proboscipedia

EVOLUTIONARY HOMOLOGS (part 3/3)

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

The hindbrain is a segmented structure divided into repeating metameric units termed rhombomeres (r). The Hox family, vertebrate homologs of the Drosophila HOM-C homeotic selector genes, are expressed in rhombomere-restricted patterns and are believed to participate in regulating segmental identities. Krox-20 (Drosophila homologs: Stripe, Huckebein and Klumpfuss), a zinc finger gene, has a highly conserved pattern of expression in r3 and r5 and is functionally required for their maintenance in mouse embryos. Krox-20 has been shown to directly regulate the Hoxb-2 gene (Drosophila homolog: Prosboscipedia) and is involved in regulating multiple Hox genes as a part of its functional role. Hoxa-2 is the only known paralog of Hoxb-2. The patterns of expression of the mouse Hoxa-2 gene were examined with particular focus on r3 and r5 in wild type and Krox-20-/- mutant embryos. There was a clear loss of expression in r3, which indicated that Hoxa-2 is downstream of Krox-20. An r3/r5 enhancer has been identified in the 5' flanking region of the Hoxa-2 gene. Mutation of these Krox-20 sites in the regulatory region specifically abolished r3/r5 activity, but did not affect neural crest and mesodermal components. This indicates that the two Krox-20 sites are required in vivo for enhancer function. Furthermore, ectopic expression of Krox-20 in r4 is able to transactivate Hoxa 2 in this rhombomere. Together these findings suggest that Krox-20 directly participates in the transcriptional regulation of Hoxa-2 during hindbrain segmentation, and is responsible for the upregulation of the r3 and r5 domains of expression of both vertebrate group 2 Hox paralogs. Therefore, the segmental phenotypes in the Krox-20 mutants are likely to reflect the role of Krox-20 in directly regulating multiple Hox genes (Nonchev, 1996).

The mouse and chicken Hoxb-2 genes are dependent for their expression in rhombdomere 3 and rhombdomere 5 on homologous enhancer elements and on the binding to this enhancer of the r3/r5-specific transcriptional activator Krox-20. Among the three Krox-20 binding sites of the mouse Hoxb-2 enhancer, only the high-affinity site is absolutely necessary for activity. In contrast, an additional cis-acting element, Box1, has been identified as essential for r3/r5 enhancer activity. It is conserved both in sequence and in position respective to the high-affinity Krox-20 binding site within the mouse and chicken enhancers. A short 44 bp sequence spanning the Box1 and Krox-20 sites can act as an r3/r5 enhancer when oligomerized. Box1 may therefore constitute a recognition sequence for another factor cooperating with Krox-20. Taken together, these data demonstrate the conservation of Hox gene regulation and of Krox-20 function during vertebrate evolution (Vesque, 1996).

The molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4) of the hindbrain has been examined by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments show that the vertebrate Labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and their Drosophila homolog labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrates that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with Labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments it is concluded that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene (Maconochie, 1997).

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).

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

It has been shown by using the quail/chick chimera system that Hox gene expression in the hindbrain is influenced by positional signals arising from the environment. To decipher the pathway that leads to Hox gene induction, an investigation was carried out to determine whether a Hox gene regulator, the leucine zipper transcription factor MafB/Kr, is itself transcriptionally regulated by the environmental signals. This gene is normally expressed in rhombomeres (r) 5 and 6 and their associated neural crest cells. MafB/Kr expression is maintained in r5/6 when grafted into the environment of r3/4. On the contrary, the environment of rhombomeres 7/8 represses MafB/Kr expression. Thus, as previously shown for the expression of Hox genes, MafB/Kr expression is regulated by a posterior-dominant signal, which in this case induces the loss of expression for this gene. The posterior signal can be transferred to the r5/6 neuroepithelium by posterior somites (somites 7 to 10) grafted laterally to r5/6. At the r4 level, the same somites induce MafB/Kr in r4, leading it to behave like r5/6. The posterior environment regulates MafB/Kr expression in the neural crest as it does in the corresponding hindbrain level, showing that some positional regulatory mechanisms are shared by neural tube and neural crest cells. Retinoic acid beads mimic the effect produced by the somites in repressing MafB/Kr in r5/6 and progressively induce it more rostrally as the retinoic acid concentration increases. It is therefore proposed that the MafB/Kr expression domain is defined by a molecule unevenly distributed in the paraxial mesoderm. This molecule would allow the expression of the MafB/Kr gene in a narrow concentration range by activating its expression at a definite threshold and repressing it at higher levels, accounting for its limited domain of expression in only two rhombomeres. It thus appears that the regulation of MafB/Kr expression in the rhombomeres could be controlled by the same posteriorizing factor(s) as Hox genes (Grapin-Botton, 1998).

