Zerknüllt homologs in vertebrates

Hox genes control regional identity during segmentation of the vertebrate hindbrain into rhombomeres. A transgenic analysis as used to investigate the upstream mechanisms for regulation of Hoxb-3 in rhombomere (r)5. Enhancers were identified from the promoter sequences of mouse and chick Hoxb-3 sufficient for r5-restricted expression. Sequence comparisons reveal two blocks of similarity (of 19 and 45 base pairs), which each contain in vitro binding sites for the kreisler protein (Kmrl1), a Maf/b-Zip protein expressed in r5 and r6. Kreisler (Krml1) is a member of a distinct basic leucine zipper family with some sequence homology in the basic DNA binding region to fos, jun, CREB and C/EBP. Both kreisler binding sites are required for r5 activity, suggesting that Hoxb-3 is a direct target of kreisler. Multimers of the 19-base-pair (bp) block recreate a Krml1-like pattern in r5/r6, but the 45-bp block mediates expression only in r5. Therefore elements within the 45-bp block restrict the response to Krml1. Additional sequences have been identified that contain an Ets-related activation site, required for both the activation and restriction to r5. These studies demonstrate that Krml1 directly activates expression of Hoxb-3 in r5 in combination with an Ets-related activation site, and suggest that kreisler plays a primary role in regulating segmental identity through Hox genes (Manzanares, 1997).

Hox genes encode transcription factors that are used to regionalize the mammalian embryo. Analysis of mice carrying targeted mutations in individual and multiple Hox genes is beginning to reveal a complex network of interactions among these closely related genes which is responsible for directing the formation of spatially restricted tissues and structures. This paper presents an analysis of the genetic interactions between all members of the third paralogous group, Hoxa3, Hoxb3, and Hoxd3. Previous analysis has shown that although mice homozygous for loss-of-function mutations in either Hoxa3 or Hoxd3 have no defects in common, mice mutant for both genes demonstrate that these two genes strongly interact in a dosage-dependent manner. To complete the analysis of this paralogous gene family, mice with a targeted disruption of the Hoxb3 gene were generated. Homozygous mutants have minor defects at low penetrance in the formation of both the cervical vertebrae and the IXth cranial nerve. Analysis and comparison of all double-mutant combinations demonstrate that all three members of this paralogous group interact synergistically to affect the development of both neuronal and mesenchymal neural crest-derived structures, as well as somitic mesoderm-derived structures. Surprisingly, with respect to the formation of the cervical vertebrae, mice doubly mutant for Hoxa3 and Hoxd3 or Hoxb3 and Hoxd3 show an indistinguishable defect: loss of the entire atlas. This suggests that the identity of the specific Hox genes that are functional in a given region may not be as critical as the total number of Hox genes operating in that region (Manley, 1997).

Hox genes encode transcription factors that are used to regionalize the mammalian embryo. Analysis of mice carrying targeted mutations in individual and multiple Hox genes is beginning to reveal a complex network of interactions among these closely related genes that is responsible for directing the formation of spatially restricted tissues and structures. An analysis is presented of the genetic interactions between all members of the third paralogous group, Hoxa3, Hoxb3, and Hoxd3. Previous analysis has shown that although mice homozygous for loss-of-function mutations in either Hoxa3 or Hoxd3 have no defects in common, mice mutant for both genes demonstrate that these two genes strongly interact in a dosage-dependent manner. To complete the analysis of this paralogous gene family, mice with a targeted disruption of the Hoxb3 gene were generated. Homozygous mutants have minor defects at low penetrance in the formation of both the cervical vertebrae and the IXth cranial nerve. Analysis and comparison of all double-mutant combinations demonstrate that all three members of this paralogous group interact synergistically to affect the development of both neuronal and mesenchymal neural crest-derived structures, as well as somitic mesoderm-derived structures. Surprisingly, with respect to the formation of the cervical vertebrae, mice doubly mutant for Hoxa3 and Hoxd3 or Hoxb3 and Hoxd3 show an indistinguishable defect, loss of the entire atlas. This suggests that the identity of the specific Hox genes that are functional in a given region may not be as critical as the total number of Hox genes operating in that region (Manley, 1998a).

