Role of Deformed homologs in the subdivision of the hindbrain and neural tube

The developing hindbrain is organized into a series of segments termed rhombomeres; these represent lineage restricted compartments correlating with domains of gene expression and neuronal differentiation. In this study, the processes of hindbrain segmentation is investigated as well as the acquisition of segmental identity by analyzing the expression of zebrafish hox genes in the hindbrains of normal fish and fish with a loss-of-function mutation in the segmentation gene valentino (val, the homolog of mouse kreisler (note: Kreisler and Valentino have no known Drosophila homolog), a bZIP factor. Zebrafish hox genes generally have similar expression profiles to their murine and avian counterparts, although there are several differences in timing and spatial extent of expression which may underlie some of the functional changes that have occurred along the separate evolutionary lineages of teleosts and tetrapods. Analysis of hox gene expression in val- embryos confirms that the val gene product is important for subdivision of the presumptive rhombomere 5 and 6 territory into definitive rhombomeres, suggests that the val gene product plays a critical role in regulating hox gene transcription, and indicates that some neural crest cells are inappropriately specified in val- embryos. Analysis of gene expression at several developmental stages has allowed the inference of differences between primary and secondary defects in the val mutant: it is found that extended domains of expression for some hox genes are secondary, late phenomena potentially resulting from inappropriate cell mixing or lack of normal inter-rhombomeric interactions in the caudal hindbrain (Prince, 1998a).

The autoregulatory enhancer of Deformed , which supports expression in subregions of posterior head segments of Drosophila embryos, is capable of conferring reporter gene expression to a discrete subregion of the hindbrain in transgenic mouse embryos. Remarkably, this anterior-posterior subregion lies within the common anterior expression domain of the Dfd cognate Hox genes in the postotic hindbrain. Indications are that the Dfd autoregulatory enhancer is part of a highly conserved mechanism for establishing region-specific gene expression along the anterior-posterior axis of the embryo (Awgulewitsch, 1992).

Hoxd-4 is expressed in the presumptive hindbrain and spinal cord, pre-vertebrae, and other tissues. In the adult, Hoxd-4 transcripts are expressed predominantly in the testis and kidney, and to a lesser extent in intestine and heart. Mice heterozygous or homozygous for a Hoxd-4 mutation exhibit homeotic transformations of the second cervical vertebrae (C2) to the first cervical vertebrae (C1) and malformations of the neural arches of C1 to C3 and of the basioccipital bone. This suggests that Hoxd-4 plays a role in conferring position information along the anteroposterior axis in the skeleton ( Horan, 1995).

The chicken and mouse Hoxb-4 genes have similar patterns of expression. The anterior boundaries of expression for both genes in segmented tissues, such as the hindbrain and paraxial mesoderm, map to the same rhombomere (r) (r6/r7) and somite (s) (s6/s7) limits, showing a direct correlation between expression of a specific Hox gene and patterning identical axial structures in both species. The functional activity of cis-regulatory regions from the chicken Hoxb-4 gene was tested in transgenic mice to identify and map components conserved between the species. Enhancers were identified which contain conserved blocks of sequence identity and which are necessary to mediate mesodermal and neural restricted patterns of expression. However, only the neural enhancer directs the proper anterior boundary of expression (r6/r7), indicating that only a subset of the underlying molecular components regulating Hoxb-4 expression are functionally conserved between species (Morrison, 1995).

The expression of Hoxb-4, Hoxb-1, Hoxa-3, Hoxb-3, Hoxa-4 and Hoxd-4 was analyzed in the neural tube of chick and quail embryos after rhombomere (r) heterotopic transplantations within the rhombencephalic area. Grafting experiments were carried out at the 5-somite stage, i.e. before rhombomere boundaries are visible. They were preceded by the establishment of the precise fate map of the rhombencephalon in order to determine the presumptive territory corresponding to each rhombomere. When a rhombomere is transplanted from a caudal to a more rostral position it expresses the same set of Hox genes as in situ. By contrast in many cases, if rhombomeres are transplanted from rostral to caudal their Hox gene expression pattern is modified. They express genes normally activated at the new location of the explant, as evidenced by unilateral grafting. This induction occurs whether transplantation is carried out before or after rhombomere boundary formation. Moreover, the fate of the cells of caudally transplanted rhombomeres is modified: the rhombencephalic nuclei in the graft develop according to the new location as shown for an r5/6 to r8 transplantation. Transplantation of 5 consecutive rhombomeres (i.e. r2 to r6), to the r8 level leads to the induction of Hoxb-4 in the two posteriormost rhombomeres but not in r2,3,4. Transplantations to more caudal regions (posterior to somite 3) result in some cases in the induction of Hoxb-4 in the whole transplant. Neither the mesoderm lateral to the graft nor the notochord is responsible for the induction. Thus, the inductive signal emanates from the neural tube itself, suggesting that planar signalling and predominance of posterior properties are involved in the patterning of the neural primordium (Grapin-Botton, 1995).

