Fish Deformed homologs

The vertebrate Hox genes have been shown to confer regional identity along the anteroposterior axis of the developing embryo, especially within the central nervous system (CNS) and the paraxial mesoderm. The notochord has been shown to play vital roles in patterning adjacent tissues along both the dorsoventral and mediolateral axes. However, the notochord's role in imparting anteroposterior information to adjacent structures is less well understood, especially since the notochord shows no morphological distinctions along the anteroposterior axis and is not generally described as a segmental or compartmentalized structure. Four zebrafish hox genes (hoxb1, hoxb5, hoxc6 and hoxc8) are regionally expressed along the anteroposterior extent of the developing notochord. Notochord expression for each gene is transient, but maintains a definite, gene-specific anterior limit throughout its duration. The hox gene expression in the zebrafish notochord is spatially colinear with those genes lying most 3' in the hox clusters having the most anterior limits. The expression patterns of these hox cluster genes in the zebrafish are the most direct molecular evidence for a system of anteroposterior regionalization of the notochord in any vertebrate studied to date (Prince, 1998b).

Amphibian Deformed homologs

As a further investigation of vertebrate head morphogenesis, expression patterns of several homeobox-containing genes were examined using whole-mount in situ hybridization in a sensory system considered to be primitive for the vertebrate subphylum: the axolotl (class: Amphibia, order: Urodela) lateral lines and the placodes from which they develop. The lateral line system develops from the ectodermal placodes. The lateral line placodes develop in a dorsolateral series parallel to the main body axis; it has been hypothesized that the dorsolateral and ventrolateral placode series may be patterned by a mechanism similar to the Hox code described for the head and branchial regions of amniote embryos. Axolotl Msx-2 and Dlx-3 are expressed in all of the lateral line placodes. Both genes are expressed throughout development of the lateral line system and their expression continues in the fully developed neuromasts. Expression within support cells is highly polarized. In contrast to most other observations of Msx genes in vertebrate organogenesis, expression of Msx-2 in developing lateral line organs is exclusively epithelial and is not associated with epithelial-mesenchymal interactions. A Hox-complex gene, Hoxb-3, is shown to be expressed in the embryonic hindbrain and in a lateral line placode at the same rostrocaudal level, but not in other placodes nor in mature lateral line organs. A Hox gene of a separate paralog group, Hoxa-4, is expressed in a more posterior hindbrain domain in the embryo, but is not expressed in the lateral line placode at that rostrocaudal level. These data provide the first test of the hypothesis that the neurogenic placodes develop in two rostrocaudal series aligned with the rhombomeric segments and are patterned by combinations of Hox genes in parallel with the central nervous system (Metscher, 1997).

Avian Deformed homologs

Classic studies have shown that the presomitic mesoderm is already committed to a specific morphological fate, for example, the ability to generate a rib. Hox gene expression in the paraxial mesoderm has also been shown to be fixed early and is not susceptible to modulation by an ectopic environment. This is in contrast to the plasticity of Hox expression in neuroectodermal derivatives. The potential of somites for morphological plasticity was reexamined by transplanting the cranial (occipital) somites 1-4, that normally produce small contributions to the skull, to the trunk of avian embryos. Surprisingly, the transposed cranial somites are able to form reasonably normal vertebral anlage. In addition, the cranial somitic mesoderm produces intervertebral disks, structures not normally found in the skull. These somites are however unable to generate some elements of the vertebrae, such as the costal process. In contrast to the morphogenetic plasticity of the occipital somites, their characteristic inability to support survival of dorsal root ganglia is not significantly modified by posterior transplantation. Dorsal root ganglia initially develop and then degenerate with the same morphological stages as normally observed. In striking contrast to the plasticity of morphology, it was found that all four members of the fourth paralogous group of Hox genes (Deformed homologs), expressed endogenously at the level of the graft, are not upregulated in the caudad-transposed cranial mesoderm. It therefore appears that genes other than those of the Hox family normally expressed at this axial level control the position-specific morphogenesis of ectopic vertebrae formed from cranial somites. In evolutionary terms, the present results imply that occipital somites that were incorporated into the 'New Head' retain the ability to develop into vertebrae, according to their original morphogenetic fate (Kant, 1999).

