HOXD10 Expression Research Articles

The limb field mesoderm determines initial limb bud anteroposterior asymmetry and budding independent of sonic hedgehog or apical ectodermal gene expressions.

We have analyzed the pattern of expression of several genes implicated in limb initiation and outgrowth using limbless chicken embryos. We demonstrate that the expressions of the apical ridge associated genes, Fgf-8, Fgf-4, Bmp-2 and Bmp-4, are undetectable in limbless limb bud ectoderm; however, FGF2 protein is present in the limb bud ectoderm. Shh expression is undetectable in limbless limb bud mesoderm. Nevertheless, limbless limb bud mesoderm shows polarization manifested by the asymmetric expression of Hoxd-11, -12 and -13, Wnt-5a and Bmp-4 genes. The posterior limbless limb bud mesoderm, although not actually expressing Shh, is competent to express it if supplied with exogenous FGF or transplanted to a normal apical ridge environment, providing further evidence of mesodermal asymmetry. Exogenous FGF applied to limbless limb buds permits further growth and determination of recognizable skeletal elements, without the development of an apical ridge. However, the cells competent to express Shh do so at reduced levels; nevertheless, Bmp-2 is then rapidly expressed in the posterior limbless mesoderm. limbless limb buds appear as bi-dorsal structures, as the entire limb bud ectoderm expresses Wnt-7a, a marker for dorsal limb bud ectoderm; the ectoderm fails to express En-1, a marker of ventral ectoderm. As expected, C-Lmx1, which is downstream of Wnt-7a, is expressed in the entire limbless limb bud mesoderm. We conclude that anteroposterior polarity is established in the initial limb bud prior to Shh expression, apical ridge gene expression or dorsal-ventral asymmetry. We propose that the initial pattern of gene expressions in the emergent limb bud is established by axial influences on the limb field. These permit the bud to emerge with asymmetric gene expression before Shh and the apical ridge appear. We report that expression of Fgf-8 by the limb ectoderm is not required for the initiation of the limb bud. The gene expressions in the pre-ridge limb bud mesoderm, as in the limb bud itself, are unstable without stimulation from the apical ridge and the polarizing region (Shh) after budding is initiated. We propose that the defect in limbless limb buds is the lack of a dorsal-ventral interface in the limb bud ectoderm where the apical ridge induction signal would be received and an apical ridge formed. These observations provide evidence for the hypothesis that the dorsal-ventral ectoderm interface is a precondition for apical ridge formation.

Activation of Fgf-4 and HoxD gene expression by BMP-2 expressing cells in the developing chick limb.

Bone morphogenetic protein-2 (BMP-2) has been implicated in the polarizing region signalling pathway, which specifies pattern across the antero-posterior of the developing vertebrate limb. Retinoic acid and Sonic Hedgehog (SHH) can act as polarizing signals; when applied anteriorly in the limb bud, they induce mirror-image digit duplications and ectopic Bmp-2 expression in anterior mesenchyme. In addition, the two signals can activate Fgf-4 expression in anterior ridge and HoxD expression in anterior mesenchyme. We tested the role of BMP-2 in this signalling cascade by ectopically expressing human BMP-2 (hBMP-2) at the anterior margin of the early wing bud using a replication defective retroviral vector, and found that ectopic expression of Fgf-4 was induced in the anterior part of the apical ectodermal ridge, followed later by ectopic expression of Hoxd-11 and Hoxd-13 in anterior mesenchyme. This suggests that BMP-2 is involved in regulating Fgf-4 and HoxD gene expression in the normal limb bud. Ectopically expressed hBMP-2 also induced duplication of digit 2 and bifurcation of digit 3, but could not produce the mirror-image digit duplications obtained with SHH-expressing cells. These results suggest that BMP-2 may be involved primarily in maintenance of the ridge, and in the link between patterning and outgrowth of the limb bud.

Analysis of limb patterning in BMP-7-deficient mice.