Hox gene expression in the rhombencephalon is controlled by environmental cues. Thus posterior transposition of anterior rhombomeres to the r7/8 level results in the activation of Hox genes of the four first paralog groups and in homeotic transformations of the neuroepithelial fate according to cellular position along the anteroposterior axis. Although cells in r2 to r6 express Hox genes they do not have inducing activity on more anterior territories. If transposed at the posterior rhombencephalon and trunk level, however, the same anterior regions are able to express Hox genes such as Hoxa-2 and a-3 (both proboscipedia homologs) or b-4 (a Deformed homolog). These signals are transferred by two paths: one vertical, arising from the paraxial mesoderm, and one planar, travelling in the neural epithelium. Hoxb-4 can be induced in anterior neural alar plate tissue when that tissue is transplanted to the posterior, into the rhombdomere 8 region. The competence to express Hox genes extends in the anterior to the forebrain and midbrain neural tissues but expression of Hox genes does not preclude Otx2 expression in the posterior territories (suggesting that transformation is incomplete) and results only in slight changes in their phenotypes. Whereas only somites posterior to somite 5 can induce Hoxb-4, only somites posterior to somites 3/4 can induce Hoxa-2 and Hoxa-3. Thus the environmental cues capable of switching on genes of the four first paralog groups can be present from the level of r7 caudalward and not in the pre-otic and otic rhombencephalon. Rhombomeres transplanted to posterior truncal levels turn out to be able to express posterior genes of the first eight paralog groups to the exclusion of others located downstream in the Hox genes genomic clusters. Thus from the 9th paralogous group and higher, the competence to be induced does not exist. This suggests that the neural tube is divided into large territories characterized by different Hox gene regulatory features (Grapin-Botton, 1997).

Normal expression of the murine Hoxa4 gene during development requires both autoregulatory and retinoic acid-dependent modes of regulation. When introduced into a Hoxa4 null background, expression of a lacZ reporter gene driven by the Hoxa4 regulatory region (Hoxa4/lacZ) is either abolished or significantly reduced in all tissues at E10.5-E12.5. Thus, the observed autoregulation of the Drosophila Deformed gene is conserved in a mouse homolog in vivo, and is reflected in a widespread requirement for positive feedback in order to maintain Hoxa4 expression. Three potential retinoic acid response elements are identified in the Hoxa4 5' flanking region, one of which is identical to a well-characterized element flanking the Hoxd4 gene. Administration of retinoic acid to Hoxa4/lacZ transgenic embryos results in stage-dependent ectopic expression of the reporter gene in the neural tube and hindbrain. When administered to Hoxa4 null embryos, however, persistent ectopic expression is not observed, suggesting that autoregulation is required for maintenance of the retinoic acid-induced expression. Mutation of the consensus retinoic acid response element eliminates the response of the reporter gene to exogenous retinoic acid, and abolishes all embryonic expression in untreated embryos, with the exception of the neural tube and prevertebrae. These data add to the evidence that Hox gene expression is regulated, in part, by endogenous retinoids and autoregulatory loops (Packer, 1998).

