Even-skipped homologs in mammals

The mouse Evx-2 gene is located in the immediate vicinity of the Hoxd-13 gene, the most posteriorly expressed gene of the HOXD complex. While the Evx-1 gene is also physically linked to the HOXA complex, it is more distantly located from the corresponding Hoxa-13 gene. The expression of Evx-2 during development has been analyzed and it has been compared to that of Evx-1 and Hoxd-13. Even though Evx-2 is expressed in the developing CNS in a pattern resembling that of other Evx-related genes, the overall expression profile is similar to that of the neighboring limbs and genitalia. It is proposed that the acquisition of expression features typical of Hox genes, together with the disappearance of some expression traits common to Evx genes, is due to the close physical linkage of Evx-2 to the HOXD complex, which results in Evx-2 expression being partly controlled by mechanisms acting in the HOX complex. This transposition of the Evx-2 gene next to the Hoxd-13 gene may have occurred soon after the large scale duplications of the HOX complexes. A scheme is proposed to account for the functional evolution of eve-related genes in the context of their linkage to the HOM/Hox complexes (Dolle, 1994).

Vertebrate gene members of the HoxD complex are essential for proper development of theappendicular skeletons. Inactivation of these genes induces severe alterations in the size and number ofbony elements. Evx-2, a gene related to the Drosophila even-skipped, is located close to Hoxd-13 (Drosophila homolog Abdominal-B) and is expressed in limbs like the neighbouring Hoxd genes. To investigate whether this tight linkage reflects a functional similarity, a null allele of Evx-2 was produced. Furthermore, and because Hoxd-13 function is prevalent over that of nearby Hoxd genes, two different doublemutant loci were generated wherein both Evx-2 and Hoxd-13 were inactivated in cis. The analysis of these variousgenetic configurations reveals the important function of Evx-2 during the development of the autopodas well as its genetic interaction with Hoxd-13. These results show that, in limbs, Evx-2 functions like aHoxd gene. A potential evolutionary scenario is discussed, in which Evx-2 was recruited by the HoxDcomplex in conjunction with the emergence of digits in an ancestral tetrapod (Hérault, 1996).

Evx-1, a murine homolog of even-skipped is first translated shortly before the onset of gastrulation in a region of ectoderm containing cells that will soon be found in the primitive streak. This localized expression of Evx-1 provides the first molecular evidence for regional differences in the mouse embryonic ectoderm before gastrulation. Throughout gastrulation, Evx-1 expression is limited to cells near and within the streak; this expression is graded, with a posterior-to-anterior decrease in the level of RNA (Dush, 1992).

Evx-1 is involved in the developing mouse limb bud. Evx-1 RNA is first detected in distal limb mesenchyme shortly after the formation of the apical ectodermal ridge. The level of Evx-1 RNA increases during the next 24 hours of development, and then decreases in the subsequent 24 hours, such that by the time the ridge regresses Evx-1 RNA is undetectable. At all these stages, Evx-1 RNA is localized primarily to the posterior distal mesenchyme, in the region immediately underlying that portion of the ridge in which the Fgf-4 gene is expressed. The ridge is required for both the induction and maintenance of Evx-1 expression in the distal mesenchyme (Niswander, 1993).

Posterior neuropore (PNP) closure coincides with the end of gastrulation, marking the end of primary neurulation and primary body axis formation. Secondary neurulation and axis formation involve differentiation of the tail bud mesenchyme. Genetic control of the primary-secondary transition is not understood. A detailed analysis of gene expression in the caudal region of day 10 mouse embryos during primary neuropore closure is reported. Embryos were collected at the 27-32 somite stage, fixed, processed for whole mount in situ hybridization, and subsequently sectioned for a more detailed analysis. Genes selected for study include those involved in the key events of gastrulation and neurulation at earlier stages and more cranial levels. Patterns of expression within the tail bud, neural plate, recently closed neural tube, notochord, hindgut, mesoderm, and surface ectoderm are illustrated and described. Specifically, continuity of expression of the genes Wnt5a, Wnt5b, Evx1, Fgf8, RARgamma, Brachyury, and Hoxb1 from primitive streak and node into subpopulations of the tail bud and caudal axial structures is reported. Within the caudal notochord, developing floorplate, and hindgut, HNF3alpha, HNF3beta, Shh, and Brachyury expression domains correlate directly with known genetic roles and predicted tissue interdependence during induction and differentiation of these structures. The patterns of expression of Wnt5a, Hoxb1, Brachyury, RARgamma, and Evx1, together with observations on proliferation, reveal that the caudal mesoderm is organized at a molecular level into distinct domains delineated by longitudinal and transverse borders before histological differentiation. Expression of Wnt5a in the ventral ectodermal ridge supports previous evidence that this structure is involved in epithelial-mesenchymal interaction. These results provide a foundation for understanding the mechanisms facilitating transition from primary to secondary body axis formation, as well as the factors involved in defective spinal neurulation (Gofflot, 1999).

