Hoxb-8 role in formation of ZPA
Ectopic expression of Hoxb-8 results in the formation of ectopic ZPA tissue along the anterior margin of the limb bud (Charité et al., 1994 ). Hoxb-8 might normally play a role in localizing the ZPA to discrete limb bud cells. It might function by interfering with a Shh repressor, by directly activating Shh transcription, or by giving competence to respond to other positive regulators of Shh transcription. Hoxb-8 is not, however, sufficient to induce Shh expression, as it is only the Hoxb-8-expressing cells at the distal margin that produce Shh, either in the endogenous or ectopic situation. It is very likely that this reflects a requirement for signals from the AER for Shh expression, most probably members of the FGF family that are necessary for maintenance of Shh.
At least one key upstream determinant forthe proper expression domain of Hoxb-8 gene appears to be retinoic acid. When retinoic acid is introduced into the anterior of a limb bud, it produces mirror-image limb duplications by inducing formation of an ectopic ZPA (Noji et al., 1991 ; Wanek et al., 1991 ) and Shh expression (Riddle et al., 1993 ). Hoxb-8 is rapidly induced in the anterior limb cells as a direct response to retinoic acid, in the absence of protein synthesis (Lu et al., 1997 ), thus likely giving them the competence to express Shh in the presence of FGFs from the AER. This likely recapitulates part of the process by which the endogenous ZPA is established since inhibitors of retinoid synthesis (Stratford et al., 1996 ) or activity (Helms et al., 1996 ) applied to the flank prevent the initial induction of Shh and block endogenous expression of Hoxb-8 (Lu et al., 1997 ).
[Randy L. Johnson, Clifford J. Tabin, Molecular Models for Vertebrate Limb Development, Cell, Vol. 90, 979-990, 1997]
"The expression of Hox-1.8, Hox-1.9, Hox-1.10, and a paralog of the Hox-4 cluster (Hox-4.5) were analyzed during stages when the developing limb pattern was being laid down. Each of these Hox genes was expressed in a restricted domain along the proximodistal axis of the limb at stage E 12.5."
[Haack H; Gruss P; Dev Biol 157: 410-22 (1993)]
The five most 5' paralogous groups (Hox 9-13) pattern the posterior region of the vertebrate embryo and the appendicular skeleton. Mice with targeted disruptions in the paralogous genes hoxa-11 and hoxd-11 have been generated by Davis et all  to evaluate the role of the genes in determining the body plan. Breeding these two strains together generated double mutants which have dramatic phenotypes: the radius and the ulna of the forelimb are almost entirely eliminated, the axial skeleton shows homeotic transformations.
In this picture from Mario Capecchi's lab at the University of Utah, you can see the effects of mutating two of the three genes from one paralogous group in the mouse HOX cluster. These pictures show the forelimb bones from mice with mutations in two genes from the Hox-11 group.
The capitol letters 'A' and 'D' represent a normal copy of the HOX A-11 or HOX D-11 genes. The small letters 'a' and 'd' represent a mutated copy of these genes. For each picture, the genotype of the animal at these two genes is shown (i.e. aa; DD indicates a mouse that was mutant at both copies of the HOX A-11 gene and normal at both copies of the HOX D-11 gene).
(a) is a normal limb. The letters h, r, u indicate the three main bones of the forelimb: r=radius, u=ulna, h=humerus.
(b) shows the type of limb observed when both copies of HOX A-11 are mutant, and both copies of HOX D-11 are normal.
(c) shows the type of limb observed when both copies of HOX D-11 are mutant, and both copies of HOX A-11 are normal.
Note that the structure and arrangement of the bones in (b) and (c) are both approximately normal. Therefore, mutation at a single gene in this paralogous group has a relatively mild effect on the animal.
(d) shows the type of limb observed when both copies of both genes are mutated.