The rae28 gene is a mouse homolog of the Drosophila polyhomeotic gene, which is a member of the Polycomb group (Pc-G) of genes. The Pc-G genes are required for the correct expression of the Homeotic complex genes and segment specification during Drosophila embryogenesis and larval development. To study the role of the rae28 gene in mouse development, rae28-deficient mice were generated by gene targeting in embryonic stem cells. The rae28-/- homozygous mice exhibit perinatal lethality, posterior skeletal transformations and defects in neural crest-related tissues, including ocular abnormalities, cleft palate, parathyroid and thymic hypoplasia and cardiac anomalies. The anterior boundaries of Hoxa-3, a-4, a-5, b-3, b-4 and d-4 expression are shifted rostrally in the paraxial mesoderm of the rae28-/- homozygous embryos, and those of Hoxb-3 and b-4 expression are also similarly altered in the rhombomeres and/or pharyngeal arches. These altered Hox codes are presumed to be correlated with the posterior skeletal transformations and neural crest defects observed in the rae28-/- homozygous mice. These results indicate that the rae28 gene is involved in the regulation of Hox gene expression and segment specification during paraxial mesoderm and neural crest development (Takihara, 1997).

The mouse kreisler gene is expressed in rhombomeres (r) 5 and 6 during neural development; also, kreisler mutants have patterning defects in the hindbrain that are not fully understood. This phenotype has been analyzed with a combination of genetic, molecular, and cellular marking techniques. Using Hox/lacZ transgenic mice as reporter lines and by analyzing Eph/ephrin expression, it has been found that while r5 fails to form in these mice, r6 is present. This shows that kreisler has an early role in the formation of r5. Patterning defects were also observed in r3 and r4 that are outside the normal domain of kreisler expression. In both heterozygous and homozygous kreisler embryos some r5 markers are induced in r3, suggesting that there is a partial change in r3 identity that is not dependent upon the loss of r5. Defects in r5 were analyzed by examining Hox gene enhancers known to be targets of kreisler. An r5-specific enhancer from Hoxb3 requires kreisler for establishment and maintenence of gene expression. The paralogous Hoxa3 gene enhancer that directs expression in r5 and r6 is a transcriptional target of kreisler. In homozygous mutant embryos, expression in r5 and r6 is completely absent, showing that in these domains the Hoxa3 enhancer activity is dependent upon kreisler. Analysis of kr mutants reveals an ectopic induction of some Hoxa3-positive cells in the r3 territory. There appear to be progressive changes in the identity of r3 dependent on the dosage of kr. In kr mutants, Hoxb4 expression has shifted anteriorly by a single rhombomere and a sharp anterior boundary is maintained (Manzanares, 1999).

To investigate the cellular character of r6 in kreisler embryos, heterotopic grafting experiments were performed in the mouse hindbrain to monitor its mixing properties. Control experiments reveal that cells from even- or odd-numbered segments only mix freely with themselves, but not with cells of opposite character. Transposition of cells from the r6 territory of kreisler mutants reveals that they adopt mature r6 characteristics, as they freely mix only with cells from even-numbered rhombomeres. Analysis of Phox2b expression shows that some aspects of later neurogenesis in r6 are altered, which may be associated with the additional roles of kreisler in regulating segmental identity. Together these results suggest that the formation of r6 has not been affected in kreisler mutants. This analysis has revealed phenotypic and mechanistic differences between kreisler and its zebrafish equivalent valentino. While valentino is believed to subdivide preexisting segmental units, in the mouse kreisler specifies a particular segment. The formation of r6 independent of r5 argues against a role for kreisler in prorhombomeric segmentation of the mouse hindbrain. It is concluded that the mouse kreisler gene regulates multiple steps in segmental patterning involving both the formation of segments and their A-P identity (Manzanares, 1999).