The novel murine Sax-1 gene, a member of the NK-1 class of homeobox genes has been isolated, and its expression pattern in the developing central nervous system (CNS) is reported in comparison to two other homeobox genes, Evx-1 and Pax-6. Sax-1 is found to be transiently expressed in the developing posterior CNS. First seen in the ectoderm, lateral to the primitive streak, the signal later encompasses the neural plate. Posteriorly, the expression domain overlaps with the Evx-1 expression in the streak, while anteriorly it is delimited by the Pax-6 signal in the neural tube. During this early phase starting at day 9.5 pc, Sax-1 is expressed in distinct areas of spinal cord, hindbrain and forebrain. Particularly strong signals are detected in rhombomere 1 and in the pretectum. In these areas, subsets of neurons may be marked and specified. In addition to the normal pattern of Sax-1 during development, the expression in different mouse mutants has been analyzed. In Brachyury curtailed homozygotes, the expression of Sax-1 is found to be reduced during neurulation and even lost at day 9.0 pc. Ventral shift and finally loss of the signal in the ventral spinal cord is observed in Danforth's short tail homozygotes (Schubert, 1995).

Cytotactin or tenascin (Drosophila homolog: Tenascin major) is a morphoregulatory molecule of the extracellular matrix affecting cell shape, division, and migration that appears in a characteristic and complex site-restricted pattern during embryogenesis. The promoter region of the gene that encodes chicken cytotactin contains a variety of potential regulatory sequences. These include putative binding sites for homeodomain proteins and a phorbol response element (TRE)/AP-1 element, a potential target for transcription factors thought to be involved in growth-factor signal transduction. To determine the effects of homeobox-containing genes on cytotactin promoter activity, a series of cotransfection experiments were carried out on NIH 3T3 cells using cytotactin promoter- reporter gene constructs and plasmids driving the expression of mouse homeobox genes Evx-1 and Hox-1.3. Cotransfection with Evx-1 stimulates cytotactin promoter activity whereas cotransfection in control experiments with Hox-1.3 have no effect. To localize the sequences required for Evx-1 activation, a series of deletions in the cytotactin promoter were tested. An 89-base-pair region containing a consensus TRE/AP-1 element was found to be required for activation. An oligonucleotide segment containing this TRE/AP-1 site was found to confer Evx-1 inducibility on a simian virus 40 minimal promoter. Mutation of the TRE/AP-1 site abolishes this activity. To explore the potential role of growth factors in cytotactin promoter activation, chicken embryo fibroblasts, which are known to synthesize cytotactin, were first transfected with cytotactin promoter constructs and cultured under minimal conditions in 1% fetal bovine serum. Although the cells exhibit only low levels of reporter activity under these conditions, cells exposed for 12 h to 10% fetal bovine serum show a marked increase in reporter activity. Cotransfection with Evx-1 and cytotactin promoter constructs of cells cultured in 1% fetal bovine serum is sufficient, however, to produce high levels of reporter activity. These findings are consistent with the hypothesis that Evx-1, a homeobox-containing gene, may activate the cytotactin promoter by a mechanism involving a growth-factor signal transduction pathway. More generally, the results support the hypothesis that the place-dependent expression of morphoregulatory molecules may depend upon local cues provided by homeobox genes and their encoded proteins (Jones, 1992).