Bone morphogenetic proteins (BMPs) are polypeptide signaling molecules, belonging to the TGF-beta superfamily. They were originally identified by their ability to induce ectopic bone formation, but their expression patterns in embryos suggest multiple functions. BMP-7-deficient mice show among other mesodermal and skeletal patterning defects, polydactyly in the hindlimbs [Luo G, Hofmann C, Bronckers ALJJ, Sohocki M, Bradley A, Karsenty G (1995): Genes Dev 9:2808-2820; Dudley AT, Lyons KM, Robertson EJ (1995): Genes Dev 9:2795-2807]. Here we report a more detailed analysis of the limb phenotype in BMP-7-deficient mice using in situ hybridization to monitor expression of molecules implicated in patterning processes of the developing vertebrate limb. In previous studies we showed that Sonic hedgehog (Shh) was expressed normally, but Hoxd-13 expression in limb mesenchyme was lower in BMP-7 mutant limbs. Here we show that Hoxd-11 expression domains are also contracted and decreased in intensity in mutant limbs, suggesting that 5' genes of the Hoxd cluster are coordinately downregulated, while another Bmp, Bmp-2, which can be activated by Shh, is similarly expressed. The mutant limb buds are broader than normal buds, and fibroblast growth factor Fgf-8 is expressed throughout the extended ridge. However, expression of the homeobox gene Msx-1, which has been shown to be involved in epithelial-mesenchymal interactions during limb development, was decreased in the mesenchyme of BMP-7 mutant limbs. Taken together, our data suggest that BMP-7 is involved in regulating proliferation and/or epithelial-mesenchymal interactions in the developing limb.

Effects of all-trans-retinoic acid on skeletal pattern, 5′HoxD gene expression, and RAR β2/β4 promoter activity in embryonic mouse limbs

Mouse embryos were exposed to all-trans-retinoic acid on day 11 or day 12 of development and the resulting skeletal pattern alterations compared with early effects on Hoxd-11 and Hoxd-13 expression domains and RAR-beta 2/beta 4 promoter activity. The effects on skeletal pattern showed a clear correlation between the timing of retinoic acid exposure and the sequence of mesenchymal condensation. Ectopic RAR-beta 2/beta 4 promoter activity was detected within 2 hr of exposure to retinoic acid, and was present throughout the limb bud after 5 hr; it remained high in the apical ectodermal ridge and proximal mesenchyme after 12 hr, by which time the abnormal digital pattern could be seen. HoxD gene expression domains in the distal handplate were narrowed by 5 hr after maternal retinoic acid administration on day 11. Following retinoic acid treatment on both day 11 and day 12, the normal downregulation of Hoxd-11 and Hoxd-13 in the digital mesenchymal condensations was retarded. There was no evidence to suggest that RAR-beta 2/beta 4 promoter activity mediates the effects of RA on HoxD gene expression, but ectopic promoter activity is a useful indicator of at least some of the sites in which RA levels are raised. We suggest (1) that the apical ectodermal ridge is the most functionally significant of these sites, (2) that raised retinoic acid levels in the ridge result in altered gene expression and/or altered cell proliferation within this epithelium, (3) that both altered HoxD gene expression domains and altered skeletal pattern formation are secondary to this effect. There was a good correlation between the effects of retinoic acid on Hoxd-11 and Hoxd-13 expression and delay of skeletal differentiation, suggesting that this may be a direct effect.

Expression of the Homeotic Gene Hox-d13 in the Developing and Adult Mouse Prostate

Purpose To examine expression of the homeotic gene Hox-d13 in the developing mouse prostate. Materials and Methods Expression was assayed by Northern analysis, reverse-transcriptase polymerase chain reaction and in situ hybridization. Results Expression is present in the urogenital sinus in late gestation and is localized postnatally in the urethral epithelium and prostatic ducts. Expression continues throughout development and persists in the adult prostate. Conclusion A restricted domain of Hox-d13 expression in the developing urogenital tract is consistent with the role of the Hox genes in specifying regional identity during development. Expression in the prostate postnatally suggests additional roles in prostate ductal morphogenesis and/or cell differentiation.

