Transcriptional regulation of Proboscipedia homologs

Direct auto- and cross-regulatory interactions between Hox genes serve to establish and maintain segmentally restricted patterns in the developing hindbrain. Rhombomere r4-specific expression of both Hoxb1 and Hoxb2 depends upon bipartite cis Hox response elements for the group 1 paralogous proteins, Labial-like Hoxa1 and Hoxb1. The DNA-binding ability and selectivity of these proteins depend upon the formation of specific heterodimeric complexes with members of the PBC homeodomain protein family (Pbx genes). The r4 enhancers from Hoxb1 and Hoxb2 have the same activity, but differ with respect to the number and organisation of bipartite Pbx/Hox (PH) sites required, suggesting the intervention of other components/sequences. Another family of homeodomain proteins, TALE (Three-Amino acids-Loop- Extension: Prep1, Meis, HTH), capable of dimerizing with Pbx/EXD, is involved in the mechanisms of r4- restricted expression. TALE/Pbx complexes bind both PH and specific Prep/Meis (PM) motifs. TALE/Pbx and Pbx/Hox interactions are not mutually exclusive, since they utilize different dimerization surfaces, allowing the formation of ternary Prep1/Pbx/Hoxb1 complexes in vitro on bipartite PH motifs. The interaction between Pbx and Hox proteins requires both homeodomains, a stretch of 20 amino acids C-terminal to the Pbx homeodomain, and the conserved pentapeptide sequence YPWMX or a similar ANW amino acid motif N-terminal to the Hox homeodomain. In contrast, Prep1 or Meis1 interaction with Pbx requires conserved amino-terminal sequences in both proteins. Therefore, by combining with Hox and Pbx, TALE proteins may also play an in vivo role in the mechanisms that serve to establish and maintain control of r4 identity. It has been shown that: (1) the r4-specific Hoxb1 and Hoxb2 enhancers are complex elements containing separate PH and Prep/Meis (PM) sites; (2) the PM site of the Hoxb2 enhancer, but not that of the Hoxb1 enhancer, is essential in vivo for r4 expression and also influences other sites of expression; (3) both PM and PH sites are required for in vitro binding of Prep1-Pbx and formation and binding of a ternary Hoxb1-Pbx1a (or 1b)-Prep1 complex. (4) A similar ternary association forms in nuclear extracts from embryonal P19 cells, but only upon retinoic acid induction. This requires synthesis of Hoxb1 and also contains Pbx with either Prep1 or Meis1. Together these findings highlight the fact that PM sites are found in close proximity to bipartite PH motifs in several Hox responsive elements shown to be important in vivo and that such sites play an essential role in potentiating regulatory activity in combination with the PH motifs (Ferretti, 2000).

Hoxa-1 and Hoxa-2 are homologs of the labial and proboscipedia Drosophila genes. In both mouse and Drosophila, these genes have been shown to play a critical role in head development. There are three independent enhancers which direct distinct portions of the Hoxa-1 and Hoxa-2 expression domains during early murine embryogenesis. Two enhancers mediate hindbrain-specific expression. The third enhancer contains a retinoic acid response element. Point mutations within the retinoic acid response element abolish expression in neuroepithelium caudal to rhombomere 4, supporting a natural role for endogenous retinoids in patterning of the hindbrain and spinal cord. Analysis of the murine Hoxa-2 rhombomere 2-specific enhancer in Drosophila embryos reveals a distinct expression domain within the fly head segments, which parallels the expression domain of proboscipedia. These results suggest an evolutionary conservation between HOM-C/Hox family members, including a conservation of certain DNA regulatory elements and possible regulatory cascades (Frasch, 1995).

Transcriptional targets of Proboscipedia homologs

Angiogenesis is characterized by distinct phenotypic changes in vascular endothelial cells (EC). Evidence is provided that the Hox D3 homeobox gene mediates conversion of endothelium from the resting to the angiogenic/invasive state. Stimulation of EC with basic fibroblast growth factor (bFGF) resultes in increased expression of Hox D3, integrin alphavbeta3, and the urokinase plasminogen activator (uPA). Hox D3 antisense blocks the ability of bFGF to induce uPA and integrin alphavbeta3 expression, yet has no effect on EC cell proliferation or bFGF-mediated cyclin D1 expression. Expression of Hox D3, in the absence of bFGF, results in enhanced expression of integrin alphavbeta3 and uPA. In fact, sustained expression of Hox D3 in vivo on the chick chorioallantoic membrane retains EC in this invasive state and prevents vessel maturation leading to vascular malformations and endotheliomas. Therefore, Hox D3 regulates EC gene expression associated with the invasive stage of angiogenesis (Boudreau, 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).

Little is known about the molecular mechanisms that integrate anteroposterior (AP) and dorsoventral (DV) positional information in neural progenitors that specify distinct neuronal types within the vertebrate neural tube. In ventral rhombomere (r)4 of Hoxb1 and Hoxb2 mutant mouse embryos, Phox2b expression is not properly maintained in the visceral motoneuron progenitor domain (pMNv), resulting in a switch to serotonergic fate. Phox2b has been shown to be a direct target of Hoxb1 and Hoxb2. A highly conserved Phox2b proximal enhancer has been found that mediates rhombomere-restricted expression and contains separate Pbx-Hox (PH) and Prep/Meis (P/M) binding sites. Both the PH and P/M sites are essential for Hox-Pbx-Prep ternary complex formation and regulation of the Phox2b enhancer activity in ventral r4. Moreover, the DV factor Nkx2.2 enhances Hox-mediated transactivation via a derepression mechanism. Induction of ectopic Phox2b-expressing visceral motoneurons in the chick hindbrain requires the combined activities of Hox and Nkx2 homeodomain proteins. This study takes an important first step to understand how activators and repressors, induced along the AP and DV axes in response to signaling pathways, interact to regulate specific target gene promoters, leading to neuronal fate specification in the appropriate developmental context (Samad, 2004)