During development of the vertebrate hindbrain, Hox genes play multiples roles in the segmental processes that regulate anteroposterior (AP) patterning. Paralogous Hox genes, such as Hoxa3, Hoxb3 and Hoxd3, generally have very similar patterns of expression, and gene targeting experiments have shown that members of paralogy group 3 can functionally compensate for each other. Hence, distinct functions for individual members of this family may primarily depend upon differences in their expression domains. The earliest domains of expression of the Hoxa3 and Hoxb3 genes in hindbrain rhombomeric (r) segments are transiently regulated by kreisler, a conserved Maf b-Zip protein, but the mechanisms that maintain expression in later stages are unknown. In this study, the segmental expression and regulation of Hoxa3 and Hoxb3 in mouse and chick embryos have been compared to investigate how they are controlled after initial activation. The patterns of Hoxa3 and Hoxb3 expression in r5 and r6 in later stages during mouse and chick hindbrain development are differentially regulated. Hoxa3 expression is maintained in r5 and r6, while Hoxb3 is downregulated. Regulatory comparisons of cis-elements from the chick and mouse Hoxa3 locus in both transgenic mouse and chick embryos have identified a conserved enhancer that mediates the late phase of Hoxa3 expression through a conserved auto/cross-regulatory loop. This block of similarity is also present in the human and horn shark loci, and contains two bipartite Hox/Pbx-binding sites that are necessary for its in vivo activity in the hindbrain. These HOX/PBC sites are positioned near a conserved kreisler-binding site (KrA) that is involved in activating early expression in r5 and r6, but their activity is independent of kreisler. This work demonstrates that separate elements are involved in initiating and maintaining Hoxa3 expression during hindbrain segmentation, and that it is regulated in a manner different from Hoxb3 in later stages. Together, these findings add further strength to the emerging importance of positive auto- and cross-regulatory interactions between Hox genes as a general mechanism for maintaining their correct spatial patterns in the vertebrate nervous system (Manzanares, 2001).

In vertebrates, certain Hox genes are known to control cellular identities along the anterior-posterior (A-P) axis in the developing hindbrain. In mouse Hoxa3 mutants, truncation of the glossopharyngeal (IXth) nerve or the fusion of the IXth and vagus (Xth) nerves occurs, although its underlying mechanism is largely unknown. To elucidate the mechanism of the IXth nerve defects, the phenotype of Hoxa3 mutant embryos has been reexamined. In Hoxa3 mutants, an abnormal caudal stream of the migrating Hoxa3-expressing neural crest cells is observed at the prospective IXth nerve-forming area. Dorsomedial migration of the placode-derived neuronal precursor cells of the IXth nerve is also affected. Motor neurons at rhombomere 6 (r6), where those of the IXth nerve were positioned, often projected axons to the Xth nerve. In summary, the Hoxa3 gene has crucial roles in ensuring the correct axon projection pattern of all three components of the IXth nerve, i.e., motor neurons and sensory neurons of the proximal and distal ganglia (Watari, 2001).

During anteroposterior (AP) patterning of the developing hindbrain, the expression borders of many transcription factors are aligned at interfaces between neural segments called rhombomeres (r). Mechanisms regulating segmental expression have been identified for Hox genes, but for other classes of AP patterning genes there is only limited information. The murine retinoic acid receptor ß gene (Rarb) was analyzed and shown to be induced prior to segmentation, by retinoic-acid (RA) signalling from the mesoderm. Induction establishes a diffuse expression border that regresses until, at later stages, it is stably maintained at the r6/r7 boundary by inputs from Hoxb4 and Hoxd4. Separate RA- and Hox-responsive enhancers mediate the two phases of Rarb expression: a regulatory mechanism remarkably similar to that of Hoxb4. By showing that Rarb is a direct transcriptional target of Hoxb4, this study identifies a new molecular link, completing a feedback circuit between Rarb, Hoxb4 and Hoxd4. It is proposed that the function of this circuit is to align the initially incongruent expression of multiple RA-induced genes at a single segment boundary (Serpente, 2005).

The mechanism regulating Rarb in the presegmented hindbrain is similar to that of Hoxb4. Both genes are transcriptionally induced by a Raldh2-dependent RA source and both possess RARE-containing enhancers [for Hoxb4, this is termed the early neural enhancer (ENE) that directs neural expression with borders that recede after E8.5. These caudal shifts presumably reflect regression of the inducing ability of the paraxial mesoderm with increasing embryonic age. Although there are strong parallels between the RARE enhancers of Rarb and Hoxb4, there are also some differences. For example, proximal enhancer activity begins at around the two-somite stage, whereas ENE activity begins at the nine-somite stage. In addition, at E8.5, the anterior border of the Rarb proximal enhancer is at presumptive r5/6 but that of the Hoxb4 ENE is at presumptive r6/r7. Although the DNA element responsible for these expression differences is undefined, it may be relevant that the DR5 class of RARE, present in both enhancers, differs at 3/12 nucleotide positions (Serpente, 2005).