A direct involvement of Kreisler in Hox gene regulation was recently attested to by Manzanares (1997), who found two sites in the Hoxb-3 promoter able to bind the Kreisler protein and to direct Hoxb-3 expression in r5 and r6 under certain conditions. Although no studies have been carried out yet with respect to a MafB/Kr function on other Hox gene promoters, modifications in the gene expression pattern in Kr/Kr mutant mice suggest that it might also act upstream to Hox genes of the fourth paralog group. In the Kreisler mutant mice, Hoxb-4 is extended rostrally to the anteroposterior level of r6 instead of r7; in Hoxd-4, the extension is to the level of r5. Hoxb-1, which is normally expressed in r4, r7 and r8 is extended to r5. These modifications can be interprpreted either as a loss of r5/6 cells or as a change in r5/6 cell specification. In the latter interpretation, MafB/Kr would downregulate Hoxb-4 and Hoxd-4 expression in r5/6 under normal conditions and the absence of Kreisler expression in Kr/Kr mutants would allow the anterior extension of paralog group four Hox genes. The transplantation experiments carried out in the current study are in agreement with this view, since the upregulation of paralog group four genes correlates with the inhibition of MafB/Kr expression. Timing considerations are also consistent with the hypothesis that absence of the MafB/Kr gene product in vivo might derepress the expression of paralog group four genes (Grapin-Botton, 1998 and references).

Experiments carried out mostly in Xenopus embryos have shown that several molecules are able to posteriorize the neural tube and, in particular, to induce Hox genes. Downstream molecules have this capacity, as well as some members of the FGF and Wnt families. It is shown here that under these conditions, bFGF is not able to regulate MafB/Kr expression. This does not exclude the possibility that FGFs may be involved in anteroposterior patterning at an earlier step or in more caudal regions. Nevertheless these studies clearly support the general concept of the gradient hypothesis in the regulation of gene expression along the anteroposterior axis in the hindbrain (Grapin-Botton, 1998).

Little is known about how the generation of specific neuronal types at stereotypic positions within the hindbrain is linked to Hox gene-mediated patterning. During neurogenesis, Hox paralog group 2 genes control both anteroposterior (A-P) and dorsoventral (D-V) patterning. Hoxa2 and Hoxb2 differentially regulate, in a rhombomere-specific manner, the expression of several genes in broad D-V-restricted domains or narrower longitudinal columns of neuronal progenitors, immature neurons, and differentiating neuronal subtypes. Moreover, Hoxa2 and Hoxb2 can functionally synergize to control the development of ventral neuronal subtypes in rhombomere 3 (r3). Thus, in addition to their roles in A-P patterning, Hoxa2 and Hoxb2 have distinct and restricted functions along the D-V axis during neurogenesis, providing insights into how neuronal fates are assigned at stereotypic positions within the hindbrain (Davenne, 1999).

Early in its development, the vertebrate hindbrain is transiently subdivided into a series of compartments called rhombomeres. Genes have been identified whose expression patterns distinguish these cellular compartments. Two of these genes, Hoxa1 and Hoxa2, have been shown to be required for proper patterning of the early mouse hindbrain and the associated neural crest. To determine the extent to which these two genes function together to pattern the hindbrain, mice simultaneously mutant at both loci were generated. The hindbrain patterning defects were analyzed in embryos individually mutant for Hoxa1 and Hoxa2 in greater detail and extended to embryos mutant for both genes. From these data a model is proposed to describe how Hoxa1, Hoxa2, Hoxb1, Krox20 (Egr2) and kreisler function together to pattern the early mouse hindbrain. Critical to the model is the demonstration that Hoxa1 activity is required to set the anterior limit of Hoxb1 expression at the presumptive r3/4 rhombomere boundary. Failure to express Hoxb1 to this boundary in Hoxa1 mutant embryos initiates a cascade of gene misexpressions that result in misspecification of the hindbrain compartments from r2 through r5. Subsequent to misspecification of the hindbrain compartments, ectopic induction of apoptosis appears to be used to regulate the aberrant size of the misspecified rhombomeres (Barrow, 2000).