According to the New Head hypothesis (Gans and Northcutt, 1983), the expansion of neural derivatives at the anterior end of the embryo caused the most rostral somites to be incorporated into the primarily neural-crest derived skull. Experiments with gain-of-function mice overexpressing caudal Hox genes and mice treated with retinoic acid have supported this contention. In both types of experiments, posterior transformations of portions of occipital (skull) bones have produced vertebra-like structures (a 'pro-atlas'). The results presented here complement these observations, by showing that cranial somites are able to give rise to most elements of vertebrae when exposed to the environment of the trunk. Some aspect of this environment is apparently able to reprogram the morphogenetic plan of the sclerotome to 'remember' its vertebral origins, even without induction of the set of Hox genes appropriate to the new position of the somite (Kant, 1999 and references therein).

Expression and mutation of Deformed homologs

Hoxa-4 (previously known as Hox-1.4) is a mouse homeobox-containing gene homologous to Deformed. Hoxa-4 is expressed in the presumptive hindbrain and spinal cord, prevertebrae, and other tissues during embryogenesis. Hoxa-4 mutant mice are viable and fertile. Analysis of neonatal skeletons revealed the development of ribs on the seventh cervical vertebra at variable penetrance and expressivity. A low frequency of alterations in sternal morphogenesis is also observed. The skeletons of transgenic mice that overexpress Hoxa-4 were analyzed. The formation of the small rib anlagen that often develop on the seventh cervical vertebra is suppressed. Analysis of adult homozygous mutant skeletons revealed that the dorsal process normally associated with the second cervical vertebra was also found on the third cervical vertebra. These results demonstrate that Hoxa-4 plays a role in conferring positional information along the anteroposterior axis to specify the identity of the third and the seventh cervical vertebrae (Horan, 1994).

Mice with a disruption in the hoxb-2 locus (homolog: Drosophila proboscipedia) were generated by gene targeting. 75% of the hoxb-2 mutant homozygotes died within 24 hours of birth. While a majority of these mice have severe sternal defects that compromise their ability to breathe, some have relatively normal sternum morphology, suggesting that one or more additional factor(s) contribute to neonatal lethality. At 3-3.5 weeks of age, half of the remaining hoxb-2 homozygotes become weak and subsequently die. All of the mutants that survive to 3 weeks of age show marked facial paralysis similar to, but more severe than, that reported for hoxb-1 mutant homozygotes (homolog: Drosophila labial). As for the hoxb-1 mutations, the facial paralysis observed in mice homozygous for the hoxb-2 mutation results from a failure to form the somatic motor component of the VIIth (facial) nerve which controls the muscles of facial expression. Features of this phenotype closely resemble the clinical signs associated with Bell's Palsy and Moebius Syndrome in humans. The sternal defects seen in hoxb-2 mutant mice are similar to those previously reported for hoxb-4 mutant mice (homolog: Drosophila Deformed). The above results suggest that the hoxb-2 mutant phenotype may result in part from effects of the hoxb-2 mutation on the expression of both hoxb-1 and hoxb-4. Consistent with this proposal, it was found that the hoxb-2 mutation disrupts the expression of hoxb-1 in cis. In addition, the hoxb-2 mutation changes the expression of hoxb-4 and the hoxb-4 mutation, in turn, alters the pattern of hoxb-2 expression. hoxb-2 and hoxb-4 appear to function together to mediate proper closure of the ventral thoracic body wall. Failure in this closure results in severe defects of the sternum (Barrow, 1996).