Sequencing of the Phox2b enhancer revealed the presence of a putative bipartite PH-binding site (TGATTGAA). Notably, its nucleotide sequence was identical to that of the low-affinity PH binding site of repeat 2 (R2) of the Hoxb1 autoregulatory (b1-ARE) r4 enhancer. Moreover, it shared fairly high conservation with the PH site present in the Hoxb2 r4 enhancer, also regulated by Hoxb1. Similar to the Hoxb1 and Hoxb2 r4 enhancers, a conserved P/M site (TTGTCATG), was found downstream of the PH site. The Phox2b P/M site and its flanking nucleotides exactly matched the sequence found in the Hoxb1 r4 enhancer and shared six out of eight nucleotides with that in the Hoxb2 r4 enhancer. Interestingly, unlike the previously identified PH and P/M sites lying in relative proximity to each other, the Phox2b P/M site was 147 nucleotides distant from the PH site (Samad, 2004).

There are functional differences between PH-P/M modules in the Phox2b and other Hox-regulated r4 enhancers. Similar to the Hoxb1 and Hoxb2 r4 enhancers, separate PH and P/M sites were found embedded within the Phox2b enhancer. Nevertheless, the in vivo output of Hox regulation on these three enhancers is rather different, since the Phox2b PH or P/M sites mediate a transcriptional response restricted to ventral progenitors, despite widespread Hoxb1 and Hoxb2 distribution throughout r4. This is in keeping with the observation that endogenous Phox2b expression is upregulated in sharp columns of selected progenitor domains at distinct DV levels. Comparing the nature and function of bipartite PH and P/M sites in the context of the Hoxb1, Hoxb2 and Phox2b enhancers may therefore provide clues of how Phox2b regulation is spatially constrained (Samad, 2004).

In the Hoxb2 enhancer, only one PH site is present that shows cooperative binding of Hoxb1 and Pbx/Exd proteins in vitro and is required for r4 expression in vivo. By contrast, the Hoxb1 autoregulatory (b1-ARE) r4 enhancer contains three PH motifs (R1-R3). Mutational analysis in the mouse indicates that all three PH sites are cooperatively required for high levels of r4 expression, although with distinct individual contributions. Among the three Hoxb1 PH sites, the R2 sequence precisely matches that of the Phox2b PH octamer core. Like the Phox2b PH site, the R2 repeat did not bind Hoxb1/Exd heterodimers in vitro, nor Hoxb1 or Exd alone, although it is necessary for optimal r4 activity. Thus, the Hoxb1 R2 repeat requires cooperative interactions with adjacent sequences in the b1-ARE to fully function in vivo. Similarly, a trimerized Phox2b PH site is not sufficient on its own to direct r4 restricted expression in the chick hindbrain, unlike the sufficiency for r4 expression of multimerized Hoxb1 R3 or Hoxb2 high-affinity PH sites. Nonetheless, the PH motif is necessary, in the context of the Phox2b enhancer, for mediating the transcriptional cooperation of Hox, Pbx and Prep/Meis co-factors and for in vivo regulation in ventral r4 both in chick and mouse hindbrain. Thus, the Phox2b low-affinity PH site, while representing a necessary site of integration of r4 activity, operates in vivo mainly through cooperative interactions with its surrounding regulatory environment, even in the presence of high endogenous levels of binding factors (Samad, 2004).

Subdivision of the brain, the neural crest, facial development and Proboscipedia homologs

In vertebrate embryos, streams of cranial neural crest (CNC) cells migrate to form segmental pharyngeal arches and differentiate into segment-specific parts of the facial skeleton. To identify genes involved in specifying segmental identity in the vertebrate head, a screen was performed for mutations affecting cartilage patterning in the zebrafish larval pharynx. The positional cloning and initial phenotypic characterization of a homeotic locus discovered in this screen is presented. A zebrafish ortholog of the human oncogenic histone acetyltransferase MOZ (monocytic leukemia zinc finger) is required for specifying segmental identity in the second through fourth pharyngeal arches. In moz mutant zebrafish, the second pharyngeal arch is dramatically transformed into a mirror-image duplicated jaw. This phenotype resembles a similar but stronger transformation than that seen in hox2 morpholino oligo (hox2-MO) injected animals. In addition, mild anterior homeotic transformations are seen in the third and fourth pharyngeal arches of moz mutants. moz is required for maintenance of most hox1-4 expression domains and this requirement probably at least partially accounts for the moz mutant homeotic phenotypes. Homeosis and defective Hox gene expression in moz mutants is rescued by inhibiting histone deacetylase activity with Trichostatin A. Although early patterning of the moz mutant hindbrain is found to be normal, a late defect is found in facial motoneuron migration in moz mutants. Pharyngeal musculature is transformed late, but not early, in moz mutants. Relatively minor defects are detected in arch epithelia of moz mutants. Vital labeling of arch development reveals no detectable changes in CNC generation in moz mutants, but later prechondrogenic condensations are mispositioned and misshapen. Mirror-image hox2-dependent gene expression changes in postmigratory CNC prefigure the homeotic phenotype in moz mutants. Early second arch ventral expression of goosecoid (gsc) in moz mutants and in animals injected with hox2-MOs shifts from lateral to medial, mirroring the first arch pattern. bapx1, which is normally expressed in first arch postmigratory CNC prefiguring the jaw joint, is ectopically expressed in second arch CNC of moz mutants and hox2-MO injected animals. Reduction of bapx1 function in wild types causes loss of the jaw joint. Reduction of bapx1 function in moz mutants causes loss of both first and second arch joints, providing functional genetic evidence that bapx1 contributes to the moz-deficient homeotic pattern. Together, these results reveal an essential embryonic role and a crucial histone acetyltransferase activity for Moz in regulating Hox expression and segmental identity, and provide two early targets, bapx1 and gsc, of moz and hox2 signaling in the second pharyngeal arch (Miller, 2004).

