extradenticle in other insects

Studies of the genes involved in patterning the appendages of Drosophila melanogaster have revealed a system of signaling and transcriptional regulation that is responsible for specifying the proximo-distal limb axis. The expression patterns of presumptive homologs of the Drosophila genes extradenticle, dachshund, nubbin, ventral veins lacking, and Dll in the limbs of the woodlouse Porcellio scaber and the spider Steatoda triangulosa is reported. Although the expression domains of the appendage genes roughly correspond to those of Drosophila, their relative positions and segmental affiliation are distinct. In addition, the expression patterns of the appendage genes allows a resolution of the segmental composition of different appendages within crustacean and spider embryos. It is concluded that certain limb types, e.g., mouthparts, appear to be derived from a leg-like ground-plan via the elimination/fusion of the intermediate and distal podomeres. Moreover, just such a modification is observed during the transformation of the anterior legs into mouthparts in P. scaber. Although these data do not unequivocally resolve the question of homology of the arthropod leg segments, they do provide evidence for a single conserved proximo-distal patterning system in the development of noninsect arthropod limbs (Abzhanov, 2000).

Most of the knowledge about appendage development comes from developmental genetic studies of D. melanogaster. Although the legs of adult flies and crickets are very similar in terms of metameric origin and segment number, the developmental processes by which their morphology is achieved are quite distinct. Cricket legs develop directly from the body wall and are patterned and segmented during subsequent outward growth in the embryo. Drosophila has a more derived system for adult appendage formation involving the imaginal discs, which are specified and patterned during later larval stages. The development of an appendage, such as a leg, from an imaginal disc is a multistep process involving initiation of the disc itself via signaling from neighbor tissues, prepatterning, establishment of limb field primordia, dorsal-ventral patterning, proximo-distal patterning culminating in growth, and evagination and differentiation of adult structures. It is possible that much of leg development in Drosophila is characteristic only of the higher Diptera and cannot be compared directly to that of other arthropods. Therefore, a relatively basal insect, the cricket A. domesticus (Orthoptera), which has a direct limb development, has been included in this comparative analysis. The morphology of cricket limbs, such as antennae, mouthparts, and legs, is more typical of the lower insects. The expression patterns and dynamics of the leg patterning genes, with regard to the specific podomeres, are conserved between Drosophila and Acheta. Nuclear Exd is detected in the basal part (coxa and trochanter in the legs) of the appendages in Drosophila and Acheta. In both species, the early Exd domain is immediately adjacent to that of Dll. In Acheta, Dll is detected throughout the telopodite during early development but then fades in the intermediate portion of the leg and forms two separate domains: proximally in the trochanter/proximal femur and distally in the tarsus/distal half of tibia. In Drosophila, this change in Dll pattern is associated with the growth of the imaginal disc and is accompanied by initiation and establishment of the Dac domain in the intermediate portion of the leg. It is notable that the dynamics of the Exd and Dll expression patterns in the maxillary and labial palps are very similar to those in the walking legs in Acheta. This suggests that the segmented maxillary and labial mouthparts, which belong to the basal mandibulate type, are patterned very similarly to the legs, in striking contrast to the adult proboscis in Drosophila. Another observation in Acheta is the restriction of Exd, which is required for antennal identity in Drosophila, to the two most basal antennal segments, which are sometimes homologized with the coxa and trochanter. This observation indirectly supports the hypothesis that the insect antenna is basically a three-segmented structure with the third, distal segment subdivided into subsections, forming a flagellum. However, little or no Dll and Exd coexpression is observed despite the fact that both genes have been shown to be required for the development of antennal structures in D. melanogaster. Thus it would appear that the cricket may pattern its antenna differently from flies (Abzhanov, 2000).