Megacolon occurs in neonatal and adult transgenic mice that overexpress the Hoxa-4 gene. Abnormalities, which are restricted to the terminal colon of these mice, include a hypoganglionosis, abnormal enteric ganglia [with a structure appropriate for extra-enteric peripheral nerve and not appropriate for the enteric nervous system (ENS)], and gaps in the longitudinal muscle occupied by ganglia. To investigate the developmental origin of these abnormalities, the development of the pelvis and terminal colon were examined in Hoxa-4 transgenic mice. Morphological abnormalities are detected as early as E13. These included an enlargement of the mucosa and the bowel wall, a thickening of the enteric mesenchyme, and the ectopic location of pelvic ganglion cells, which initially cluster on the dorsolateral wall of the hindgut. As the bowel enlarges, these ectopic cells become ventrolateral: between days E17 and E18.5, they appear to become incorporated into the gut, leaving neuron-filled gaps in the longitudinal muscle layer. The ectopic ganglia retain extra-enteric characteristics, including the presence of capillaries, basal laminae, collagen fibers, and catecholaminergic neurons, even after their incorporation into the bowel. It is proposed that the abnormal and ectopic expression of the Hoxa-4 transgene in the colon causes signaling molecule(s) of the enteric mesenchyme to be overproduced and that the overabundance of these signals leads to mucosal enlargement and misdirection of migrating pelvic neuronal progenitors (Tennyson, 1998).

Three different alleles of the Hoxb4 locus were generated by gene targeting in mice. Two alleles contained insertions of a selectable marker in the first exon in either orientation, and, in the third, the selectable marker was removed, resulting in premature termination of the protein. Presence and orientation of the selectable marker correlate with the severity of the phenotype, indicating that the selectable marker induces cis effects on neighboring genes that influence the phenotype. Homozygous mutants of all alleles had cervical skeletal defects similar to those reported for Hoxb4 mutant mice. In the most severe allele, Hoxb4PolII, homozygous mutants died either in utero at approximately E15.5 or immediately after birth, with a severe defect in ventral body wall formation. Analysis of embryos showed thinning of the primary ventral body wall in mutants relative to control animals at E11.5, before secondary body wall formation. Prior to this defect, both Alx3 and Alx4 were specifically down regulated in the most ventral part of the primary body wall in Hoxb4PolII mutants. Hoxb4loxp mutants in which the neo gene has been removed did not have body wall or sternum defects. In contrast, both the Hoxb4PolII and the previously described Hoxb2PolII alleles that have body wall defects have been shown to disrupt the expression of both Hoxb2 and Hoxb4 in cell types that contribute to body wall formation. These results are consistent with a model in which defects in ventral body wall formation require the simultaneous loss of at least Hoxb2 and Hoxb4, and may involve Alx3 and Alx4 (Manley, 2001).

A number of models attempt to explain the functional relationships of Hox genes. The functional equivalence model states that mammalian Hox-encoded proteins are largely functionally equivalent, and that Hox quantity is more important than Hox quality. In this report, the results of two homeobox swaps are described. In one case, the homeobox of Hoxa 11 was replaced with that of the very closely related Hoxa 10. Developmental function was assayed by analyzing the phenotypes of all possible allele combinations, including the swapped allele, and null alleles for Hoxa 11 and Hoxd 11. This chimeric gene provided wild-type function in the development of the axial skeleton and male reproductive tract, but served as a hypomorph allele in the development of the appendicular skeleton, kidneys, and female reproductive tract. In the other case, the Hoxa 11 homeobox was replaced with that of the divergent Hoxa 4 gene. This chimeric gene provided near recessive null function in all tissues except the axial skeleton, which developed normally. These results demonstrate that even the most conserved regions of Hox genes, the homeoboxes, are not functionally interchangeable in the development of most tissues. In some cases, developmental function tracked with the homeobox, as previously seen in simpler organisms. Homeoboxes with more 5' cluster positions were generally dominant over more 3' homeoboxes, consistent with phenotypic suppression seen in Drosophila. Surprisingly, however, all Hox homeoboxes tested did appear functionally equivalent in the formation of the axial skeleton. The determination of segment identity is one of the most evolutionarily ancient functions of Hox genes. It is interesting that Hox homeoboxes are interchangeable in this process, but are functionally distinct in other aspects of development (Zhao, 2002).

Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation

The relocalisation of some genes to positions outside chromosome territories, and the visible decondensation or unfolding of interphase chromatin, are two striking facets of nuclear reorganisation linked to gene activation that have been assumed to be related to each other. Here, in a study of nuclear reorganisation around the Hoxd cluster, it is suggested that this may not be the case. Despite its very different genomic environment from Hoxb, Hoxd also loops out from its chromosome territory, and unfolds, upon activation in differentiating embryonic stem (ES) cells and in the tailbud of the embryo. However, looping out and decondensation are not simply two different manifestations of the same underlying change in chromatin structure. In the limb bud of the embryonic day 9.5 embryo, where Hoxd is also activated, there is visible decondensation of chromatin but no detectable movement of the region out from the chromosome territory. During ES cell differentiation, decondensed alleles can also be found inside of chromosome territories, and loci that have looped out of the territories can appear to still be condensed. It is concluded that evolutionarily conserved chromosome remodelling mechanisms, predating the duplication of mammalian Hox loci, underlie Hox regulation along the rostrocaudal embryonic axis. However, it is suggested that separate modes of regulation can modify Hoxd chromatin in different ways in different developmental contexts (Morey, 2007).

To dissect the events of nuclear reorganisation, an ES cell differentiation system was used. Gene expression and nuclear reorganisation could be induced at Hoxb by triggering the differentiation of murine ES cells with retinoic acid (RA). To determine whether similar activation occurs at Hoxd RT-PCR was used to analyse the expression of Hoxd genes in undifferentiated OS25 ES cells, and during 18 days after the withdrawal of LIF and the addition of RA. As for Hoxb, there was no detectable expression of Hoxd genes in undifferentiated cells. The extinction of Oct4 expression upon differentiation is accompanied by the rapid induction (by day 2) of Hoxd1 expression, but not of the more 5' genes Hoxd3 through to Hoxd12. Expression of Hoxd3 and Hoxd4 were detected by day 6, Hoxd8 by day 8, Hoxd9 and Hoxd10 by day 10 and Hoxd12 expression was not detected until day 18. Hoxd1 expression declined at later stages of differentiation, but not as rapidly as seen for Hoxb1 (Morey, 2007).

Hoxd is flanked by structurally and functionally unrelated genes. Expression of Mtx2, located 3' of Hoxd1, is induced by day 2, suggesting that this gene might also be subject to temporal colinearity. However, at the 5' end of Hoxd, the early detection of Hoxd13 expression (by day 2) suggests a break in the temporal colinearity at this end of the cluster in this system. A large conserved noncoding region 5' of Hoxd, termed a global control region (GCR), contains digit enhancers that act on Hoxd13, Lnp and Evx2. Neural enhancers in the GCR also act on Evx2 and Lnp. As Evx2 is also activated early in the timecourse of differentiation, this suggests that the GCR may have some activity in ES cells. Lnp expression in ES cells is constitutive. This analysis shows that the colinear activation of the Hoxd cluster is mostly recapitulated upon RA-induced ES cell differentiation (Morey, 2007).

Regulatory elements of Deformed homologs

The regulatory function of the mouse introns for Hoxa-4 and Hoxb-4 was analyzed in Drosophila. Introns of both these genes contain a cluster of three homeodomain binding sites called the HB1 element, which is also found in the introns of other Hox genes ranging from fish to humans as well as in the Ultrabithorax and decapentaplegic genes of Drosophila. The enhancer of the Hoxa-4 intron responds to several homeobox genes by activating transcription in cells of Drosophila embryonic epidermis. The response is strong to Deformed and Ubx. The enhancer activity is similar to previously described autoregulatory elements of Deformed, but additional expression is observed in more posterior segments activated by Ubx and repressed by abdominal-A. Point mutations in the homeodomain binding sites in HB1 abolish the HB1 enhancer activity. A second site suppression experiment shows that UBX interacts directly with the HB1 element. When the HB1 element in the Hoxa-4 intron is replaced by that of the mesodermal enhancer of dpp, which is under contol of Ubx, Ubx-dependent activation is retained, but repression by abd-A is lost. The same result is obtained when the third binding site of HB1 is altered, suggesting that this site is reponsible of abd-A-dependent repression. Deletion of potential cofactor binding sites flanking the HB1 element reveals that they are important for enhancer function in Drosophila and that the Dfd-dependent and the Ubx-dependent expression requires different sites. The cofactor sites are likely to render cell specific expression. The Hox-b4 intron is not functional in Drosophila (Haerry, 1997).