The regulatory parallels between Rarb and Hoxb4 also extend to the later phase of segmental expression. Like Rarb, Hoxb4 uses a two-step regulatory strategy of establishment and maintenance within the hindbrain, involving two enhancer elements that are mechanistically and physically separable. For Hoxb4, the late hindbrain element is termed the late neural enhancer (LNE). Both the Rarb distal enhancer and the Hoxb4 LNE drive expression with a sharp r6/r7 border and respond to stabilizing inputs from group 4 Hox genes. In both cases, these late Hox inputs serve to halt the caudal regression of diffuse borders that were established by RARE-containing enhancers. However, when the functions of Hoxb4 and Hoxd4 are completely removed, Hoxb4 LNE activity is lost only from r7, whereas Rarb distal enhancer activity is abolished within the entire neural tube. This suggests that, although group 4-6 Hox paralogues activate the Hoxb4 LNE, only some or all of the group 4 Hox genes may be capable of activating the Rarb distal enhancer (Serpente, 2005).

Hox proteins drive cell segregation and non-autonomous apical remodelling during hindbrain segmentation

Hox genes encode a conserved family of homeodomain transcription factors regulating development along the major body axis. During embryogenesis, Hox proteins are expressed in segment-specific patterns and control numerous different segment-specific cell fates. It has been unclear, however, whether Hox proteins drive the epithelial cell segregation mechanism that is thought to initiate the segmentation process. This study investigated the role of vertebrate Hox proteins during the partitioning of the developing hindbrain into lineage-restricted units called rhombomeres. Loss-of-function mutants and ectopic expression assays reveal that Hoxb4 and its paralogue Hoxd4 are necessary and sufficient for cell segregation, and for the most caudal rhombomere boundary (r6/r7). Hox4 proteins regulate Eph/ephrins and other cell-surface proteins, and can function in a non-cell-autonomous manner to induce apical cell enlargement on both sides of their expression border. Similarly, other Hox proteins expressed at more rostral rhombomere interfaces can also regulate Eph/ephrins, induce apical remodelling and drive cell segregation in ectopic expression assays. However, Krox20, a key segmentation factor expressed in odd rhombomeres (r3 and r5), can largely override Hox proteins at the level of regulation of a cell surface target, Epha4. This study suggests that most, if not all, Hox proteins share a common potential to induce cell segregation but in some contexts this is masked or modulated by other transcription factors (Prin, 2014).

Role of Deformed homologs in the neural crest

In addition to pigment cells, and neural and endocrine derivatives, the neural crest is characterized by its ability to yield mesenchymal cells. In amniotes, this property is restricted to the cephalic region from the mid-diencephalon to the end of rhombomere 8 (the level of somites 4/5). The cephalic neural crest is divided into two domains: an anterior region corresponding to the diencephalon, mesencephalon and metencephalon in which expression of Hox genes is never observed, and a posterior domain in which neural crest cells exhibit (with a few exceptions) the same Hox code as the rhombomeres from which they originate. A determination was made of the contribution of the different transverse levels of the neural fold to the maxillary, mandibullary buds (i.e. the first branchial arch, BA1), the second (BA2), the third (BA3) and the posterior (BA4, BA5 and BA6) branchial arches. Hox genes are expressed in the neural crest as they are in the rhombomere from which they originate. Thus BA1 is colonized by neural crest cells (NCC) from the posterior mesencephalon and from r1 and r2 with a small contingent of cells from r3 that do not express any genes of the Hox clusters. BA2, which is extensively colonized by NCC originating from r4 (with a minor contribution from r3 and r5), contain Hoxa-2-positive mesectodermal cells. BA2 is invaded by NCC expressing Hox genes of the second and third paralogous groups, while in the posterior BAs, the NCC express additional genes of the fourth paralogous groups. By altering the normal distribution of neural crest cells in the branchial arches through appropriate embryonic manipulations, the relationships between Hox gene expression and the level of plasticity that neural crest cells display has been examined when these cells are led to migrate to an ectopic environment. The following observation have been made: (1) Hox gene expression is not altered in neural crest cells by their transposition to ectopic sites. (2) Expression of Hox genes by the branchial arch (BA) ectoderm does not depend upon an induction by the neural crest. This second finding further supports the concept of segmentation of the cephalic ectoderm into ectomeres. According to this concept, metameres can be defined in large bands of ectoderm including not only the CNS and the neural crest but also the corresponding superficial ectoderm fated to cover craniofacial primordia. (3) The construction of a lower jaw requires the environment provided by the ectomesodermal components of BA1 or BA2 associated with the Hox gene non-expressing neural crest cells. Hox gene-expressing neural crest cells are unable to yield the lower jaw apparatus, including the entoglossum and basihyal even in the BA1 environment. In contrast, the posterior part of the hyoid bone can be constructed by any region of the neural crest cells whether or not they are under the regulatory control of Hox genes. Such is also the case for the neural and connective tissues (including those comprising the cardiovascular system) of neural crest origin, on which no segmental restriction is imposed. The latter finding confirms the plasticity observed for the precursors of the PNS (Couley, 1998).