The thymus, thyroid, and parathyroid glands in vertebrates develop from the pharyngeal region, with contributions both from pharyngeal endoderm and from neural crest cells in the pharyngeal arches. Hoxa3 mutant homozygotes have defects in the development of all three organs. Roles for the Hoxa3 paralogs, Hoxb3 and Hoxd3, were investigated by examining various mutant combinations. The thyroid defects seen in Hoxa3 single mutants are exacerbated in double mutants with either of its paralogs, although none of the double-mutant combinations result in thyroid agenesis. The results indicate that the primary role of these genes in thyroid development is their effect on the development and migration of the ultimobranchial bodies, which contribute the parafollicular or C-cells to the thyroid. Hoxb3;Hoxd3 double mutants show no obvious defects in the thymus or parathyroids. However, the removal of one functional copy of Hoxa3 from the Hoxb3;Hoxd3 double mutants (Hoxa3 +/-, Hoxb3-/-, Hoxd3-/-) results in the failure of the thymus and parathyroid glands to migrate to their normal positions in the throat. Very little is known about the molecular mechanisms used to mediate the movement of tissues during development. These results indicate that Hoxa3, Hoxb3, and Hoxd3 have highly overlapping functions in mediating the migration of pharyngeal organ primordia. In addition, Hoxa3 has a unique function with respect to its paralogs in thymus, parathyroid, and thyroid development. This unique function may be conferred by the expression of Hoxa3, but not Hoxb3 nor Hoxd3, in the pharyngeal pouch endoderm (Manley, 1998b).

The thymus and parathyroid glands in mice develop from a thymus/parathyroid primordium that forms from the endoderm of the third pharyngeal pouch. The molecular mechanisms that promote this unique process, in which two distinct organs form from a single primordium, were investigated using mice mutant for Hoxa3 and Pax1. Thymic ectopia in Hoxa3+/-Pax1-/- compound mutants is due to delayed separation of the thymus/parathyroid primordium from the pharynx. The primordium is hypoplastic at its formation, and has increased levels of apoptosis. The developing third pouch in Hoxa3+/-Pax1-/- compound mutants initiates normal expression of the parathyroid-specific Gcm2 and thymus-specific Foxn1 genes. However, Gcm2 expression is reduced at E11.5 in Pax1-/- single mutants, and further reduces or is absent in Hoxa3+/-Pax1-/- compound mutants. Subsequent to organ-specific differentiation from the shared primordium, both the parathyroids and thymus develop defects. Parathyroids in compound mutants are smaller at their formation, and absent at later stages. Parathyroids are also reduced in Pax1-/- mutants, revealing a new function for Pax1 in parathyroid organogenesis. Thymic hypoplasia at later fetal stages in compound mutants is associated with increased death and decreased proliferation of thymic epithelial cells. These results suggest that a Hoxa3-Pax1 genetic pathway is required for both epithelial cell growth and differentiation throughout thymus and parathyroid organogenesis (Su, 2001).

HOXB3 mRNA levels are high in the earliest CD34+ lineage bone marrow cells and low to undetectable in later CD34+/CD34- cells. To gain some insight into the role this gene may play in hematopoiesis, HOXB3 was overexpressed in murine bone marrow cells using retroviral gene transfer. Thymi of HOXB3 marrow recipients are reduced in size when compared with control transplant recipients, with a 24-fold decrease in the absolute number of CD4+ CD8+ cells and a 3-fold increase in the number of CD4- CD8- thymocytes that contain a high proportion of gammadelta TCR+ cells. B cell differentiation is also perturbed in these mice, as indicated by the virtual absence of transduced IL-7-responsive pre-B clonogenic progenitors. Recipients of HOXB3-transduced cells also have elevated numbers of mature granulocyte macrophage colony-forming cells in their bone marrow and spleen. Together these results suggest roles for HOXB3 in proliferation and differentiation processes of both early myeloid and lymphoid developmental pathways (Sauvageau, 1998).