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 extent to which the spatial organization of craniofacial development is due to intrinsic properties of the neural crest is at present unclear. There is some experimental evidence supporting the concept of a prepattern established within the neural crest while it is contiguous with the neural plate. In experiments in which the neural tube and premigratory crest are relocated within the branchial region, crest cells retain patterns of gene expression appropriate for their position of origin after migration into the branchial arches, resulting in skeletal abnormalities. However, and in apparent conflict with these findings, when crest is rerouted by late deletion of adjacent crest, infilling crest alters its pattern of gene expression to match its new location, and a normal facial skeleton results. In order to reconcile these findings and thus identify processes relevant to the course of normal development, a series of neural tube and crest rotations were performed producing a more extensive reorganization of cephalic crest than has been previously described. Lineage analysis using DiI labelling of crest derived from the rotated hindbrain reveals that crest does not migrate into the branchial arch it would have colonized in normal development; rather, it simply populates the nearest available branchial arches. Crest adjacent to the grafted region contributes to a greater number of branchial arches than it would in normal development, resulting in branchial arches containing mixed cell populations that do not occur in normal development. After exchange of first and third arch crest by rotation of r1-7, crest alters its expression of hoxa-2 and hoxa-3 to match its new location within the embryo, resulting in the reestablishment of the normal branchial arch Hox code. A facial skeleton in which all the normal components are present, with some additional ectopic first arch structures, is formed in this situation. In contrast, when second and third arch crest are exchanged by rotation of r3 to 7, ectopic Hox gene expression is stable, resulting in the persistance of an abnormal branchial arch Hox code and extensive defects in the hyoid skeleton. It is suggested that the intrinsic properties of crest have an effect on the spatial organization of structures derived from the branchial arches, but that exposure to increasingly novel environments within the branchial region or "community effects" within mixed populations of cells can result in alterations to crest Hox code and morphogenetic fate. In both classes of operation there are tight links between the resulting branchial arch Hox code and a particular skeletal morphology (Hunt, 1998).

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 (Couley, 1998).

Hoxa2 is expressed in cranial neural crest cells that migrate into the second branchial arch and is essential for proper patterning of neural-crest-derived structures in this region. Transgenic analysis has been used to begin to address the regulatory mechanisms that underlie neural-crest-specific expression of Hoxa2. By performing a deletion analysis on an enhancer from the Hoxa2 gene, that is capable of mediating expression in neural crest cells in a manner similar to the endogenous gene, it is demonstrated that multiple cis-acting elements are required for neural-crest-specific activity. One of these elements consists of a sequence that binds to the three transcription factor AP-2 family members. Mutation or deletion of this site in the Hoxa2 enhancer abrogates reporter expression in cranial neural crest cells but not in the hindbrain. In both cell culture co-transfection assays and transgenic embryos AP-2 family members are able to trans-activate reporter expression, showing that this enhancer functions as an AP-2-responsive element in vivo. Reporter expression is not abolished in an AP-2alpha null mutant embryos, suggesting redundancy with other AP-2 family members for activation of the Hoxa2 enhancer. Other cis-elements identified in this study as critical for neural-crest-specific expression include an element that influences levels of expression and a conserved sequence, which when multimerized directs expression in a broad subset of neural crest cells. These elements work together to co-ordinate and restrict neural crest expression to the second branchial arch and more posterior regions. These findings have identified the cis-components that allow Hoxa2 to be regulated independently in rhombomeres and cranial neural crest cells (Maconochie, 1999).

Hox genes have been implicated in specifying positional values along the anteroposterior axis of the caudal central nervous system, but their nested and overlapping expression has complicated the understanding of how they confer specific neural identity. A direct gain-of-function approach was employed using retroviral vectors to misexpress Hoxa2 and Hoxb1 outside of the normal Hox expression domains, thereby avoiding complications resulting from possible interactions with endogenous Hox genes. Misexpression of either Hoxa2 or Hoxb1 in the anteriormost hindbrain (rhombomere1, r1) leads to the generation of motor neurons in this territory, even though this region is normally devoid of this cell type. Depending on the target tissue they innervate, motor neurons in the hindbrain can be classified into three different subtypes: visceromotor, branchiomotor and somatomotor. An attempt was made to determine whether misexpression of Hoxb1 leads to the induction of a particular subtype of motor neuron or rather to the induction of a generic type of motor neuron. The migratory behaviour displayed by the ectopic motor neurons in r1 indicate that they might be of the branchiomotor subtype. To test this at the molecular level, use was made of the observation that in the hindbrain only somatomotor neurons but not branchiomotor neurons express the LIM-homeobox gene Isl2. When analysed at HH25 stage, somatic trochlear and abducens motor neurons coexpress Isl1 and Isl2, whereas branchiomotor neurons express Isl1 but not Isl2. Ectopic motor neurons in r1 are never found to express Isl2 in addition to Isl1, consistent with their being of the branchiomotor subtype. This result demonstrated, therefore, that the activity of Hoxb1 was sufficient to selectively induce the generation of a distinct specified subtype of motor neurons (Jungbluth, 1999).

In the case of Hoxb1-induced cells, their axons leave the hindbrain either by fasciculating with the resident cranial motor axons at isthmic (trochlear) or r2 (trigeminal) levels of the axis or via novel ectopic exit points in r1. To determine the subclass of motor neurons generated, the expression profiles of Isl1 and Isl2 were examined. The ectopic motor neurons generated following Hoxa2 misexpression were found to express Isl1 but not Isl2. Therefore, as with Hoxb1, Hoxa2 misexpression also resulted in the generation of motor neurons of branchiomotor subtype identity, as shown by their lateral migration behaviour and by their Isl gene expression patterns. Next, an attempt was made to identify the precise branchiomotor subtypes that are generated after misexpression: the results suggest that the ectopic motor neurons generated following Hoxa2 misexpression are trigeminal-like, while those generated following Hoxb1 misexpression are facial-like. The data demonstrate that at least to a certain extent, and for certain cell types, the singular activities of individual Hox genes (compared to a combinatorial mode of action, for example) are sufficient to impose on neuronal precursor cells the competence to generate distinctly specified cell types. Moreover, since these particular motor neuron subtypes are normally generated in the most anterior domains of Hoxa2 and Hoxb1 expression, respectively, the data support the idea that the main site of individual Hox gene action is in the anteriormost subdomain of their expression, consistent with the phenomenon of posterior dominance (Jungbluth, 1999).