Effect of FGF on Gene Expression in Chick Limb Bud Cells in Vivo and in Vitro

Fibroblast growth factors (FGFs) are central to signaling in the developing limb. FGF-2 and FGF-4 can substitute for the apical ectodermal ridge to maintain both limb bud outgrowth and polarizing region signaling. Here, we have repeated and extended previous studies and investigated local effects of the apical ectodermal ridge on gene expression of Msx-1, 5′ members of the HoxD complex, and Bmp-2 in the limb bud mesenchyme and tested whether members of the FGF family can substitute for the ridge to maintain their expression patterns. We found that expression of Msx-1, Hoxd-13, and Bmp-2 in posterior limb bud mesenchyme is dependent on a local signal from the apical ectodermal ridge. When the apical ectodermal ridge of young chick wing buds is removed, or when posterior cells are taken from the bud and placed in culture, expression of Msx-1, Hoxd-13, and Bmp-2 is not detectable in posterior mesenchymal cells. Local application of FGF-soaked beads to posterior limb mesenchyme following ridge removal or addition of FGF to cultured cells maintains expression of Msx-1, Hoxd-13, and Bmp-2. In contrast, expression of Hoxd-11 in posterior mesenchyme appears to be stable in the absence of either the apical ectodermal ridge or FGF. Expression of Msx-1 in anterior and apical cells is also locally maintained by the apical ectodermal ridge and this effect can be reproduced by local application of FGF. Furthermore, the addition of FGF to cultured anterior limb bud cells maintains their ability to respond to positional cues when grafted back into limb buds.

Analysis of theHoxd-3Gene: Structure and Localization of Its Sense and Natural Antisense Transcripts

This study set out to investigate the structure and localized expression of the mouse homeobox-containing gene Hoxd-3. In addition to identifying a transcript of the type known from other Antennapedia (Antp)-like mammalian homeobox cDNAs, an antisense transcript was also detected. The antisense form of Hoxd-3 overlaps with 603 bp of the sense transcript including the homeobox. Active antisense transcription has been confirmed by RNA blot analysis with single-stranded probes and by the direction of splicing of an intron in the antisense transcript. The localized expression of sense and antisense transcripts was compared by in situ hybridization. Hoxd-3 expression was observed from 8.5 days p.c., in the neural tube with a sharp border in the hind brain at the level of rhombomeres 4-5. In contrast, the earliest antisense expression was detected at 10.5 days p.c. in cDNA libraries. At 12.5 days p.c., sense and antisense transcripts colocalized in the liver. The possible role of antisense homeobox transcripts during liver and the hematopoietic development is discussed.

Reciprocal Changes in Hox D13 and RAR-β2 Expression in Response to Retinoic Acid in Chick Limb Buds

The finding that retinoic acid (RA) has major effects on pattern formation in developing chick limbs has led to the conclusion that RA plays a central role in the normal development of the limb. In addition, it has been demonstrated that treatment with exogenous RA leads to changes in expression of homeobox-containing Hox complex genes within the developing limb. This has been used to argue that, since RA activates 5′ Hox D complex genes in the same temporal and spatial sequence as in normal development, Hox genes might be regulated by RA during normal limb development. In this study, we further examine the temporal and spatial changes in expression of Hox D13 and RAR-β2 transcripts in wing buds in response to local application of RA in vivo. We confirm reports that RAR-β2 expression is induced early and locally by RA. However, we find that the effect of RA on Hox D13 gene expression at the distal end of wing buds at stage 25/26 is downregulation of transcript expression. Furthermore, we find that activation of ectopic Hox D13 expression in response to implantation of RA beads along the anterior margin at stage 20/21 is indirect. Finally, the effects of RA exposure on Hox D13 expression appear to directly correlate with effects on the pattern of distal skeletal elements.

Regeneration of HoxD Expression Domains during Pattern Regulation in Chick Wing Buds

The expression domains of genes located at the 5′ end of the HoxD (formerly Hox-4) complex appear to correlate with pattern along both the proximal-distal (PrDi) and the anterior-posterior (AP) axes of the developing limb bud, and it has been suggested that the HoxD gene products are involved in the specification of positional information during limb development. The apical ectodermal ridge is required for limb outgrowth and is thought to influence mesodermal cells at the distal end of the limb bud in a region within which patterning events occur. In this paper, we examine the expression of 5′ HoxD genes during PrDi pattern regulation in chick wing buds. In limbs undergoing pattern regulation, we demonstrate that the domains of HoxD11 and HoxD13 gene expression are "regenerated" within 24 hr of removal of the distal mesenchyme. In contrast, in limbs which will not form distal structures, HoxD13 expression becomes reduced.