Note that two of the forelimb bones, the radius and ulna, are almost completely missing. A striking change in body shape has occurred due to double mutation at the HOX A-11 and D-11 genes.
[Davis AP; Witte DP; Hsieh-Li HM; Potter SS; Capecchi MR; Nature 375: 791-5 (1995)]
hoxb-5 and hoxb-6 are adjacent genes in the mouse HoxB locus. To determine the roles of these genes during development, Rancourt et all  generated mice with a targeted disruption in each gene. hoxb-5- homozygotes have a rostral shift of the shoulder girdle, analogous to what is seen in the human Sprengel anomaly. This suggests a role for hoxb-5 in specifying the position of limbs along the anteroposterior axis of the vertebrate body.
[Rancourt DE; Tsuzuki T; Capecchi MR Genes Dev 9: 108-22 (1995)]
The expression domains of Hox-9 genes in the lateral plate mesoderm correlate with the hindlimb and forelimb fields (Cohn et al., 1997).
Using gene targeting, Fromental-Ramain with co-authors has produced mice with a disruption of Hoxa-9 or Hoxd-9, two paralogous Abdominal B-related genes. During embryogenesis, these genes are expressed in limb buds and along the vertebral axis with anterior expression boundaries at the level of prevertebra #20 for Hoxa-9 and #23 for Hoxd-9. Skeletal analysis revealed homeotic transformations corresponding to anteriorisations of vertebrae. Subtle forelimb (but not hindlimb) defects, corresponding to a reduction of the humerus length and malformation of its deltoid crest, were also observed in Hoxd-9-/-, but not in Hoxa-9-/-, mutant mice. Hoxa-9/Hoxd-9 double mutants exhibit synergistic limb and axial malformations consisting of: (i) an increase of penetrance and expressivity of abnormalities present in the single mutants, and (ii) novel limb alterations at the level of the forelimb stylopod and additional axial skeleton transformations. These observations demonstrate that the two paralogous genes Hoxa-9 and Hoxd-9 have both specific and redundant functions in lumbosacral axial skeleton patterning and in limb morphogenesis at the stylopodal level.
[Catherine Fromental-Ramain, Xavier Warot, Sudhakar Lakkaraju, Bertrand Favier, Herbert Haack, Céline Birling, Andrée Dierich, Pascal Dollé and Pierre Chambon, Development, 122 (2): 461-472, 1996]
Using gene targeting Davis andCapecchi , have created mice with a disruption in the gene hoxd-11. Skeletons of mutant mice show a homeotic transformation that repatterns the sacrum such that each vertebra adopts the structure of the next most anterior vertebra. Defects are also seen in the bones of the limb (regional malformations in the shapes, length and segmentation of bones). These results are discussed in the context of two other recent gene targeting studies involving the paralogous gene hoxa-11 and another member of the Hox D locus, hoxd-13. The position of these limb deformities reflects the temporal and structural colinearity of the Hox genes, such that inactivation of 3' genes has a more proximal phenotypic boundary than that of the more 5' genes.
[Davis AP; Capecchi MR; Development 120: 2187-98 (1994)]
"The 5' genes of the vertebrate Hox clusters are expressed in complex patterns during limb morphogenesis. Various models suggest that the Hoxd genes specify positional identity along the anteroposterior axis of the limb. Close examination of the pattern of Hoxd gene expression in the limb suggests that a distinct combination of Hoxd gene expressed in different digit primordia is unlikely to specify each digit independently."
[Morgan BA; Tabin C; Dev Suppl 36: 181-6 (1994)]
"The apical ectodermal ridge (AER) is a specialized thickening of the distal limb mesenchyme that has been demonstrated to support limb outgrowth and proper limb development. The homeobox gene, Msx-1, is associated with the distal limb mesenchyme (progress zone) and its expression depends upon the presence of the AER in chick limbs."