The rhombencephalic neural crest play several roles in craniofacial development. This structure gives rise to the cranial sensory ganglia and much of the craniofacial skeleton, and is vital for patterning of the craniofacial muscles. The loss of Hoxa1 or Hoxa2 function affects the development of multiple neural crest-derived structures. To understand how these two genes function together in craniofacial development, an allele was generated that disrupts both of these linked genes. Some of the craniofacial defects observed in the double mutants are additive combinations of those that exist in each of the single mutants, indicating that each gene functions independently in the formation of these structures. Other defects are found only in the double mutants, demonstrating overlapping or synergistic functions. Multiple defects were uncovered in the attachments and trajectories of the extrinsic tongue and hyoid muscles in Hoxa2 mutants. Interestingly, the abnormal trajectory of two of these muscles, the styloglossus and the stylohyoideus, blocks the attachment of the hyoglossus to the greater horn of the hyoid, which in turn correlates exactly with the presence of cleft palate in Hoxa2 mutants. It is suggested that the hyoglossus, whose function is to depress the lateral edges of the tongue, when unable to make its proper attachment to the greater horn of the hyoid, forces the tongue to adopt an abnormal posture which blocks closure of the palatal shelves. Unexpectedly, in Hoxa1/Hoxa2 double mutants, the penetrance of cleft palate is dramatically reduced. Two compensatory defects, associated with the loss of Hoxa1 function, restore normal attachment of the hyoglossus to the greater horn, thereby allowing the palatal shelves to lift and fuse above the flattened tongue (Barrow, 1999).

The external ear or pinna is derived from six mesenchymal swellings that flank the first branchial cleft. Three of the swellings are located on the first arch adjacent to the cleft, whereas the other three are found on the second arch. All mutant classes exhibit defects in the pinnae. Mutants lacking Hoxa1, but possessing at least one copy of Hoxa2, demonstrated reductions (usually unilaterally) of the external ear, although there are no significant changes in the patterning of the pinna. Hoxa2-/- mutants and Hoxa1 +/-:Hoxa2-/- mutants possess severe external ear anomalies. The pinnae are almost entirely absent, leaving the underlying external auditory meatus exposed. Interestingly, at 100% penetrance two folds of tissue dorsal and posterior to the external auditory meatus that correspond to the pinnae have been found. It has been reported that Hoxa2 mutants demonstrate several duplications of first arch structures. It is possible therefore that these two outgrowths represent duplications of the first arch portions of the external ear. As further evidence that these outgrowths represent duplications, a single ectopic vibrissum was observed to be associated with each pinna structure. Hoxa1/Hoxa2 double mutant mice do not possess any remnants of the pinnae or ectopic vibrissae. The only structure that is visible is the external auditory meatus (Barrow, 1999).

The middle ear ossicles are derived from neural crest that populates the first and second branchial arches. The proximal first arch structures, the malleus and incus, are derived from neural crest that originates from rhombomeres (r) 1 and 2. The gonial bone and tympanic ring are also derived from neural crest that migrates to the first arch. The stapes, however, is derived from r4 neural crest that populates the second arch. Hoxa1 -/- and Hoxa1 -/- /Hoxa2 +/- mutants exhibit reductions in the middle ear apparatus. Similar to the situation with the external ear, these reductions are not associated with major changes in patterning. In most cases, the stapes is absent; however, in one specimen, it was fused to the styloid process. In contrast, Hoxa2 (and Hoxa1+/- ::Hoxa2 -/-) mutants possess severe patterning defects of the middle ear ossicles and the associated skeleton. Mutants of these classes exhibit mirror-image duplications of Meckel’s cartilage, the malleus and incus, as well as the tympanic ring and gonial bone. In addition, the orthotopic and duplicated gonial bones are connected by an ossified bridge. The second arch-derived stapes is not present. The collection of these defects is identical to those previously reported for two other Hoxa2 alleles. The middle ear ossicles of Hoxa1/Hoxa2 double mutants exhibit the patterning defects of the Hoxa2 mutants and the hypoplastic characteristics of Hoxa1 mutants. For example, the double mutants possess mirror-image duplications of Meckel’s cartilage and the malleus and incus; however, both the orthotopic and duplicated structures are markedly reduced in size. The stapes and gonial bone are always absent. The tympanic ring is almost always absent although a small vestige could sometimes be found. Thus, Hoxa2 appears to not only affect the patterning of the tympanic ring and gonial bone but synergizes with Hoxa1 in controlling the growth of these structures as well (Barrow, 1999).

Hox genes are required to pattern neural crest (NC) derived craniofacial and visceral skeletal structures. However, the temporal requirement of Hox patterning activity is not known. An inducible system has been used to establish Hoxa2 activity at distinct NC migratory stages in Xenopus embryos. Stage-specific effects of Hoxa2 gain-of-function have been uncovered, suggesting a multistep patterning process for hindbrain NC. Most interestingly, Hoxa2 induction at postmigratory stages results in mirror image homeotic transformation of a subset of jaw elements, which are normally devoid of Hox expression, toward hyoid morphology. This is the reverse phenotype to that observed in the Hoxa2 knockout. These data demonstrate that the skeletal pattern of rhombomeric mandibular crest is not committed before migration and further implicate Hoxa2 as a true selector of hyoid fate. Moreover, the demonstration that the expression of Hoxa2 alone is sufficient to transform the upper jaw and its joint selectively may have implications for the evolution of jaws (Pasqualetti, 2000).