The pattern and regulation of Hoxa3 expression in the hindbrain and associated neural crest cells was investigated in the chick embryo, using whole mount in situ hybridization in conjunction with DiI labeling of neural crest cells and microsurgical manipulations. Hoxa3 is expressed in the neural plate and later in the neural tube with a rostral border of expression corresponding to the boundary between rhombomeres (r) 4 and 5. Initial expression is diffuse and becomes sharp after boundary formation. Hoxa3 exhibits uniform expression within r5 after formation of rhombomeric borders. Cell marking experiments reveal that neural crest cells migrating caudally (but not rostrally) from r5 and r6 express Hoxa3 in normal embryos. Results from transposition experiments demonstrate that expression of Hoxa3 in r5 neural crest cells is not strictly cell-autonomous. When r5 is transposed with r4 by rostrocaudal rotation of the rhomobomeres, Hoxa3 is expressed in cells migrating lateral to transposed r5 and for a short time, in condensing ganglia, but Hoxa3 is not expressed by neural crest within the second branchial arch. Since DiI-labeled cells from transposed r5 are present in the second arch, Hoxa3-expressing neural crest cells from r5 appear to down-regulate their Hoxa3 expression in their new environment. In contrast, when r6 is transposed to the position of r4 after boundary formation, Hoxa3 is maintained in both migrating neural crest cells and those positioned within the second branchial arch and associated ganglia. These results suggest that Hoxa3 expression is cell-autonomous in r6 and its associated neural crest. These results suggest that neural crest cells expressing the same Hox gene are not equivalent; they respond differently to environmental signals and exhibit distinct degrees of cell autonomy depending on their rhombomere of origin (Saldivar, 1996).

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 (a proboscipedia homolog) and a-3 (a zerknüllt homolog) 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).

Vertebrate Hox and Otx genes encode homeodomain-containing transcription factors thought to transduce positional information along the body axis in the segmental portion of the trunk and in the rostral brain, respectively. Moreover, Hox and Otx2 genes show a complementary spatial regulation during embryogenesis. A 1821-base pair (bp) upstream DNA fragment of the Otx2 gene is positively regulated by co-transfection with expression vectors for the human HOXB1, HOXB2, and HOXB3 proteins in an embryonal carcinoma cell line (NT2/D1) and a shorter fragment of only 534 bp is able to drive this regulation. The HOXB1, HOXB2, and HOXB3 DNA-binding region on the 534-bp Otx2 genomic fragment has been demonstrated using nuclear extracts from Hox-transfected COS cells and 12.5 days postcoitum mouse embryos or HOXB3 homeodomain-containing bacterial extracts. HOXB1, HOXB3, and nuclear extracts from 12.5 day mouse embryos bind to a sequence containing two palindromic TAATTA sites, which bear four copies of the ATTA core sequence, a common feature of most HOM-C/HOX binding sites. HOXB2 protects an adjacent site containing a direct repeat of an ACTT sequence, quite divergent from the ATTA consensus. The region bound by the three homeoproteins is strikingly conserved through evolution and necessary (at least for HOXB1 and HOXB3) to mediate the up-regulation of the Otx2 transcription. Taken together, the data support the hypothesis that anteriorly expressed Hox genes might play a role in the refinement of the Otx2 early expression boundaries in vivo (Guazzi, 1998).

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?

The anterior-posterior identities of cells in the hindbrain and cranial neural crest are thought to be determined by their Hox gene expression status, but how and when cells become committed to these identities remain unclear. This question has been addressed using cell transplantation in zebrafish, to test plasticity in hox expression in single cells. Cells were transplanted alone, or in small groups, between hindbrain rhombomeres or between the neural crest primordia of pharyngeal arches. Transplanted cells regulate hox expression according to their new environments. Single cells or small groups transplanted heterotopically along the hindbrain or neural crest primordium change their patterns of hoxa2 and hoxb3 expression. The degree of plasticity, however, depends on both the timing and the size of the transplant. At later stages transplanted cells are more likely to be irreversibly committed and maintain their hox expression, demonstrating a progressive loss of responsiveness to the environmental signals that specify segmental identities. Individual transplanted cells also showed greater plasticity than those lying within the center of larger groups, suggesting that a community effect normally maintains hox expression within segments. Experimental embryos were raised to larval stages to analyze transplanted cells after differentiation and it was found that neural crest cells contribute to pharyngeal cartilages appropriate to the anterior-posterior level of the new cellular environment. Thus, consistent with models implicating hox expression in control of segmental identity, plasticity in hox expression correlates with plasticity in final cell fate (Schilling, 2001).