The intron of the mouse Hoxa-4 gene acts as a strong homeotic response element in Drosophila melanogaster leg imaginal discs. This activity depends on homeodomain binding sites present within a 30 bp conserved element, HB1, in the intron. A similar arrangement of homeodomain binding sites is found in many other potential homeotic target genes, for example, mouse Hox b-4, mouse Hox d-4, mouse Hox a-7, Drosophila Ultrabithorax and Drosophila decapentaplegic. HB1 activity in Drosophila imaginal discs is activated by Antennapedia and more posterior homeotic genes, but is not activated by more anterior genes. The eye-antennal disc shows no expression, but strong, specific expression is seen in the leg discs of all three thoracic segments. No expression is seen in the wing or haltere discs. Expression is seen in three pairs of rings in the thoracic segments that correspond to the leg imaginal discs in the embryo. Three homeodomain binding sites within a 30 bp region occur within the mouse Hoxb-4 intron. Neither Even-skipped, Engrailed, Fushi-tarazu, Distal-less nor Bicoid alter HB1 expression. Four specificity-determining residues in the amino terminal arm of the ANTP homeodomain confer the capacity of ANTP to activate transcription from the HB1 element. Testing a reporter gene construct with mutated binding sites in mouse embryos shows that HB1 is also active in the expression domains of posterior Hox genes in the mouse neural tube. Similarly, posterior Hox genes in Drosophila can also activate HB1 (Keegan, 1997).

Transgenic analysis of the human Hoxd4 locus was used to identify one neural and two mesodermal 3' enhancers that are capable of mediating, respectively, the proper anterior limits of expression in the hindbrain and paraxial mesoderm (somites). In addition to directing expression in the central nervous system (CNS) up to the correct rhombomere 6/7 boundary in the hindbrain, the neural enhancer also mediates a three rhombomere anterior shift from this boundary in response to retinoic acid (RA), mimicking the endogenous Hoxd4 response. The transgenic analysis was extended to Hoxa4; mesodermal, neural and retinoid responsive components were identified in the 3' flanking region of that gene that reflect aspects of endogenous Hoxa4 expression. Comparative analysis of the retinoid responses of Hoxd4, Hoxa4 and Hoxb4 reveals that while they can be rapidly induced by RA, there is a window of competence for this response, which is different from that of more 3' Hox genes. Mesodermal regulation involves multiple regions with overlapping or related activity and is complex, but with respect to neural regulation and response to RA, Hoxb4 and Hoxd4 appear to be more closely related to each other than to Hoxa4. The Hoxd4 neural and mesodermal enhancers are located in the 3' flanking region of the gene. Regulatory elements are conserved between mouse and human. The 3' neural enhancer, unlike the 5' retinoic acid response element (RARE), has no typical consensus RARE. Hence it is not clear if the RA response is direct. These results illustrate that much of the general positioning of 5' and 3' flanking regulatory regions has been conserved between three of the group 4 paralogs during vertebrate evolution, which most likely reflects the original positioning of regulatory regions in the ancestral Hox complex (Morrison, 1997).

A lacZ reporter spanning 12.5 kb of murine Hoxd4 genomic DNA contains the major regulatory elements controlling Hoxd4 expression in the mouse embryo. Mutational analysis reveals multiple regulatory regions both 5' and 3' to the coding region. These include a 3' enhancer region required for expression in the central nervous system (CNS) and setting the anterior border in the paraxial mesoderm, and a 5' mesodermal enhancer that directs expression in paraxial and lateral plate mesoderm. A previously defined retinoic acid response element (RARE) is a component of the 5' mesodermal enhancer. These results support a model in which retinoic acid receptors (RARs) and HOX proteins mediate the initiation and maintenance of Hoxd4 expression (Zhang, 1997).