The human homeobox protein EVX1 (EVX1) is thought to play an important role during embryogenesis. The effect of EVX1 on gene transcription has been investigated in transfected mammalian cells. EVX1 expression represses transcription of a reporter gene directed by either cell-specific or viral promoter/enhancer sequences in a variety of mammalian cell lines and in a concentration-dependent manner. Transcriptional repression is independent of the presence of DNA-binding sites for EVX1 in all the promoters tested. Furthermore, repression by EVX1 is evident also using a TATA-less minimal promoter in the reporter construct. A carboxyl-terminal proline/alanine-rich region of EVX1 seems to be responsible for the transcriptional repression activity, as suggested by transfection of EVX1 mutants. It is speculated that the repressor function of EVX1 contributes to its proposed role in embryogenesis (Briata, 1995).

The human homeodomain protein EVX1 is a transcriptional repressor in transfected mammalian cells and this function depends on a region carboxyl-terminal to the homeodomain. Several deletions of the EVX1 C-terminal region were transiently expressed in mammalian cells and their effect on the transcription of a reporter gene directed by different promoters was investigated. The repressor activity maps to a region of 51 amino acids with a high abundance of alanine and proline residues. This region is able to transfer the repressor function to either the entire HOXC6 or CREB transcription factors, or to the GAL4 DNA binding domain (Briata, 1997).

Interneurons in the ventral spinal cord are essential for coordinated locomotion in vertebrates. During embryogenesis, the V0 and V1 classes of ventral interneurons are defined by expression of the homeodomain transcription factors Evx1/2 and En1, respectively. Evx1 V0 interneurons are locally projecting intersegmental commissural neurons. In Evx1 mutant embryos, the majority of V0 interneurons fail to extend commissural axons. Instead, they adopt an En1-like ipsilateral axonal projection and ectopically express En1, indicating that V0 interneurons are transfated to a V1 identity. Conversely, misexpression of Evx1 represses En1, suggesting that Evx1 may suppress the V1 interneuron differentiation program. These findings demonstrate that Evx1 is a postmitotic determinant of V0 interneuron identity and reveal a critical postmitotic phase for neuronal determination in the developing spinal cord (Moran-Rivard, 2001).

Transcriptional regulation of vertebrate Hox genes involves enhancer sequences located either inside or outside the gene clusters. In the mouse Hoxd complex, for example, series of contiguous genes are coordinately controlled by regulatory sequences located at remote distances. However, in different cellular contexts, Hox genes may have to be insulated from undesirable external regulatory influences to prevent ectopic gene activation, a situation that would likely be detrimental to the developing embryo. The presence is shown of an insulator activity, at one extremity of the Hoxd complex, that is composed of at least two distinct DNA elements, one of which is conserved throughout vertebrate species. However, deletion of this element on its own does not detectably affect Hoxd gene expression, unless another DNA fragment located nearby is removed in cis. These results suggest that insulation of this important gene cluster relies, at least in part, upon a sequence-specific mechanism that displays some redundancy (Kmita, 2002).

The requirement for a context-dependent insulation is best exemplified by the presence of the Evx2 gene in the immediate 5' neighbourhood of the Hoxd cluster. Evx2 indeed displays specific expression features that are not shared by any Hoxd genes, not even by Hoxd13, whose promoter lies close to that of Evx2. This is best illustrated by discrete cell types of the developing central nervous system, in both spinal cord and more rostral parts of the brain, in various vertebrate species. In the spinal cord, transcripts are localized in the ventrally located V0 interneurons, as well as in a population of dorsal interneurons. In the developing brain, Evx2 expression is detected in the rhombencephalic isthmus area (the metencephalic-mesencephalic transition) and extends into the superficial layer of the entire midbrain. It is also expressed in the developing hindbrain and in part of the future cerebellum (Kmita, 2002).