A key evolutionary innovation in the history of vertebrates has been the appearance of the gnathostome biting jaws and of their articulation, the primary jaw joint. It is still controversial whether upper and lower jaws derive from a transformation of a mouth structure (e.g. the velum of lampreys) or a modification of the anterior gill-bearing visceral arches of an agnathan ancestor. The results of this study are not only relevant for the understanding of patterning of jaws and their articulation, but they may also provide a conceptual framework to investigate jaw evolution in the transition between agnathans and gnathostomes. The existence of a default 'ground pattern' program for rhombencephalic NC has been suggested, corresponding to the Hox-negative r1+r2 program and shared by r4-derived hyoid Hoxa2-expressing NC. The present work conclusively demonstrates the existence of such a default developmental pathway for rostral rhombencephalic NC and also shows that Hoxa2 is sufficient to modify it, acting as a true selector gene. Most interestingly, these results provide further support for the proposal that the mandibular arch does not represent a homogeneous default state of morphogenetic specification. In fact, Hoxa2 is not able to re-pattern mesencephalic NC contributing to the distal portion of the lower jaw and the ethmoidal plate. These structures appear to be genetically specified by Otx2. The mandibular arch is therefore a complex patterning system in which NC cells of different axial origins are under the control of distinct genetic programs that differentially segregate them within the arch, yet they are integrated in a combinatorial fashion on the jaw elements. In particular, the articulation between palatoquadrate and lower jaw (primary jaw joint) is confined to the skeletal regions derived from rhombomeric 1st arch crest. Strikingly, the expression of Hoxa2 alone is sufficient to suppress the primary jaw joint and results in the fusion of upper and lower jaw cartilages into a single element. Conversely, the lack of Hoxa2 leads to the appearance of a second set of articulated jaw structures in the 2nd arch. In this context, the study of the Hoxa2 expression pattern in the mandibular and branchial arches of lampreys, as compared with that of Otx2, will be particularly informative about the agnathan to gnathostome transition. This analysis will show whether the downregulation of Hoxa2 in r2-derived NC may have been a prerequisite for the acquisition of a palatoquadrate and a primary jaw joint, providing insights into the evolution of jaws (Pasqualetti, 2000).

The diverse neuronal subtypes in the adult central nervous system arise from progenitor cells specified by the combined actions of anteroposterior (AP) and dorsoventral (DV) signaling molecules in the neural tube. Analyses of the expression and targeted disruption of the homeobox gene Hoxb1 demonstrate that it is essential for patterning progenitor cells along the entire DV axis of rhombomere 4 (r4). Hoxb1 accomplishes this function by acting very early during hindbrain neurogenesis to specify effectors of the sonic hedgehog and Mash1 signaling pathways. In the absence of Hoxb1 function, multiple neurons normally specified within r4 are instead programmed for early cell death. The findings reported here provide evidence for a genetic cascade in which an AP-specified transcription factor, Hoxb1, controls the commitment and specification of neurons derived from both alar and basal plates of r4 (Gaufo, 2000).

The most prominent mutant phenotype associated with disruption of Hoxb1 is the absence of a functional facial branchiomotor (FBM) nucleus. As a consequence, Hoxb1 mutant homozygous adults show complete paralysis of the muscles of facial expression, which are normally innervated by this nerve. Consistent with the hypothesis that the absence of a FBM nucleus results from a failure to specify FBM neurons, early postmitotic molecular markers that normally label these neurons, such as Isl1 and Phox2a, fail to do so in r4 and r5 of these mutant embryos. Even more informative, one of the earliest transcription factors known to be required for specification of all branchial and visceral motor neurons of the brainstem, Phox2b, is not expressed in a distinct pool of progenitor cells in the ventral progenitor domain of r4 of Hoxb1 mutant embryos, at any stages examined. Instead, cells expressing effector molecules of Shh, such as HNF3b and Nkx2.2, that are normally associated with early, dividing neural progenitors, are reduced and continue to be expressed ectopically in the mantle layer normally occupied by postmitotic neurons. This aberrant cellular phenotype is very similar to the phenotype that occurs with mis-specified FBM neurons resulting from disruption of Phox2b. This is accompanied in Hoxb1 mutant embryos by induction of a wave of ectopic apoptosis that begins at E9.5, and corresponds directly to the time of normal onset of FBM neuron generation and Phox2b expression. The ectopic apoptosis is, however, not restricted to the ventral region of r4 in Hoxb1 mutant embryos, but extends across the three regions of high Hoxb1 expression. And indeed, failure to specify the r4-component of specific neuronal columns is observed throughout the DV extent of the neural tube (Gaufo, 2000).

Hoxa2 plays an important role in the DV patterning of neurons within r2 and r3. As observed for the Hoxb1 mutation, disruption of Hoxa2 selectively affects the formation of the r2/r3-component of neuronal columns that express transcription factors, such as Pax6 and Phox2b, that are in turn involved in the specification of neuronal subtypes. Together, these observations emphasize that the neuronal columns that extend longitudinally across multiple rhombomeres and even into the spinal cord, are built in modules, with different Hox genes being responsible for the formation of the separate modules. Concomitant with this early role of Hox genes in neuronal specification, the progenitor cells automatically acquire a positional value along the AP axis, that allows them to be distinguished from similar cells within a contiguous longitudinal functional column. These observations also emphasize that these Hox genes are epistatic to the set of transcription factors that are used to specify neuronal subtype differentiation. The obvious advantage of this strategy is that positional value can be assigned to multiple neuronal subtypes within an AP region, rather than having to ascribe positional cues individually to each subtype subsequent to its specification (Gaufo, 2000).