In situ analysis of hox mRNA expression during rhombomere formation indicates that the dynamic changes in hox expression within the neural tube reflect plasticity in the hox expression status of individual cells. Boundaries of hoxa2 and hoxb3 expression progressively sharpen between 12 and 18 h and weaker expression spreads to additional segments. It is likely that these changes reflect cells initiating de novo synthesis or downregulation of hox transcription rather than the alternative -- that cells move in or out of existing early hox expression domains. In support of this model, AP movements of cells at these stages within the neural tube are extremely restricted. In addition, by 17 h and later, expression of both genes becomes more DV restricted within the hindbrain, suggesting distinct DV-restricted functions in specification of neuronal subtypes within the CNS. The observation that hoxa2 and hoxb3 expression remains plastic as late as 18 h indicates that signals in the environment continue to determine AP identities during head segmentation. These results contrast with many grafting experiments in the avian embryo which have shown that expression of Hoxb-1 and Hoxa-2 in the hindbrain remains stable following whole rhombomere transposition. Similar types of grafts, however, have provided seemingly contradictory evidence for plasticity in the expression of different Hox-a, Hox-b, and Hox-d paralogs. Recent grafting experiments in mice have demonstrated plasticity for several Hox genes, including Hoxa-2, in small groups of cells, implicating the cranial mesoderm as a possible source of regulative influences that maintain Hox expression. The results suggest that such environmental signals are conserved. Further, they define the spatial and temporal natures of such plasticity and suggest that differences in previous studies may reflect the timing and size of their grafts. Based on these results it would be predicted that hox gene expression in individual cells at the edges of large rhombomere grafts in chick would show plasticity (Schilling, 2001).

The products of PBX homeobox genes, which were initially discovered in reciprocal translocations occurring in human leukemias, have been shown to cooperate in the in vitro DNA binding with HOX proteins. Despite the growing body of data implicating Hox genes in the development of various cancers, little is known about the role of HOX-PBX interactions in the regulation of proliferation and induction of transformation of mammalian cells. Both cellular transformation and proliferation induced by Hoxb4 and Hoxb3 are greatly modulated by the levels of available PBX1 present in these cells. The transforming capacity of these two HOX proteins depends on their conserved tetrapeptide and homeodomain regions, which (respectively) mediate binding to PBX and DNA. Taken together, results of this study demonstrate that cooperation between HOX and PBX proteins modulates cellular proliferation and strongly suggest that cooperative DNA binding by these two groups of proteins represents the basis for Hox-induced cellular transformation (Krosl, 1998).

The expression pattern of the mouse Hoxb3 gene is exceptionally complex and dynamic compared with that of other members of the Hoxb cluster. There are multiple types of transcripts for Hoxb3 gene, and the anterior boundaries of its expression vary at different stages of development. Two enhancers flanking Hoxb3 on the 3' and 5' sides regulate Hoxb2 and Hoxb4, respectively, and these control regions define the two ends of a 28-kb interval in and around the Hoxb3 locus. To assay the regulatory potential of DNA fragments in this interval transgenic analysis with a lacZ reporter gene was used to locate cis-elements for directing the dynamic patterns of Hoxb3 expression. Detailed analysis has identified four new and widely spaced cis-acting regulatory regions that can together account for major aspects of the Hoxb3 expression pattern. Elements Ib, IIIa, and IVb control gene expression in neural and mesodermal tissues; element Va controls mesoderm-specific gene expression. The most anterior neural expression domain of Hoxb3 is controlled by an r5 enhancer (element IVa); element IIIa directs reporter expression in the anterior spinal cord and hindbrain up to r6, and the region A enhancer (in element I) mediates posterior neural expression. Hence, the regulation of segmental expression of Hoxb3 in the hindbrain is different from that of Hoxa3, since two separate enhancer elements contribute to expression in r5 and r6. The mesoderm-specific element (Va) directs reporter expression to prevertebra C1 at 12.5 dpc, which is the anterior limit of paraxial mesoderm expression for Hoxb3. When tested in combinations, these cis-elements appear to work as modules in an additive manner to recapitulate the major endogenous expression patterns of Hoxb3 during embryogenesis. Together this study shows that multiple control elements direct reporter gene expression in diverse tissue-, temporal-, and spatially restricted subset of the endogenous Hoxb3 expression domains and work in concert to control the neural and mesodermal patterns of expression (Kwan, 2001).