Hoxa1 and Hoxb1 are coexpressed up to the presumptive r3/4 boundary. Hoxa1 is required to establish Hoxb1 expression in anterior r4. Hoxa1 and Hoxb1 activate the transcription of r4-specific downstream targets including a signal that, in turn, induces Krox 20 expression in cells just anterior to the r3/4 boundary (in cells that are not expressing Hoxa1 or Hoxb1). Krox 20 is repressed, however, in r4 and r5 cells that are expressing Hoxa1 and Hoxb1. Hoxa1 is required for kreisler expression in r5. Without Hoxa1, the anterior limit of Hoxb1 is established in the posterior region of r4. Because of this posterior shift, neither Hoxa1 nor Hoxb1 is expressed in the anterior portion of r4 and Krox 20 is no longer repressed there. Furthermore, the signal downstream of Hoxb1 must be propagated a longer distance causing a delay in the induction of Krox 20 expression in presumptive r3. Due to the absence of Hoxa1, kreisler expression is not activated in r5 (Barrow, 2000).

Without Hoxa1 and Hoxb1 expression, Krox 20 expression is no longer repressed in r4 and r5. In addition, the signal downstream from Hoxa1 and Hoxb1 required to induce Krox 20 expression in r3 is not activated. By E8.5, Hoxa1 expression has completely retreated from the hindbrain. Hoxb1 has also retreated with the exception of the strong autoregulatory expression in r4. Once Hoxa1 and Hoxb1 expression has fully retreated from r5, Krox 20 expression commences at this level. Krox 20 expression also expands into r3. This expansion requires activation of its downstream target(s) Hoxa2 and possibly Hoxb2. Strong kreisler expression in r5 maintains Hoxb1 autoregulated expression at the r4/5 boundary. In Hoxa1 mutants Hoxb1 expression retreats from the caudal hindbrain leaving autoregulated expression in caudal r4. Because kreisler is not activated in r5, autoregulated Hoxb1 expression extends into r5 as well. Krox 20 expansion into r3 although delayed (due to the fewer number of cells that were induced at E8.0) occurs somewhat normally due to the fact that Krox 20 and its downstream target(s) Hoxa2 (and perhaps Hoxb2) are functioning. As a consequence of the larger expression domains of follistatin (r2 and part of r3) and Krox 20 (part of r3 and r4), a regulatory event driven by apoptosis commences in these regions of the neural tube. The hindbrain is similar to that of Hoxa1 single mutants except that Krox 20 expansion into r3 is severely delayed. Hoxa2 is a downstream target of Krox 20 and if absent, cripples the expansion of Krox 20-expressing cells into r3. In double Hoxa1/Hoxa2 mutants Krox 20 is never induced in r3 and thus never expands into r3. As a result, follistatin expression extends to the r3/r4 boundary. Due to enlarged follistatin and Krox 20-expressing domains, apoptosis is activated in the neural tube at this level. Due to the apoptosis at the levels of r2 and r3 in HoxA1 mutants, there is not only a reduction in the number of neural crest cells that will populate the first arch, but also the abnormally large r3 is reduced to almost normal proportions. There is also a reduction in the number of neural crest cells that reach the second arch due to the reduced size of r4 and the fact that the otocyst may act as a barrier to prevent normal migration of the crest. The otocysts do not shift anteriorly to the level of r4; instead, r4 is specified more posteriorly. Double HoxA1/HoxA2 mutants are very similar to Hoxa1 single mutants except that, due to the lack of Hoxa2, the r4 neural crest takes on an r1/r2 identity. In addition, the lack of Hoxa1 causes a reduction in r4 neural crest contributing to the second arch. In HoxA1/HoxA2 double mutants r4 is never specified. Therefore, there is no r4 neural crest to populate the second arch (Barrow, 2000).

