275:1301-1304.

Johnson, R.L.  and Tabin, C.J., 1995.  The long and short of hedgehog signalling, Cell 81:313- 316.

Johnson, R.L.  and Tabin, C.J., 1997.  Molecular models for vertebrate limb development, Cell 90:979-990.

Laufer et al., 1994.  Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud, Cell 79:993-1003.

Laufer, E., R. Dahn, O.E. Orozco, C.-Y. Yeo, J. Pisenti, D. Henrique, U.K. Abbott, J.F. Fallon and C. Tabin, 1997.  Expression of Radical Fringe in limb-bud ectoderm regulates apical ectodermal ridge formation, Nature 386:366-373.

Martin, G.R., 1995.  Why thumbs are up, Nature 374:410-411.

Parr and McMahon, 1995.  Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb, Nature 374:350-353.

Riddle et al., 1993.  Sonic hedgehog mediates the polarizing activity of the ZPA, Cell 75:1401- 1416.

Robertson, E.J., 1997.  Left-right asymmetry (Perspectives), Science 275:1280

Rodrigues-Esteban, C., J.W.R. Schwabe, J. DeLa Pena, B. Foys, B. Eshelman and J.C.I. Belmonte, 1997.  Radical fringe positions the apical ectodermal ridge at the dorsoventral boundary of the vertebrate limb, Nature 386:360-366.

Shubin, N., C. Tabin and S. Carroll, 1997.  Fossils, genes and the evolution of animal limbs, Nature 388:639-648.

Yang and Niswander, 1995.  Interacation between the signalling molecules WNT7a and SHH during vertebrate limb development:  dorsal signals regulate anteroposterior patterning, Cell 80:939-947.