In the segmented vertebrate hindbrain, the Hoxa3 and Hoxb3 genes are expressed, respectively, at high relative levels in the rhombomeres (r) 5 and 6, and 5. The single enhancer elements responsible for these activities constitute direct targets of the transcription factor kreisler, which is expressed in r5 and r6. The contribution of the transcription factor Krox20, present in r3 and r5 has been anayzed. Genetic analyses demonstrate that Krox20 is required for activity of the Hoxb3 r5 enhancer, but not of the Hoxa3 r5/6 enhancer. Mutational analysis of the Hoxb3 r5 enhancer, together with ectopic expression experiments, reveals that Krox20 binds to the enhancer and synergizes with kreisler to promote Hoxb3 transcription, restricting enhancer activity to r5, their domain of overlap. These analyses also suggest contributions from an Ets-related factor and from putative factors likely to heterodimerize with kreisler. The integration of multiple independent inputs present in overlapping domains by a single enhancer is likely to constitute a general mechanism for the patterning of subterritories during vertebrate development (Manzanares, 2002).

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).

Homeobox gene Hoxa3 is strongly expressed in the third pharyngeal arch and pouch. Hoxa3 homozygous null mutant mice lack the carotid body, a chemosensory organ that reacts sensitively to hypoxia, hypercapnia, and acidic pH in blood. In all late-term mutant embryos examined, no carotid body was present. The carotid body rudiment is formed in the wall of the third branchial artery, which develops into the common carotid artery and the first part of the internal carotid artery. The symmetrical patterns of the third, fourth, and sixth arch arteries were observed in wild-type littermates at embryonic day (E) 10.5-12.5. In Hoxa3 homozygous mutant embryos, however, the third arch artery begins to degenerate at E10.5 and almost disappears at E11.5. Furthermore, the bifurcation of the common carotid artery at the normal position, i.e., at the upper end of the larynx, is never detected in the mutant embryos at E16.5-E18.5. The common carotid artery of the homozygous mutants is separated into the internal and external carotid arteries immediately after its origin. Thus, the present study evidenced that the absence of the carotid body in Hoxa3 homozygous mutants is due to the defect of development of the third arch artery, resulting in malformation of the carotid artery system. During fetal development, the carotid body of mice is in close association with the superior cervical ganglion of the sympathetic trunk. The superior cervical ganglion shows hypertrophic features in Hoxa3 homozygous mutants lacking the carotid body (Kameda, 2002).

The complex and dynamic pattern of Hoxb3 expression in the developing hindbrain and the associated neural crest of mouse embryos is controlled by three separate cis-regulatory elements: element I (region A), element IIIa, and the r5 enhancer (element IVa). The cis-regulatory element IIIa has been examined by transgenic and mutational analysis to determine the upstream trans-acting factors and mechanisms that are involved in controlling the expression of the mouse Hoxb3 gene in the anterior spinal cord and hindbrain up to the r5/r6 boundary, as well as the associated neural crest that migrates to the third and posterior branchial arches and to the gut. By deletion analysis, the sequence requirements have been identified within a 482-bp element III482. Two Hox binding sites are identified in element III482 and in vitro both Hoxb3 and Hoxb4 proteins can interact with these Hox binding sites, suggesting that auto/cross-regulation is required for establishing the expression of Hoxb3 in the neural tube domain. Interestingly, a novel GCCAGGC sequence motif has been identified within element III482, that is also required to direct gene expression to a subset of the expression domains except for rhombomere 6 and the associated neural crest migrating to the third and posterior branchial arches. Element III482 can direct a higher level of reporter gene expression in r6, which led to an investigation to see whether kreisler is involved in regulating Hoxb3 expression in r6 through this element. However, transgenic and mutational analysis has demonstrated that, although kreisler binding sites are present, they are not required for the establishment or maintenance of reporter gene expression in r6. These results have provided evidence that the expression of Hoxb3 in the neural tube up to the r5/r6 boundary is auto/cross-regulated by Hox genes and expression of Hoxb3 in r6 does not require kreisler (Yau, 2003).