[Wang Y; Sassoon D; Dev
Biol 168: 374-82 (1995)]
The interactions between the apical ectodermal ridge (AER) and the underlying mesenchyme seem to be mediated, in part, by the products of the msx-1 and msx-2 (Msx1 and Msx2) genes. These homeobox-containing genes are not related to the HOM/Hox gene complex, but are related to another homeotic Drosophila gene, muscle segment homeobox (msh), which is not in the cluster.
The msx-1 gene is initially expressed throughout the early limb bud, but its expression becomes limited to the progress zone mesenchyme directly beneath the AER. In limbless mutants that lack AERs, no msx-1 message is induced in this mesenchyme, and in polydactylous limb buds with an overly broad AER, the mesenchyme transcribes the msx-1 gene over an extended area beneath the AER (Figure). In mutants such as eudiplopodia wherein two AERs form, each giving a limb axis, msx-1 gene expression is seen under both AERs. In experimental manipulations, removal of the AER causes the cessation of msx-1 expression, while the addition of an AER (even a mouse AER to a chick limb bud) causes the expression of msx-1 in the mesenchyme beneath it and directs the formation of a secondary limb axis (Robert et al., 1991; Coelho et al., 1991; 1993). Therefore, it appears that msx-1 is induced in the mesenchyme as a direct response to signals from the AER.
FIGURE. Diagrammatic representation of the msx-1 and msx-2 expression patterns in normal chick wing buds. The msx-1 mRNA is seen primarily in the distal mesenchyme below the AER, but also in the anterior region of the limb bud and in the anterior and posterior necrotic zones where cells will die. The mRNA from msx-2 is expressed in a similar fashion to that for msx-1, except that the main region of expression is in the AER. (After Coelho et al., 1993.)
Since msx-1 probably encodes a transcription factor, this protein may be critical for keeping this population of cells proliferating. When myogenic cells are transfected with actively transcribing msx-1 genes, they lose their differentiated phenotype and divide without the need of further growth factors (Song et al., 1992). When the regeneration blastema forms, the appearance of msx-1 is correlated with the dedifferentiation and proliferation of the mesenchyme (Simon et al., 1995). The ability of the AER to maintain a zone of proliferating cells immediately beneath it is critical to limb development, and msx-1 may be providing this key function in limb development. The retention of msx-1 may also be important for regenration in mammals. Mice, like most mammals, do not regenerate their limbs well, and murine limb regeneration is confined to the fingertips of newborn mice. But fetal mice do regenerate their limbs well. Reginelli and colleagues (1995) have correlated this regeneration to the presence of msx-1 gene expression in the distalmost mesenchyme.
Recently, FGFs have been seen to be critical in maintaining the progress zone mesenchyme proliferating, so the FGFs looked like good candidates for the AER signal to the mesenchyme. Indeed, when the AER is removed, the mesenchymal msx-1 levels decline to undetectable levels. However, if a bead soaked in FGF replaces the AER, the msx-1 expression is restored (Vogel et al., 1995; Wang and Sassoon, 1995).
Although msx-1 is expressed in the progress zone mesenchyme, the msx-2 gene is primarily expressed in the AER and anterior mesenchyme. In chick mutations giving rise to polydactylous limbs, the expression of msx-2 expands throughout the broad AER and leaves the anterior mesenchyme (Coelho et al., 1993). There are other nonchondrogenic regions of limb mesenchyme that express these two genes but are probably not involved in the proximal-distal axis. In humans, a deficiency of the msx-2 gene causes a syndrome characterized by the premature fusion of the skull sutures and a failure of complete limb growth (Jabs et al., 1993).
Coelho, C. N. D., Krabbenhoft, K. M., Upholt, W. B., Fallon, J. F. and Kosher, R. A. 1991. Altered expression of the chick homeobox-containing genes GHox-7 and GHox-8 in the limb buds of limbless mutant chick embryos. Development 113: 1487-1493.