During hindbrain development, segmental regulation of the paralogous Hoxa2 and Hoxb2 genes in rhombomeres (r) 3 and 5 involves Krox20-dependent enhancers that have been conserved during the duplication of the vertebrate Hox clusters from a common ancestor. Examining these evolutionarily related control regions could provide important insight into the degree to which the basic Krox20-dependent mechanisms, cis-regulatory components, and their organization have been conserved. Toward this goal a detailed functional analysis has been performed of a mouse Hoxa2 enhancer capable of directing reporter expression in r3 and r5. The combined activities of five separate cis-regions, in addition to the conserved Krox20 binding sites, are involved in mediating enhancer function. A CTTT (BoxA) motif adjacent to the Krox20 binding sites is important for r3/r5 activity. The BoxA motif is similar to one (Box1) found in the Hoxb2 enhancer and indicates that the close proximity of these Box motifs to Krox20 sites is a common feature of Krox20 targets in vivo. Two other rhombomeric elements (RE1 and RE3) are essential for r3/r5 activity and share common TCT motifs, indicating that they interact with a similar cofactor(s). TCT motifs are also found in the Hoxb2 enhancer, suggesting that they may be another common feature of Krox20-dependent control regions. The two remaining Hoxa2 cis-elements, RE2 and RE4, are not conserved in the Hoxb2 enhancer and define differences in some of components that can contribute to the Krox20-dependent activities of these enhancers. Furthermore, analysis of regulatory activities of these enhancers in a Krox20 mutant background has uncovered differences in their degree of dependence upon Krox20 for segmental expression. Together, this work has revealed a surprising degree of complexity in the number of cis-elements and regulatory components that contribute to segmental expression mediated by Krox20 and sheds light on the diversity and evolution of Krox20 target sites and Hox regulatory elements in vertebrates (Maconochie, 2001).

The mechanisms involved in generating hindbrain motoneuron subtypes has been investigated, focusing on somatic motoneurons: these cells are confined to the caudal hindbrain within rhombomeres 5-8. Following heterotopic transplantation of rhombomeres along the rostrocaudal axis at various developmental stages, it has been found that the capacity of rhombomeres to generate somatic motoneurons is labile at the neural plate stage but becomes fixed just after neural tube closure, at stage 10-11. Grafting of somites or retinoic acid-loaded beads beneath the rostral hindbrain induces the formation of somatic motoneurons in rhombomere 4 only, and Hox genes normally expressed more caudally (Hoxa3, Hoxd4) were induced in this region. Targeted overexpression of Hoxa3 in the rostral hindbrain leads to the generation of ectopic somatic motoneurons in ventral rhombomeres 1-4, and is accompanied by the repression of the dorsoventral patterning gene Irx3. Taken together, these observations suggest that the somites, retinoic acid and Hox genes play a role in patterning somatic motoneurons in vivo (Guiato, 2003).

Hox genes are instrumental in assigning segmental identity in the developing hindbrain. Auto-, cross- and para-regulatory interactions help establish and maintain their expression. To understand to what extent such regulatory interactions shape neuronal patterning in the hindbrain, neurogenesis, neuronal differentiation and motoneuron migration were examined in Hoxa1, Hoxb1 and Hoxb2 mutant mice. This comparison revealsthat neurogenesis and differentiation of specific neuronal subpopulations in r4 are impaired in a similar fashion in all three mutants, but with different degrees of severity. In the Hoxb1 mutants, neurons derived from the presumptive r4 territory are re-specified towards an r2-like identity. Motoneurons derived from that territory resemble trigeminal motoneurons in both their migration patterns and the expression of molecular markers. Both migrating motoneurons and the resident territory undergo changes consistent with a switch from an r4 to r2 identity. Abnormally migrating motoneurons initially form ectopic nuclei that are subsequently cleared. Their survival can be prolonged through the introduction of a block in the apoptotic pathway. The Hoxa1 mutant phenotype is consistent with a partial misspecification of the presumptive r4 territory that results from partial Hoxb1 activation. The Hoxb2 mutant phenotype is a hypomorph of the Hoxb1 mutant phenotype, consistent with the overlapping roles of these genes in facial motoneuron specification. Therefore, the functional requirements in hindbrain neuronal patterning that follow the establishment of the genetic regulatory hierarchy between Hoxa1, Hoxb1 and Hoxb2 have been functionally delineated (Gavalas, 2003).