To examine the role of Hoxb1 during early neural differentiation, the effects of the Hoxb1 mutation on cells expressing the neural-specific bHLH transcription factors were studied. It is apparent that the broad longitudinal columns expressing Mash1, Ngn1 and Ngn2 are juxtaposed in r4 with columns of cells expressing high levels of Hoxb1. In the absence of Hoxb1, the width of these columns expands in r4, suggesting that Hoxb1 normally restricts the domains of these early neural progenitor cells. In both Drosophila and vertebrates the early neural progenitor domains are restricted and reinforced by the Delta/Serrate/Notch signaling pathways. It is thus attractive to consider that the restriction of the Mash1, Ngn1 and Ngn2 domains within r4 by Hoxb1 is also mediated by the same signaling pathway. On the basis of this hypothesis, it will be of interest to determine whether the production of successively more refined Hoxb1- expressing columns within r4 is involved in restricting and/or reinforcing increasing numbers of neuronal subtype columns within this rhombomere (Gaufo, 2000).

In Hoxb1 mutant mice, there is a loss of r4-dorsal and intermediate columns expressing Phox2b and Phox2a, respectively. Interestingly, in Mash1 mutant mice, there is also a loss of the same Phox2b-expressing column, but in contiguous dorsal columns along the hindbrain. Together, these data suggest that these two transcriptional systems work in parallel with each other during neural determination of common progenitors, and provide further support for Hoxb1 contributing the AP-specific information to progenitor cells that may otherwise be similar along the length of the hindbrain. In conclusion, the present study provides evidence that Hoxb1 is required for the formation of multiple neuronal subtypes along the full extent of the DV axis of r4. The role of Hoxb1 appears to be required very early during hindbrain neurogenesis in parallel with molecules required for DV patterning and neural determination, and prior to the activation of the transcription factors such as Nkx2.2, Isl1, Phox2b and Phox2a, which are used to specify neuronal subtypes. In the absence of Hoxb1, the r4-component of multiple neuronal subtypes fails to be properly specified and is then destined for aberrant programmed cell death (Gaufo, 2000).

Overexpression of Hoxa2 in the chick first branchial arch leads to a transformation of first arch cartilages, such as Meckel’s and the quadrate, into second arch elements, such as the tongue skeleton. These duplicated elements are fused to the original in a similar manner to that seen in the Hoxa2 knockout, where the reverse transformation of second to first arch morphology is observed. This confirms the role of Hoxa2 as a selector gene specifying second arch fate. When first arch neural crest alone is targeted, first arch elements are lost, but the Hoxa2-expressing crest is unable to develop into second arch elements. This is not due to Hoxa2 preventing differentiation of cartilages. Upregulation of a second arch marker in the first arch, and homeotic transformation of cartilage elements is only produced after global Hoxa2 overexpression in the crest and the surrounding tissue. Thus, although the neural crest appears to contain some patterning information, it needs to read cues from the environment to form a coordinated pattern. Hoxa2 appears to exert its effect during differentiation of the cartilage elements in the branchial arches, rather than during crest migration, implying that pattern is determined quite late in development (Grammatopoulos, 2000).

It appears that the neural crest alone does not contain all the patterning information necessary to form a complete jaw and tongue skeleton. It may be that the whole neural tube, and not just the crest, needs to be targeted with Hoxa2 in order to achieve transformation. The neural tube plays a role in controlling the patterning of the overlying neural crest. Duplicated first arch structures after grafting of midbrain to more caudal positions only occurs if the underlying neural tube is also grafted. Thus grafts of midbrain neural folds alone to more caudal positions lead to the formation of ectopic cartilages, but not duplicated first arch structures. In the embryos injected with viral particles, both the neural tube and crest would have been targeted, while with the electroporation into virus-resistant embryos only the neural crest and dorsal neural tube would have been hit, perhaps supporting this view that Hoxa2 would need to be expressed in the whole neural tube in order to produce a transformation (Grammatopoulos, 2000).

A role for the ectoderm in conveying patterning information to cells once in the arch is also indicated. Sensory organ homeobox 1 (Soho1) upregulation in the first branchial arch is only seen in conjunction with high levels of Hoxa2 expression in the adjacent epithelium. In control embryos, the ectoderm surrounding the second and more caudal arches expresses Hoxa2 after crest migration. The Hoxa2 expression in the second arch ectoderm was initially proposed to be induced by Hoxa2-expressing crest, but has recently been shown to be independently induced, such that Hox is still expressed in the epithelium despite the fact that the Hox-expressing neural crest has been replaced with non-Hox-expressing neural crest. This independence of the ectoderm from the neural crest has also been shown by following the expression of other ectoderm/endoderm markers after mass ablation of the neural crest. It is possible that instead of the migrating neural crest inducing the ectodermal expression of Hoxa2, the initial plan of all the tissues at a particular level is set very early in development (Grammatopoulos, 2000).

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).

The pharyngeal arches are one of the defining features of the vertebrates, with the first arch forming the mandibles of the jaw and the second forming jaw support structures. The cartilaginous elements of each arch are formed from separate migratory neural crest cell streams, which derive from the dorsal aspect of the neural tube. The second and more posterior crest streams are characterized by specific Hox gene expression. The zebrafish has a larger overall number of Hox genes than the tetrapod vertebrates, as the result of a duplication event in its lineage. However, in both zebrafish and mouse, there are just two members of Hox paralogue group 2 (PG2): Hoxa2 and Hoxb2. Morpholino-mediated 'knock-down' of both zebrafish Hox PG2 genes results in major defects in second pharyngeal arch cartilages, involving replacement of ventral elements with a mirror-image duplication of first arch structures, and accompanying changes to pharyngeal musculature. In the mouse, null mutants of Hoxa2 have revealed that this single Hox gene is required for normal second arch patterning. By contrast, loss-of-function of either zebrafish Hox PG2 gene individually has no phenotypic consequence, showing that these two genes function redundantly to confer proper pattern to the second pharyngeal arch. hoxb1a mis-expression was used to induce localized ectopic expression of zebrafish Hox PG2 genes in the first arch; using this strategy, it has been found that ectopic expression of either Hox PG2 gene can confer second arch identity onto first arch structures, suggesting that the zebrafish Hox PG2 genes act as 'selector genes' (Hunter, 2002).