While the enhancer sequences driving Evx2 expression in the CNS have not yet been precisely identified, experiments involving targeted genomic rearrangements around the Evx2 locus have revealed some of their properties: (1) targeted deletions have shown that these enhancer sequences are located at a remote position, upstream the Hoxd complex; (2) a Hoxd9/lacZ transgene is able to respond to the Evx2 CNS-specific enhancer sequences, whenever it was relocated upstream the Hoxd complex, 3' to Evx2. However, the same transgene is unable to respond similarly when placed within the complex, even when positioned immediately next to the Evx2 promoter. These results demonstrated that the Evx2 CNS enhancers have a weak specificity for Evx2 itself, i.e., they are able to interact with other promoters. In addition, Hoxd promoters can respond to such regulatory controls provided they are relocated in the proper genomic environment, i.e., in 3' of the Evx2 transcription unit. These observations raise the question of which mechanism prevent Hoxd genes from responding to these CNS enhancers, in the wild-type context. In other words, why a promoter able to respond to a given regulatory sequence, when placed outside the cluster, is unable to do so from within the Hoxd complex, even when localized right next to the Evx2 promoter (Kmita, 2002).

In this set of experiments, potential sequences, located between Evx2 and the Hoxd cluster, were sought that would be able to isolate this latter cluster from the surrounding regulatory influences. An evolutionary conserved DNA stretch participates in the insulation of the cluster, as revealed by novel genomic rearrangements in this locus. However, even though this sequence is sufficient to ensure proper insulation of the cluster, additional sequences, located nearby, were also found to be involved in this process. The requirement for a combined deletion in cis of these sequences in order to bypass the insulation of the cluster, raises the possibility that some functional redundancy exists between these regulatory sequences (Kmita, 2002).

During limb development, coordinated expression of several Hoxd genes is required in presumptive digits. A search for the underlying control sequences upstream from the cluster resulted in the discovery of Lunapark (Lnp: Drosophila homolog: CG8735, located at 44D4), a gene that shares limb and CNS expression specificities with both Hoxd genes and Evx2, another gene located nearby. A targeted enhancer-trap approach was used to identify a DNA segment capable of directing reporter gene expression in both digits and CNS, following Lnp, Evx2, and Hoxd-specific patterns. This DNA region shows an unusual interspecies conservation, including with its pufferfish counterpart. It contains a cluster of global enhancers capable of controlling transcription of several genes unrelated in structure or function, thus defining large regulatory domains. These domains were interrupted in the Ulnaless mutation, a balanced inversion that modifies the topography of the locus. The heuristic value of these results is discussed in term of locus specific versus gene-specific regulation (Spitz, 2003).

A functionally conserved boundary element from the mouse HoxD locus requires GAGA factor in Drosophila

Hox genes are necessary for proper morphogenesis and organization of various body structures along the anterior-posterior body axis. These genes exist in clusters and their expression pattern follows spatial and temporal co-linearity with respect to their genomic organization. This colinearity is conserved during evolution and is thought to be constrained by the regulatory mechanisms that involve higher order chromatin structure. Earlier studies, primarily in Drosophila, have illustrated the role of chromatin-mediated regulatory processes, which include chromatin domain boundaries that separate the domains of distinct regulatory features. In the mouse HoxD complex, Evx2 and Hoxd13 are located ∼ 9 kb apart but have clearly distinguishable temporal and spatial expression patterns. This study reports the characterization of a chromatin domain boundary element from the Evx2-Hoxd13 region that functions in Drosophila as well as in mammalian cells. The Evx2-Hoxd13 region has sequences conserved across vertebrate species including a GA repeat motif, and the Evx2-Hoxd13 boundary activity in Drosophila is dependent on GAGA factor that binds to the GA repeat motif. These results show that Hox genes are regulated by chromatin mediated mechanisms and highlight the early origin and functional conservation of such chromatin elements (Vasanthi, 2010).

The role of chromatin organization in developmental gene regulation has been well established. In particular, chromatin organization that involves domain boundary elements has been shown to be a key feature of the regulation of homeotic genes in Drosophila . As the organization of Hox genes is well conserved among bilatarians, it is reasonable to speculate that the constraint that led to this conservation of organization is due to chromatin elements that regulate Hox genes. In general, when differentially expressed genes are in close proximity, as is often the case in Hox complexes, boundary elements are likely to be present between the genes to establish and maintain their distinct expression states. In the mouse HoxD complex, Evx2 and Hoxd13 are ∼9 kb apart and they are expressed in distinct regions in the developing embryo. This suggests the presence of a boundary within this 9 kb region that prevents the crosstalk between regulatory elements of the two flanking genes (Vasanthi, 2010).