Hindbrain development is a well-characterized segmentation process in vertebrates. The bZip transcription factor MafB/kreisler is specifically expressed in rhombomeres (r) 5 and 6 of the developing vertebrate hindbrain and is required for proper caudal hindbrain segmentation. Evidence is provided that the mouse protooncogene c-jun, which encodes a member of the bZip family, is coexpressed with MafB in prospective r5 and r6. Analysis of mouse mutants suggests that c-jun expression in these territories is dependent on MafB but independent of the zinc-finger transcription factor Krox20, another essential determinant of r5 development. Loss- and gain-of-function studies, performed in mouse and chick embryos, respectively, demonstrate that c-Jun participates, together with MafB and Krox20, in the transcriptional activation of the Hoxb3 gene in r5. The action of c-Jun is likely to be direct, since c-Jun homodimers and c-Jun/MafB heterodimers can bind to essential regulatory elements within the transcriptional enhancer responsible for Hoxb3 expression in r5. These data indicate that c-Jun acts both as a downstream effector and a cofactor of MafB and belongs to the complex network of factors governing hindbrain patterning (Mechta-Grigoriou, 2003).

Thus, c-Jun is involved in the early phase of Hoxb3 expression in r5 and it acts through the previously identified Hoxb3 r5 enhancer. This enhancer has been shown to carry both MafB and Krox20 binding sites that are essential for its activity. Furthermore, MafB and Krox20 have been shown to synergistically cooperate in regulating enhancer activity and Hoxb3 expression. The domain of Hoxb3 expression in the hindbrain reflects these requirements, since it corresponds precisely to the intersection of the territories where MafB (r5 and r6) and Krox20 (r3 and r5) are expressed. These data add further complexity to the regulation of Hoxb3, since it is shown that the expression of Hoxb3 in r5 can be divided in two phases -- an early phase (around 5 s stage) that is dependent on c-Jun, and a late phase (around 10-12 s stage) that is c-Jun-independent. Other AP-1 members, such as JunB and JunD, are not expressed in the hindbrain during these stages, suggesting that they are not involved in the late upregulation of Hoxb3 in c-jun mutant embryos. Moreover, it has been shown that these AP-1 members are not upregulated in the c-jun knock-out. Electroporation studies in chick embryos confirm and extend the mouse data. They demonstrate that c-Jun can cooperate with Krox20 in the activation of the Hoxb3 r5-enhancer (Mechta-Grigoriou, 2003).

It is unclear why c-Jun is required for the early phase of Hoxb3 expression, while c-jun is itself under the control of MafB. However, the following points are noteworthy: (1) although one of the two MafB binding sites (the Kr2 site) in the Hoxb3 r5 enhancer is absolutely required for activity, it interacts relatively poorly with MafB homodimers; (2) during the early phase of Hoxb3 expression in r5, the level of MafB in this rhombomere is likely to be much lower than at later stages (judging from the levels of mRNA). (3) MafB can form heterodimers with c-Jun, as shown by coimmunoprecipitations and bandshift experiments. On the basis of these observations, the following model is proposed for Hoxb3 regulation in r5: at the onset of Hoxb3 expression, the level of MafB in r5 is sufficient to allow transcriptional activation of c-jun, but not to sustain Hoxb3 activation. In contrast, c-Jun itself has sufficient affinity to bind efficiently to the Kr2 site of the Hoxb3 r5-enhancer, either as a homodimer or heterodimer with MafB, and to activate Hoxb3 expression in r5 synergistically with Krox20. At later stages, the level of MafB in r5 increases dramatically and can replace c-Jun, either as a homodimer or a heterodimer with other bZip proteins. At this stage, c-Jun is no longer required for expression of Hoxb3 in r5 (Mechta-Grigoriou, 2003).

In conclusion, c-Jun appears to accelerate the activation of Hoxb3 by MafB, allowing transcriptional activation when MafB alone is insufficient. In addition, the identification of c-Jun as a novel upstream regulator of Hoxb3 supports an important role for MafB partners in hindbrain patterning. The complexity of interactions involved in this latter process may offer additional levels of regulation for fine tuning of gene expression (Mechta-Grigoriou, 2003).

The delay in Hoxb3 activation in r5 is the only phenotype so far identified associated with c-jun inactivation at early developmental stages. The normal expression of r5 markers, such as Hoxa3, MafB, Krox20, and EphA4, indicates that the patterning of r5 and r6 is not dramatically affected and that the restored expression of Hoxb3 in r5 at later stages does not result from a delay in maturation of this rhombomere. Furthermore, the lack of modification in the Phox2B and neurofilament expression patterns suggests that specification of regional identity and neurogenesis in r5 and r6 are not dramatically perturbed. Hoxb3-/- mice survive until adulthood, but show minor defects in the cervical vertebrae. The early lethality of the c-jun mutants at 13.5 dpc prevents determination of whether the delayed expression of Hoxb3 leads to similar defects (Mechta-Grigoriou, 2003).