The perception of environmental stimuli is mediated through a diverse group of first-order sensory relay interneurons located in stereotypic positions along the dorsoventral (DV) axis of the neural tube. These interneurons form contiguous columns along the anteroposterior (AP) axis. Like neural crest cells and motoneurons, first-order sensory relay interneurons also require specification along the AP axis. Hox genes are prime candidates for providing this information. In support of this hypothesis, distinct combinations of Hox genes in rhombomeres (r) 4 and 5 of the hindbrain are shown to be required for the generation of precursors for visceral sensory interneurons. Since Hoxa2 is the only Hox gene expressed in the anterior hindbrain (r2), disruption of this gene allowed for the demonstration that the precursors for somatic sensory interneurons are under the control of Hox genes. Surprisingly, the Hox genes examined are not required for the generation of proprioceptive sensory interneurons. Furthermore, the persistence of some normal rhombomere characteristics in Hox mutant embryos suggests that the loss of visceral and somatic sensory interneurons cannot be explained solely by changes in rhombomere identity. Hox genes may thus directly regulate the specification of distinct first-order sensory relay interneurons within individual rhombomeres. More generally, these findings contribute to an understanding of how Hox genes specifically control cellular diversity in the developing organism (Gaufo, 2004).

Hoxb1, Hoxa3 and Hoxb3, are required for the segment-specific formation of Mash1-dependent noradrenergic visceral sensory interneurons. By analogy to the sensory system of Drosophila, the Hox gene Ubx appears to control the segmental pattern of achaete-scute complex-dependent sensory bristles (Rozowski, 2002). In contrast to mouse, where Hox genes positively influence the specification of Mash1-dependent noradrenergic interneurons, Ubx suppresses the differentiation of progenitors or proneural clusters into sensory bristles. The regulation of analogous sensory structures in the mouse is therefore opposite that observed in Drosophila. However, the stage by which the mouse and Drosophila Hox genes regulate this differentiation process appears to be similar. In both mouse and Drosophila, the formation of progenitors appears to be independent of Hox gene function. However, subsequent activation or suppression of noradrenergic visceral sensory interneuron or sensory bristle formation, respectively, is dependent on Hox genes (Gaufo, 2000; Rozowski, 2002). This study in the mouse suggests that Hox genes regulate noradrenergic visceral sensory interneurons at the level of Phox2b expression. However, direct evidence for this hypothesis will require testing the functional relevance of a conserved Hox/Pbx regulatory element in the promoter/enhancer region of Phox2b. Nevertheless, the similarities in the segmental regulation of sensory structures by Mash1 and achaete-scute complex in mouse and Drosophila, respectively, support an evolutionarily conserved regulatory process in neuronal subtype specification (Gaufo, 2004).

The present study also shows that the expression of homeobox protein Rnx (related to Drosophila C15), a known determinant of noradrenergic visceral sensory interneurons important for gustatory, cardiovascular and respiratory control, is also subject to Hox gene regulation. In contrast to Phox2b RNA, however, the expression of Rnx RNA appears to be completely eliminated in Hoxb1-/- embryos. The loss of Rnx RNA expression is consistent with the absence of dopamine ß-hydroxylase (Dbh) RNA. The presence of Phox2b RNA in Hoxb1-/- embryos, however, suggests that the identity of r4 is initially intact and therefore, the loss of noradrenergic visceral sensory interneurons is not solely due to a secondary effect resulting from changes in rhombomere identity. From these observations, Hox, Phox2b and Rnx genes may be placed in a hierarchical order to broadly define a regulatory cascade in the specification of noradrenergic visceral sensory interneurons within a hindbrain segment. Furthermore, the convergence of these genes on a common function is supported by central respiratory defects in mice with targeted mutations for Hoxa3 and Rnx and in humans with heterozygous mutations for PHOX2B. Altogether, these observations showing the segment-specific control of sensory structures and the convergence of genes on a common physiological function provides evidence for an evolutionary conserved pathway (Gaufo, 2004).

The Hoxa2 transcription factor acts during development of the second branchial arch. As for most of the developmental processes controlled by Hox proteins, the mechanism by which Hoxa2 regulates the morphology of second branchial arch derivatives is unclear. Six2, another transcription factor, is genetically downstream of Hoxa2. High levels of Six2 are observed in the Hoxa2 loss-of-function mutant. By using a transgenic approach to overexpress Six2 in the embryonic area controlled by Hoxa2, a phenotype is observed that is reminiscent of the Hoxa2 mutant phenotype. Furthermore, Hoxa2 regulation of Six2 is confined to a 0.9 kb fragment of the Six2 promoter and Hoxa2 binds to this promoter region. These results strongly suggest that Six2 is a direct target of Hoxa2 (Kutejova, 2005).

Little is known about the spatiotemporal requirement of Hox gene patterning activity in vertebrates. In Hoxa2 mouse mutants, the hyoid skeleton is replaced by a duplicated set of mandibular and middle ear structures. Hoxa2 is selectively required in cranial neural crest cells (NCCs). Moreover, a Cre-ERT2 recombinase system was used to induce a temporally controlled Hoxa2 deletion in the mouse. Hoxa2 inactivation after cranial NCC migration into branchial arches results in homeotic transformation of hyoid into mandibular arch skeletal derivatives, reproducing the conventional Hoxa2 knockout phenotype, and induces rapid changes in Alx4, Bapx1, Six2 and Msx1 expression patterns. Thus, hyoid NCCs retain a remarkable degree of plasticity even after their migration in the arch, and require Hoxa2 as an integral component of their morphogenetic program. Moreover, subpopulations of postmigratory NCCs required Hoxa2 at discrete time points to pattern distinct derivatives. This study provides the first temporal inactivation of a vertebrate Hox gene and illustrates Hox requirement during late morphogenetic processes (Santagati, 2005).