In order to identify this putative boundary, sequence comparison of the Evx2-Hoxd13 region from different vertebrates were carried out, and a cluster of conserved sites along with a GA repeat motif was identified in all the species checked, from fish to mammals. The ∼3 kb fragment that included the GA repeats showed enhancer-blocking activity in Drosophila embryos, as well as in a human cell line, indicating the presence of a complex evolutionarily conserved boundary between Evx2 and Hoxd13 genes. The boundary activity was shown by both overlapping fragments, ED1a and ED1b, suggesting that the Evx2-Hoxd13 boundary is spread over several kilobases, unlike Drosophila boundaries that tend to be smaller, often less than 1 kb. Spread out boundary function in this region has also been suggested by an earlier study (Yamagishi, 2007). The complex nature of the Evx2-Hoxd13 boundary is also indicated by the observation that only early enhancers of ftz are effectively blocked, whereas late enhancers are able to drive expression of the lacZ reporter gene even in the presence of this boundary. This boundary activity was examined in the adult eye using a white gene enhancer and promoter interaction assay, and the results clearly showed no enhancer blocking activity in this tissue. These observations indicate that Evx2-Hoxd13 is a developmentally regulated boundary that functions in early embryos but not in late embryonic CNS and adult eye (Vasanthi, 2010).

It was also found that the boundary activity shown by the fragment containing GA-repeat motif is dependent on GAF in Drosophila. This indicates that the conserved GA sites are functionally relevant in Drosophila. Evx2 is the homolog of the even skipped (eve) gene of Drosophila, and both are thought to have evolved from a common ancestral gene Evx. In vertebrates, Evx is located near Hox clusters: Evx1 near HoxA and Evx2 near HoxD. In Drosophila, eve has moved away from the Hox cluster. The finding that a GAF-dependent boundary is present in the Evx2-Hoxd13 region is of particular interest in the light of a previous study showing that the eve gene in fly is also associated with a GAF-dependent boundary. These observations suggest that the boundary function evolved early on near the ancestral Evx gene and that the same combination has been conserved during evolution even in the organisms where the linkage between eve to Hox complex has been lost (Vasanthi, 2010).

Although several boundary-interacting factors are known in Drosophila, in vertebrates, CTCF is the only protein that has been well studied for its role in boundary function. A CTCF homolog is also present in Drosophila and is known to play a role in the Fab-8 boundary function in the BX-C. Interestingly, however, the Fab-7 boundary of the BX-C does not involve CTCF, and instead GAF plays an important role in its function and regulation. In the case of the Evx2-Hoxd13 boundary, and in agreement with earlier studies, no CTCF-binding sites are found. As in Fab-7, this boundary appears to be dependent on GAF. These observations suggest that although several factors act together to establish a boundary, some of them may be mutually exclusiv. Further studies in this direction will help in understanding the function and regulation of boundaries during development (Vasanthi, 2010).

These results strongly indicate the presence of GAGA-binding protein in vertebrates with functional similarity to that of Drosophila GAF. Earlier studies have also indicated that transcription of st-3 gene in Xenopus is regulated by GAGA sequences and GAGA factor, but the identity of vertebrate GAF has been elusive. In a separate study, c-krox/Th-POK was identified as the vertebrate homolog of GAF and was shown to binds to Evx2-Hoxd13 region in vertebrates (Matharu, 2010). These findings suggest that eve/Evx2 dependence on GAF is a feature acquired early in evolution and that even after eve separated from the Hox context, it retained this association and the functional features as seen in Drosophila. This work indicates that, in vertebrates, the ancient organization (as well as the GAF-dependent regulation) has been maintained at least at one of the Hox complexes. Finally, it is suggested that using this approach, other evolutionarily conserved cis elements and trans-acting factors involved in genomic organization and developmental gene regulation can be explored (Vasanthi, 2010).

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Interactive Fly, Drosophila even-skipped: Biological Overview | Transcriptional regulation | Post-transcriptional regulation | Targets of activity | Protein interactions | Developmental Biology | Effects of Mutation | References

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