Coelho, C. N. D., Upholt, W. B. and Kosher, R. A. 1993. The expression pattern of the chicken homeobo-containing gene GHox-7 in developing polydactylous limb buds suggests its involvement in apical ectodermal ridge-directed outgrowth of limb mesoderm and in programmed cell death. Differentiation 52: 129-137.
Jabs, E. W., and several others. 1993. A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostostis. Cell 75: 443 - 450.
Reginelli, A. D., Wang, Y.-Q., Sassoon, D. and Muneoka, K. 1995. Digit tip regeneration correlates with regions of Msx1 (Hox7 ) expression in fetal and newborn mice. Development 121: 1065 - 1075.
Robert, B., Lyons, G., Simandl, B. K., Kuroiwa, A. and Buckingham, M. 1991. The apical ectodermal ridge regulates Hox-7 and Hox-8 gene expression in developing chick limb buds. Genes Dev. 5: 2363-2374.
Simon, H. G., Nelson, C., Goff, D., Laufer, E., Morgan, B. A., and Tabin, C. 1995. The differential expression of myogenic regulatory genes and msx-1 during dedifferentiation and redifferentiation of regenerating amphibian limbs. Devel. Dynam. 202: 1 - 12.
Song, K., Wang, Y. and Sassoon, D. 1992. Expression of Hox-7.1 in myoblasts inhibits terminal differentiation and induces cell transformation. Nature 360: 477-481.
Vogel, A., Roberts-Clarke, D., and Niswander, L. 1995. Effect of FGF on gene expression inb chick limb buds in vivo and in vitro. Devel. Biol. 171: 507 - 520.
Wang, Y. Q. and Sassoon, D. 1995. Ectodermal-mesenchyme and mesenchyme-mesenchyme interactions regulate msx-1 expression and cellular differentiation in murine limb bud. Devel. Biol. 168: 374 - 382.
Ranson et all (1995) have identified Gnot1 as a member of a new homeobox gene subfamily. Gnot1 is expressed in a dynamic temporospatial distribution in the developing limb, initially correlating with regions destined to form distal structures and then becoming progressively more restricted to specific regions determined to give rise to wrist and ankle.
[Ranson M; Tickle C; Mahon KA; Mackem S; Mech Dev 51: 17-30 (1995)]
Proper limb growth and patterning requires signals from the zone of polarizing activity and from the apical ectodermal ridge. Sonic hedgehog and Fgf-4, respectively, have recently been identified as candidates for these signals. Laufer et all  have dissected the roles of these secreted proteins in early limb development. The results indicate that Sonic hedgehog initiates expression of secondary signaling molecules, including Bmp-2 in the mesoderm and Fgf-4 in the ectoderm.
[Laufer E; Nelson CE; Johnson RL; Morgan BA; Tabin C Cell
79: 993-1003 (1994)]
Strong's Luxoid (1stD) is a semidominant mouse mutation in which heterozygotes show preaxial hindlimb polydactyly, and homozygotes show fore- and hindlimb polydactyly. The digit patterns of these polydactylous limbs resemble those caused by polarizing grafts, since additional digits with posterior character are present at the anterior side of the limb. Such observations suggest that 1stD limb buds might contain a genetically determined ectopic region of polarizing activity.
[Chan DC; Laufer E; Tabin C; Leder P; Development 121: 1971-8 (1995)]
The positional signaling along the anteroposterior axis of the developing vertebrate limb is provided by the zone of polarizing activity (ZPA) located at the posterior margin. Recently, it was established that the Sonic hedgehog (Shh) mediates ZPA activity. New Recombination induced mutant 4 (Rim4), and two old mutants, Hemimelic extra toes (Hx) and Extra toes (Xt), exhibit mirror-image duplications of the skeletal pattern of the digits. All results indicate the presence of an additional ZPA at the anterior margin of limb buds in these mutants.
[Masuya H et al.; Genes Dev 9: 1645-53 (1995)]