Diencephalic, mesencephalic and metencephalic neural crest cells are skeletogenic and derive from neural folds that do not express Hox genes. In order to examine the influence of Hox gene expression on skull morphogenesis, expression of Hoxa2, Hoxa3 and Hoxb4 in conjunction with that of the green fluorescent protein has been selectively targeted to the Hox-negative neural folds of the avian embryo prior to the onset of crest cell emigration. Hoxa2 expression precludes the development of the entire facial skeleton. Transgenic Hoxa2 embryos (such as those from which the Hox-negative domain of the cephalic neural crest has been removed) have no upper or lower jaws and no frontonasal structures. Embryos subjected to the forced expression of Hoxa3 and Hoxb4 show severe defects in the facial skeleton but not a complete absence of facial cartilage. Hoxa3 prevents the formation of the skeleton derived from the first branchial arch, but allows the development (albeit reduced) of the nasal septum. Hoxb4, by contrast, hampers the formation of the nasal bud-derived skeleton, while allowing that of a proximal (but not distal) segment of the lower jaw. The combined effect of Hoxa3 and Hoxb4 prevents the formation of facial skeletal structures, comparable with Hoxa2. None of these genes impairs the formation of neural derivatives of the crest. These results suggest that over the course of evolution, the absence of Hox gene expression in the anterior part of the chordate embryo is crucial in the vertebrate phylum for the development of a face, jaws and brain case, and, hence, also for that of the forebrain (Creuzet, 2002).

Deformed homologs and somite development

At the cellular level, retinoic acid (RA) regulates gene expression through nuclear receptors, which act as ligand-dependent transcription factors. There are two families of retinoid nuclear receptors, the Retinoic Acid Receptors (RARs), which are activated by all-trans and 9-cis retinoic acid, and the Retinoid X Receptors (RXRs), which are activated by 9-cis retinoic acid only. Each family is represented by three genes, Rara, Rarb, and Rarg, and Rxra, Rxrb and Rxrg. Single and compound null mutants for all of these receptors have revealed both unique and redundant functions during development. All combined, the congenital malformations presented by the Rar and Rxr single or compound mutants recapitulate the vitamin A phenotypes induced in rats deprived of vitamin A in utero. Relative to single null mutants, mice bearing mutations in both Hoxd4 and Rarg display malformations of the basioccipital bone, and first (C1) and second cervical vertebrae (C2) at increased penetrance and expressivity, demonstrating synergy between Hoxd4 and Rarg in the specification of the cervical skeleton. In contrast to Rarg mutants, retinoic acid (RA) treatment on embryonic day 10.5 of Hoxd4 single or Hoxd4;Rarg double mutants does not rescue normal development of C2. Somitic expression of Hoxd4 is not altered in wild-type or Rarg mutant animals before or after RA treatment on day 10.5, suggesting that Hoxd4 and Rarg act in parallel to regulate the expression of target genes directing skeletogenesis (Folberg, 1999).