During mouse hindbrain development, Hoxb3 and Hoxb4 share an expression domain caudal to the boundary between rhombomeres 6 and 7. Murine Hoxb3 is a homolog of Drosophila zerknüllt, an Antennapedia cluster gene whose function has diverged in insects; Hoxb4 is a functional homolog of Drosophila Deformed. An enhancer (CR3), shared between both murine genes, specifies the domain of transcriptional overlap in the hindbrain. Both the position of CR3 within the complex and its sequence are conserved from fish to mammals, suggesting it has a common role in regulating the vertebrate HoxB cluster. This study investigates the regulation of transcriptional overlap between Hoxb3 and Hoxb4 in the hindbrain. In Drosophila there are instances of Hox overlaps most often resulting in the down-regulation of anterior genes through negative cross-regulation by loci expressed more posteriorly. For example, posterior to parasegment 4, Ultrabithorax and abdominal-A repress Antennapedia and posterior to parasegement 6, abd-A and Abd-B repress Ubx (Gould, 1997 and references). How does cross-regulation function in vertebrates, and how are adjacent Hox genes regulated from a common promoter?

CR3 mediates transcriptional activation by multiple Hox genes Hoxb4, Hoxd4, and Hoxb5 but not Hoxb1. The overlap between Hoxb3 and Hoxb4 expression in the hindbrain occurs relatively late in the timetable of Hoxb3 expression, on day 9.5 after fertilization in rhombomere 4/5 boundary and only from day 10.5 onward at the rhombdomere 6/7 boundary. CR3 regulation of Hoxb3 and Hoxb4 appears to be involved in the maintenance and not the establishment of the Hoxb4 neural domain. CR3 is subject to autoregulation, that is, it is dependent on endogenous Hoxb4 activity. CR3 is also subject to Hoxd4 regulation specifying the important role for cross-regulatory interactions between these two paralogs. In double Hoxb4/Hoxd4 double mutants, CR3 expression is not abolished completely, rather the rostral limit is shifted posteriorly. It is found that Hoxb5 can induce expression from CR3 in a manner similar to both Hoxb4 and Hoxd4 (Gould, 1997).

Transformant Drosophila carrying murine CR3 linked to a minimal promoter-lacZ construct express lacZ in groups of cell in the posterior maxillary and anterior thoracic (T1-3) segments. Staining is most intense in the maxillary domain and weaker in anterior T1, T2 and T3. The domain is a subset of the normal Deformed expression domain. CR3 was shown to be responsive to Deformed. Ectopic Dfd expression causes expression of CR3 outside the normal expression domain of CR3 directed expression in transgenic CR3 flies. In Deformed mutants, despite loss of the maxillary domain, thoracic expression of CR3 is unaffected. CR3 is responsive to Antennapedia and Sex combs reduced. In the absence of these Hox genes thoracic expression is abolished, whereas the maxillary domain remains unaffected. Therefore, Dfd, Scr, and Antp are all required for activating different aspects of the CR3 expression pattern (Gould, 1997).

A 61-bp region within the enhancer contains two closely spaced and highly conserved TAAT motifs that are the direct targets for Hox gene regulation of CR3. These two sites are capable of mediating all the Hox regulatory inputs to CR3 that were observed. One of the motifs is part of a bipartite HOX/PBC motif, which has been shown to serve as a target site for cooperative binding between multiple Hox and PBX/Extradenticle family members. Removing both maternal and xygotic exd contributions from flies results in a somewhat reduced level of maxillary and thoracic expression of CR3. It is concluded that CR3 activity is not completely dependent on Extradenticle in Drosophila. Mutating the two motifs shows that both are required for both Hoxb3 and Hoxb4 expression in transgenic mice (Gould, 1997).