Rhombomeric and neuronal patterning defects are milder in Hoxb2 mutants, compared with Hoxb1^–/– embryos. Furthermore, there are no r4-derived phenotypes in Hoxb2 mutants that are not detected in Hoxb1 mutants. This is consistent with Hoxb2 being a direct transcriptional target of Hoxb1 and raises a number of possibilities concerning the precise regulatory and functional relationships between Hoxb1 and Hoxb2. Hoxb2 may act synergistically with Hoxb1 by regulating either distinct target genes or a set of common target genes in r4, so that their combined activities are required for the normal differentiation of r4-derived motoneurons. An alternative mechanism whereby Hoxb2 may synergise with Hoxb1 would be through a role for Hoxb2 in maintaining Hoxb1 expression (Gavalas, 2003).

To begin to distinguish between these possibilities, the r4 status was monitored in the Hoxb2 mutants by assaying Hoxb1 expression and the expression of a the r4-specific transgene (HL5/lacz), which is known to be a direct target of Hoxb1. Endogenous Hoxb1 expression and staining for the HL5/lacZ transgene are initiated in the r4 of Hoxb2 mutants but are not maintained at appropriate levels in later stages. This demonstrates a direct or indirect requirement for Hoxb2 in maintaining Hoxb1 expression in r4. The observation that Hoxb1 expression is initiated normally in Hoxb2 mutants, but is not maintained properly could explain the mixed behavior of facial motoneurons. Those r4 motoneuron progenitors that retain sufficient Hoxb1 activity adopt a normal fbm identity, while the rest adopt trigeminal motoneuron characteristics. The idea is favored that the effect of Hoxb2 on Hoxb1 expression is most probably indirect, through regulation of general aspects of r4 identity. Hoxb2 cannot bind the Hoxb1 r4 regulatory element in vitro, although it is possible that Hoxb2 may bind to an as yet unidentified Hoxb1 r4 regulatory element. In vivo, ectopic expression of Hoxb2 does not ectopically activate Hoxb1, whereas Hoxb1 does transactivate Hoxb2 (Gavalas, 2003).

Hox2 genes are required for tonotopic map precision and sound discrimination in the mouse auditory brainstem

Tonotopy is a hallmark of auditory pathways and provides the basis for sound discrimination. Little is known about the involvement of transcription factors in brainstem cochlear neurons orchestrating the tonotopic precision of pre-synaptic input. This study found that in the absence of Hoxa2 and Hoxb2 (see Drosophila Proboscipedia) function in Atoh1-derived glutamatergic bushy cells of the anterior ventral cochlear nucleus (see Drosophila Atonal), broad input topography and sound transmission were largely preserved. However, fine-scale synaptic refinement and sharpening of isofrequency bands of cochlear neuron activation upon pure tone stimulation were impaired in Hox2 mutants, resulting in defective sound-frequency discrimination in behavioral tests. These results establish a role for Hox factors in tonotopic refinement of connectivity and in ensuring the precision of sound transmission in the mammalian auditory circuit (Karmakar, 2017).

Proboscipedia homologs interact with a vertebrate Polycomb system

To assess its function during development, M33, the murine counterpart of Drosophila Polycomb was targeted by means of homologous recombination in embryonic stem (ES) cells. Homozygous M33 mutant mice show greatly retarded growth, homeotic transformations of the axial skeleton, sternal and limb malformations and a failure of several cell types to expand in vitro of several cell types including lymphocytes and fibroblasts. Mutant mice show a posteriorisation of the thoracic vertebra T7 into T8, resulting in the presence of six vertebrosternal ribs instead of seven in nonmutated mice. In addition, 15% of the mutant mice show a transformation of the lumbar vetebra L6 into a first sacral vertebra. Hoxa-3, a homolog of Drosophila proboscipedia, is detected over the basioccipital bone anlage in mutant mice but not in wild-type mice. This anterior shift in the Hoxa-3 boundary is made wider by the fusion of the basioccipital and the first prevertebra. Other Hox genes show a similar anterior transformation. M33 null mutant mice show an aggravation of the skeletal malformations when treated with retinoic acid at embryonic day 7.5, leading to the hypothesis that during development, the M33 gene might play a role in defining access to retinoic acid response elements localised in the regulatory regions of several Hox genes, For example retinoic acid response genes have been found in Hoxa-1, Hoxb-1 (both labial homologs) and Hoxd-4 (a Deformed homolog) (Core, 1997).

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

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