Outside of the CNS, several of the early sites of c-jun expression are correlated with phenotypic consequences. The c-jun gene is expressed in the foregut and the septum transversum. This latter structure is critical for liver formation since it affects the outgrowth of hepatic ducts and also influences the vascular organization of the foregut/midgut junction. The impaired hepatogenesis observed in c-jun-/- mutants might reflect a premature deficiency in the foregut and/or result from an early defect in the septum transversum. c-jun expression is also observed in the future sclerotomal compartment of the recently formed somites. Consistently, conditional inactivation of c-jun in the sclerotome and notochord has been shown to perturb the development of intervertebral bodies. The demonstration of a causal link between these different sites of early c-jun expression and the observed phenotypes deserves further investigation (Mechta-Grigoriou, 2003).

In the developing hindbrain, the functional loss of individual Hox genes has revealed some of their roles in specifying rhombomere (r) identity. However, it is unclear how Hox genes act in concert to confer the unique identity to multiple rhombomeres. Moreover, it remains to be elucidated how these genes interact with other transcriptional programs to specify distinct neuronal lineages within each rhombomere. In r5, the combined mutation of Hoxa3 and Hoxb3 result in a loss of Pax6- and Olig2-expressing progenitors that give rise to somatic motoneurons of the abducens nucleus. In r6, the absence of any combination of the Hox3 paralogous genes results in ectopic expression of the r4-specific determinant Hoxb1. This ectopic expression in turn results in the differentiation of r4-like facial branchiomotoneurons within this rhombomere. These studies reveal that members of the Hox1 and Hox3 paralogous groups participate in a 'Hox code' that is necessary for coordinating both suppression and activation mechanisms that ensure distinction between the multiple rhombomeres in the developing hindbrain (Gaufo, 2003).

The phenotypes of mice harboring independent mutations for the Hox3 paralogs suggest that these genes are part of a common regulatory network necessary for determining the fate of somatic motorneurons (SMNs). Although these genes are necessary for the specification of SMNs, the regulatory process by which they attain this goal is qualitatively different. Hox3 genes and Pax6 are upstream of Olig2 expression. In the loss of Hoxa3 and Hoxb3, however, Pax6 is expressed at ectopically high levels in the pSMN domain, suggesting that Hoxa3 and Hoxb3 genetically suppress Pax6 expression levels in the pSMN domain. Pax6 expression is derepressed in the Nkx2.2-expressing BMN progenitor domain in r4 of Hoxb1 mutant embryos. These observations are analogous to the role of the Drosophila Hox gene, Antp, in suppressing the activity of eyeless, the homolog of Pax6 in vivo and in vitro. However, a direct interaction between the mammalian Hox and Pax genes remains to be tested (Gaufo, 2003).

Pbx1 is a TALE-class homeodomain protein that functions in part as a cofactor for Hox class homeodomain proteins. Previous analysis of the in vivo functions of Pbx1 by targeted mutagenesis in mice has revealed roles for this gene in skeletal patterning and development and in the organogenesis of multiple systems. Both RNA expression and protein localization studies have suggested a possible role for Pbx1 in pharyngeal region development. Since several Hox mutants have distinct phenotypes in this region, the potential requirement for Pbx1 in the development of the pharyngeal arches and pouches and their organ derivatives was investigated. Pbx1 homozygous mutants exhibit delayed or absent formation of the caudal pharyngeal pouches, and disorganized patterning of the third pharyngeal pouch. Formation of the third pouch-derived thymus/parathyroid primordia is also affected, with absent or hypoplastic primordia, delayed expression of organ-specific differentiation markers, and reduced proliferation of thymic epithelium. The fourth pouch and the fourth pouch-derived ultimobranchial bodies were usually absent. These phenotypes are similar to those reported in Hoxa3/ single mutants and Hoxa1/;Hoxb1/ or Hoxa3+/−;Hoxb3/;Hoxd3/ compound mutants, suggesting that Pbx1 acts together with multiple Hox proteins in the development of the caudal pharyngeal region. However, some aspects of the Pbx1 mutant phenotype included specific defects that were less severe than those found in known Hox mutant mice, suggesting that some functions of Hox proteins in this region are Pbx1-independent (Manley, 2004).

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zerknüllt: Biological Overview | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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