Deformed homologs and hematopoiesis

The extent to which primitive embryonic blood progenitors contribute to definitive lymphoid-myeloid hematopoiesis in the adult remains uncertain. In an effort to characterize factors that distinguish the definitive adult hematopoietic stem cell (HSC) and primitive progenitors derived from yolk sac or embryonic stem (ES) cells, the effect of ectopic expression of HoxB4, a homeotic selector gene implicated in self-renewal of definitive HSCs was examined. Expression of HoxB4 in primitive progenitors combined with culture on hematopoietic stroma induces a switch to the definitive HSC phenotype. These progenitors engraft lethally irradiated adults and contribute to long-term, multilineage hematopoiesis in primary and secondary recipients. These results suggest that primitive HSCs are poised to become definitive HSCs and that this transition can be promoted by HoxB4 expression. This strategy for blood engraftment enables modeling of hematopoietic transplantation from ES cells (Kyba, 2002).

Hox transcription factors have emerged as important regulators of primitive hematopoietic cell proliferation and differentiation. In particular, HOXB4 appears to be a strong positive regulator of hematopoietic stem cell (HSC) self-renewal. The potency of HOXB4 to enable high-level ex vivo HSC expansion is demonstrated in this study. Cultures of nontransduced or GFP-transduced murine bone marrow cells experience large HSC losses over 10-14 days. In sharp contrast, cultures of HOXB4-transduced cells achieve rapid, extensive, and highly polyclonal HSC expansions, resulting in over 1000-fold higher levels relative to controls and a 40-fold net HSC increase. Importantly, these HSCs retain full lympho-myeloid repopulating potential and enhanced in vivo regenerative potential, demonstrating the feasibility of achieving significant ex vivo expansion of HSCs without functional impairment (Antonchuk, 2002).

Deformed homologs and heart development

The anteroposterior (A-P) patterning of the developing heart underlies atrial and ventricular lineage specification and heart chamber morphogenesis. The posteriorization of cardiomyogenic phenotype with retinoic acid (RA) treatment of primitive streak stage chicken embryos is suggestive of a role for the clustered homeobox (Hox) genes in early heart patterning. A screen for Hox genes expressed in chick heart primordia and primitive heart led to the isolation of anterior genes of the Hox clusters expressed during cardiogenesis. Specific hoxd-3, hoxa-4, and hoxd-4 transcripts are detected at the early stages of heart formation and full-length cDNA clones have been isolated. Expression of hoxd-3 is detected in the heart forming region of embryos prior to heart tube formation. Expression of hoxa-4, hoxd-3, and hoxb-5 is increased in cardiogenic tissue treated with RA in culture conditions that also produced changes in positionally restricted cardiomyogenic phenotypes. Hox genes expressed in cardiac explants exhibit distinct sensitivities to RA and ouabain treatment, when compared to genes, such as nkx-2.5, that are involved in cardiac commitment and differentiation. These studies support a role for Hox genes in early heart patterning and suggest that positional information in the cardiogenic region is established by regulatory mechanisms distinct from early heart lineage specification (Searcy, 1998).

Vertebrate Polycomb homologs regulate Hox genes

The Polycomb group genes are required for the correct expression of the homeotic complex genes and segment specification during Drosophila embryogenesis and larval development. In mouse, inactivation studies of several Polycomb group genes indicate that they are also involved in Hox gene regulation. M33 mutants have been used to study the function of M33, the mouse homolog of the Drosophila Polycomb gene. In the absence of M33, the window of Hoxd4 retinoic acid (RA) responsiveness is opened earlier and Hoxd11 gene expression is activated earlier in development This indicates that M33 antagonizes the RA pathway and has a function in the establishment of the early temporal sequence of activation of Hox genes. Despite the early activation, A-P boundaries are correct in later stages, indicating a separate control mechanism for early aspects of Hox regulation. This raises a number of interesting issues with respect to the roles of both Pc-G proteins and Hox regulatory mechanisms. It is proposed that a function of the M33 protein is to control the accessibility of retinoic acid response elements in the vicinity of Hox genes regulatory regions by direct or indirect mechanisms or both. This could provide a means for preventing ectopic transactivation early in development and be part of the molecular basis for temporal colinearity of Hox gene expression (Bel-Vialar, 2000).