Rostral hindbrain patterning involves the direct activation of a Krox20 transcriptional enhancer by Hox/Pbx and Meis factors

The morphogenesis of the vertebrate hindbrain involves the generation of metameric units called rhombomeres (r), and Krox20 encodes a transcription factor that is expressed in r3 and r5 and plays a major role in this segmentation process. Knowledge of the basis of Krox20 regulation in r3 is rather confusing, especially concerning the involvement of Hox factors. This paper describes a study of one of the Krox20 hindbrain cis-regulatory sequences, element C, which is active in r3-r5 and which is the only initiator element in r3. Element C is shown to contains multiple binding sites for Meis and Hox/Pbx factors; these proteins synergize to activate the enhancer. Mutation of these binding sites showed that Krox20 is under the direct transcriptional control of both Meis (presumably Meis2) and Hox/Pbx factors in r3. Furthermore, the data indicate that element C functions according to multiple modes, in Meis-independent or -dependent manners and with different Hox proteins, in r3 and r5. Finally, it was shown that the Hoxb1 and Krox20 expression domains transiently overlap in prospective r3, and that Hoxb1 binds to element C in vivo, supporting a cell-autonomous involvement of Hox paralogous group 1 proteins in Krox20 regulation. Altogether, these data clarify the molecular mechanisms of an essential step in hindbrain patterning. A model is proposed for the complex regulation of Krox20, involving a novel mode of initiation, positive and negative controls by Hox proteins, and multiple direct and indirect autoregulatory loops (Wassef, 2008).

Krox20 regulation appears to constitute a complex process and this study attempted to amalgamate the observations collected in the present work with previous data to develop a molecular model. The consistent observations in mouse, chick and zebrafish allow combination of data obtained in different vertebrate species. First the regulation in r3 will be envisaged. It is proposed that, in contrast to what was previously thought, at around E8 in the mouse, when Hoxa1/Hoxb1 neural domains reach their maximal rostral extensions, their limits are located within prospective r3. This point is consistent with recent tracing data indicating that derivatives of Hoxa1-expressing cells are found in r3, and is supported by the observation of an overlap between Krox20 and Hoxb1 expression domains in r3. In addition, the existence of another factor (X, unknown) is postulated, whose expression domain extends caudally and will start to overlap with the Hox paralog group (PG) 1 domain around E8. This defines a transversal, narrow stripe of cells where Krox20 is specifically activated under the synergistic transcriptional activities of factor X, Hox PG 1, Pbx and Meis2 proteins, acting through element C. Interestingly, an essential role of Iroquois transcription factors in the activation of krox20 in r3 has been recently uncovered. Factor X might therefore be an Iroquois transcription factors or it might lie downstream to them in the regulatory cascade. A complementary involvement of Hox PG 2 proteins is also likely, although loss-of-function analyses suggest that the major role is played by PG 1 factors. An important feature of this hypothesis is that it provides an explanation for the characteristic initial expression pattern of Krox20, restricted to a very narrow stripe of cells. Krox20 activation will have multiple consequences. (1) It will lead to the progressive retraction of the rostral limit of Hox PG 1 gene expression to the future r3/r4 boundary. This is consistent with the observations that the Hoxb1-positive domain extends within prospective r3 in a Krox20-null mutant and that ectopic Krox20 expression results in Hoxb1 repression. (2) Krox20 initiates several transcriptional autoregulatory loops that are necessary for the maintenance of its own expression. One of them is direct and relies on the binding of Krox20 to element A, whereas the others involve the activation of Hoxa2 and Hoxb2, which will replace Hox PG 1 proteins on element C. These autoregulatory mechanisms are likely to be redundant, as the double mutation of Hoxa2 and Hoxb2 only marginally affects the r3 domain of Krox20 expression. (3) Expression of Krox20 also results in its activation in neighbouring Krox20-negative cells by non-cell autonomous autoregulation, a process thought to participate in the extension of r3. The caudal extension of r3 might also rely on the progression of the front of gene X expression. These processes will give rise to a moving stripe of cells co-expressing Krox20 and Hoxb1 at the caudal edge of developing r3, as was observed in mouse and zebrafish embryos. At some point (around E8.5), these processes of extension of r3 at the expense of adjacent rhombomeres will stop, delimiting the final extensions of r2, r3 and r4 (Wassef, 2008).

In r5, Krox20 is under the control of two initiation enhancer elements, B and C. The severe loss of Krox20 expression in r5 upon mutation of Mafb or vHnf1, and the fact that these factors are likely to act only via element B, suggests that element B is predominant. In r5, element C functions according to a different mode than in r3: although it still requires binding of a Hox protein, Meis factors are not necessary (Wassef, 2008).

Finally, what happens in r4, where element C is active but Krox20 is not expressed? To explain this apparent contradiction, it is proposed that Krox20, in addition to the positive regulatory mechanisms discussed above, is subject to a negative regulation, which may lie downstream of the Hox PG 1 genes and prevent Krox20 expression in r4. The existence of such a negative regulation is consistent with the inactivation of Hoxa1, which results in an extension of the anterior domain of Krox20 into prospective r4, and with the repressive activity of Nlz family members on Krox20 expression (Wassef, 2008).

In conclusion, a particularly interesting feature of this model resides in the initial phase of Krox20 expression in r3. It is proposed that a narrow band of cells is defined by the encounter of two domains extending in opposite directions. In these cells, Krox20 is very transiently activated by Hox PG 1 proteins, which disappear rapidly while Krox20 expression is maintained and propagated by different molecular mechanisms. It is proposed to use the term 'ignition' to refer to the role of Hox PG 1 proteins in this novel type of initiation of gene expression, which may occur in other developmental processes (Wassef, 2008).

Hox genes, Krox-20, Kreisler, and the segmentation of the rhombencephalon

Continued: proboscipedia Evolutionary homologs part 3/3 | back to part 1/3

proboscipedia: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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