What conclusions can be drawn from this study? A single regulatory element is shared by two neighboring Hox genes and therefore the enhancer acts bidirectionally. The bidirectional nature of CR3 implies that there are not boundary or insulatory elements restricting the activity of this enhancer to only one Hox promoter. The sharing of regulatory elements is not unique to CR3, as a single silencer element regulates both Hoxd10 and Hoxd11. Therefore, it may be that the sharing of regulatory components is a widespread and important feature of vertebrate Hox complex organization. At present there is no evidence for the sharing of cis-regulatory regions between Hox genes in the Drosophila BX-C and ANT-C; it is more difficult to envisage sharing operating over the larger distances involved in these clusters, as compared with vertebrate clusters that are much more compact. However, there is good evidence for autoregulation in Drosophila. The cross regulation observed for CR3 in vertebrates is very different from the type of cross-regulation seen in Drosophila. In vertebrates the cross-regulation is positive in character and responsible for reinforcing a posterior subset of a Hox gene expression domain. In Drosophila most instances of cross-regulation are negative and directed by loci expressed more posteriorly. Because of the importance of auto- and cross-regulation in Hox gene expression, and because of the importance of enhancer sharing, it is suggested that the clustered organization of Hox genes within a complex is essential for appropriate gene activation rather than maintenance of expression. From an evolutionary standpoint, it is possible that the interdigitation of promoters, and sharing of regulatory regions, might provide an important constraint for maintaining the tight clustering of the vertebrate Hox complexes (Gould, 1997). One is left wondering what aspect of Hox gene regulation results in the conservation of a specific enhancer so that it can direct transcription to the proper domain in both humans and flies?

A retinoic acid response element (RARE) has been identified within a neural enhancer located 3' to the Hoxd4 gene. This RARE is required for the initiation and maintenance of Hoxd4 transgene expression in neurectoderm, and for full anteriorized expression upon retinoic acid (RA) treatment. Mutations within the sequence TTTTCTG, located 2 bp downstream of the RARE, posteriorizes transgene activity. However, the onset of transgene expression and its response to RA are indistinguishable from wild type. While the TTTTCTG motif resembles a CDX binding site, human CDX1 protein does not interact with this element in vitro. Three additional regions have also been shown to control transgene expression in neurectoderm, establishing that multiple elements constitute the Hoxd4 neural enhancer (Zhang, 2000).

Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. A detailed analysis of the mouse Hoxb4 intron enhancer has been performed to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. Since these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site (Gilthorpe, 2002).

Hox genes are key determinants of anteroposterior patterning of animal embryos, and spatially restricted expression of these genes is crucial to this function. Expression of Hoxb4 in the paraxial mesoderm of the mouse embryo is transcriptionally regulated in several distinct phases, and multiple regulatory elements interact to maintain the complete expression domain throughout embryonic development. An enhancer located within the intron of the gene (region C) is sufficient for appropriate temporal activation of expression and the establishment of the correct anterior boundary in the paraxial mesoderm (somite 6/7). However, the Hoxb4 promoter is required to maintain this expression beyond 8.5 dpc. In addition, sequences within the 3' untranslated region (region B) are necessary specifically to maintain expression in somite 7 from 9.0 dpc onwards. Neither the promoter nor region B can direct somitic expression independently, indicating that the interaction of regulatory elements is crucial for the maintenance of the paraxial mesoderm domain of Hoxb4 expression. The domain of Hoxb4 expression is restricted by regulating transcript stability in the paraxial mesoderm and by selective translation and/or degradation of protein in the neural tube. Moreover, the absence of Hoxb4 3'-untranslated sequences from transgene transcripts leads to inappropriate expression of some Hoxb4 transgenes in posterior somites, indicating that there are sequences within region B that are important for both transcriptional and post-transcriptional regulation (Brend, 2003).

The zebrafish hoxd4a locus was compared to its murine ortholog, Hoxd4. The sequence of regulatory elements, including a DR5 type retinoic acid response element (RARE) required for Hoxd4 neural enhancer activity, are highly conserved. Additionally, zebrafish and mouse neural enhancers function identically in transgenic mouse embryos. Whether sequence conservation reflects functional importance was tested by altering the spacing and sequence of the RARE in the Hoxd4 neural enhancer. Stabilizing receptor-DNA interactions does not anteriorize transgene expression. By contrast, conversion of the RARE from a DR5 to a DR2 type element decreases receptor-DNA stability and posteriorizes expression. Hence, the setting of the Hox anterior expression border is not a simple function of the affinity of retinoid receptors for their cognate element (Nolte, 2003).

Role of Deformed homologs in the subdivision of the hindbrain

Continued: Deformed Evolutionary homologs part 3/3 | back topart 1/3

Deformed : Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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