A model proposes that each gene within a Hox cluster would show a graded sensitivity to RA through a repression imposed by Pc-G genes: with 3' genes being accessible earlier than the adjacent 5' gene. This differential repression contributed by M33 can be hypothesized since, in two different backgrounds in M33-/- mice, the skeletal transformations are found more frequently in the anterior (due to the deregulation of more 3' Hox genes) than in the posterior regions. This would imply that the Pc-G repression is more dependent on the M33 product at the 3' end of the cluster than at the 5' end, or/and that the nature of the complex may vary from the 3' end to the 5' end of a Hox cluster. This repression could again be mediated by direct or indirect mechanisms (Bel-Vialar, 2000).

Interaction with PBX, the mammalian homolog of Drosophila Extradenticle

The normal Pbx1 homeodomain protein (Homolog of Drosophila Extradenticle), as well as its oncogenic derivative, E2A-Pbx1, binds the DNA sequence ATCAATCAA cooperatively with the murine Hox-A5 (homolog of Drosophila Scr) and Hox-B7, Hox-B8, and Hox-C8 (Antennapedia class) homeodomain proteins, which are themselves known oncoproteins, as well as with the Hox-D4 (homolog of Deformed) homeodomain protein. Cooperative binding to ATCAATCAA required the homeodomain-dependent DNA-binding activities of both Pbx1 and the Hox partner. In cotransfection assays, Hox-B8 suppresses transactivation by E2A-Pbx1. These results suggest that (1) Pbx1 may participate in the normal regulation of Hox target gene transcription in vivo and thereby contribute to aspects of anterior-posterior patterning and structural development in vertebrates; (2) that E2A-Pbx1 could abrogate normal differentiation by altering the transcriptional regulation of Hox target genes in conjunction with Hox proteins, and (3) that the oncogenic mechanism of certain Hox proteins may require their physical interaction with Pbx1 as a cooperating, DNA-binding partner (Lu, 1995).

The labial group protein HOXA-1 has intrinsically weak DNA-binding activity due to residues in the N-terminal arm of its homeodomain. This observation, among others, suggests that HOX and HOM proteins require cofactors for stable interactions with DNA. A putative HOX cofactor, PBX1A, related to Drosophila Extradenticle, participates in cooperative DNA binding with HOXA-1 and the Deformed group protein HOXD-4. Three Abdominal-B class HOX proteins fail to cooperate with PBX1A. The interacting domain of HOXD-4 maps to the YPWMK pentapeptide motif, a conserved sequence found N terminal to the homeodomain of HOXA-1 and many other homeoproteins, but one that is absent from the Abdominal-B class. The naturally occurring fusion of the transcriptional activation domain of E2A with PBX1 creates an oncoprotein implicated in human pre-B-cell leukemias. A pentapeptide mutation that abolished cooperative interaction with PBX1A in vitro also abrogates synergistic transcriptional activation with the E2A/PBX oncoprotein. The direct contact of PBX family members by the HOX pentapeptide is likely to play an important role in developmental and oncogenic processes (Phelan, 1995).

Dimerization with Extradenticle or the mammalian Exd homologs, the PBX homeoproteins, dramatically improves DNA binding by HOX transcription factors, indicating that recognition by such complexes is important for HOX specificity. For HOX monomeric binding, a major determinant of specificity is the flexible N-terminal arm. It makes base-specific contacts via the minor groove, including one to the 1st position of a 5'-TNAT-3' core by a conserved arginine (Arg-5). Arg-5 also contributes to the stability of HOX.PBX complexes, apparently by forming the same DNA contact. Heterodimers of PBX with HOXA1 (Drosophila homolog: labial) or HOXD4 (Drosophila homolog: Deformed) proteins have different specificities at another position recognized by the N-terminal arm (the 2nd position in the TNAT core). Significantly, N-terminal arm residues 2 and 3, which distinguish the binding of HOXA1 and HOXD4 monomers, play no role in the specificity of their complexes with PBX. In addition, HOXD9 and HOXD10,(Abdominal-B homologs) which are capable of binding both TTAT and TAAT sites as monomers, can cooperate with PBX1A only on a TTAT site. These data suggest that some DNA contacts made by the N-terminal arm are altered by interaction with PBX (Phelan, 1997).

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

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