The developing leg of Drosophila is initially patterned by subdivision of the leg into proximal and distal domains by the activity of the homeodomain proteins Extradenticle (Exd) and Distal-less (Dll). These early domains of gene expression are postulated to reflect a scenario of limb evolution in which an undifferentiated appendage outgrowth was subdivided into two functional parts: the coxapodite and telopodite. The legs of most arthropods have a more complex morphology than the simple rod-shaped leg of Drosophila. The expression of Dll and Exd is documented in two crustacean species with complex branched limbs. In these highly modified limbs there is a Dll domain exclusive of Exd but there is also extensive overlap in Exd and Dll expression. While arthropod limbs all appear to have distinct proximal and distal domains, those domains do not define homologous structures throughout arthropods. In addition, a striking correlation is found throughout the proximal/distal extent of the leg between setal-forming cells and Dll expression. It is postulated that this may reflect a pleisiomorphic (ancestral) function of Dll in development of the peripheral nervous system. In addition, the results confirm previous observations that branch formation in multiramous arthropod limbs is not regulated by a simple iteration of the proximal/distal patterning module employed in Drosophila limb development (Williams, 2002).

Do data support genetically defined P/D domains? A qualified 'yes' is given to this question. The timing and position of coexpression of Exd and Dll behave as predicted and correspond with the Drosophila leg. Dll is restricted to what will presumably become the most distal parts of the limb, Exd is downregulated in that region, and Exd is expressed proximally. However, this pattern is found in only the earliest limb bud and is soon obscured by subsequent development, in part by the extensive overlap of Exd and Dll expression domains. It is therefore postulated that the initial Dll expression involved in P/D outgrowth is parallel to its well described function in P/D outgrowth in the Drosophila disc and may reflect a generic, genetically-defined P/D domain found in all arthropod limbs. This conservation is intriguing if the proximal and distal limb have not only distinct regulatory pathways but also distinct evolutionary histories. Numerous theories of limb evolution based on adult comparative morphology invoke lability between body and proximal limb structures, i.e., that there is not a fixed and inviolable boundary between the two. Theories like the evolution of wings via the recruitment of a proximal limb branch to the dorsal body wall or the origins of a biramous limb depend explicitly on such variability. Despite this conservation of generic, exclusive P/D domains for Exd and Dll in early limb buds, the combined analysis of these two genes in multiple species suggests they are best viewed as functional domains which in no way map onto specific structures of adult morphology. In general, calling the domain of Exd expression the 'coxapodite' and the domain of Dll expression the 'telopodite' in distantly related taxa implies an unwarranted extrapolation. Both Dll and Exd expression vary greatly depending on leg morphology, stage of development, and species. Although the Exd/Dll expression boundaries do not define homologous structures across taxa, it seems clear that they do define some kind of proximal and distal limb region. Therefore, instead of expecting a direct mapping onto adult limb structure, it is likely that early, exclusive Exd and Dll expression domains are used as developmental patterning tools. As with the gap gene expression domains in the early embryo, they set up developmental domains without a one-to-one relation to adult morphological structures (Williams, 2002).

Leg development in Drosophila has been studied in much detail. However, Drosophila limbs form in the larva as imaginal discs and not during embryogenesis as in most other arthropods. Appendage genes have been analyzed in the spider Cupiennius salei and the beetle Tribolium castaneum. Differences in decapentaplegic expression suggest a different mode of distal morphogen signaling suitable for the specific geometry of growing limb buds. Also, expression of the proximal genes homothorax and extradenticle (exd) is significantly altered: in the spider, exd is restricted to the proximal leg and hth expression extends distally, while in insects, exd is expressed in the entire leg and hth is restricted to proximal parts. This reversal of spatial specificity demonstrates an evolutionary shift, which is nevertheless compatible with a conserved role for this gene pair as instructor of proximal fate. Different expression dynamics of dachshund and Distal-less point to modifications in the regulation of the leg gap gene system. The significance of this finding is discussed in terms of attempts to homologize leg segments in different arthropod classes. Comparison of the expression profiles of H15 and optomotor-blind to the Drosophila patterns suggests modifications also in the dorsal-ventral patterning system of the legs. Together, these results suggest alterations in many components of the leg developmental system, namely proximal-distal and dorsal-ventral patterning, and leg segmentation. Thus, the leg developmental system exhibits a propensity to evolutionary change, which probably forms the basis for the impressive diversity of arthropod leg morphologies (Prpic, 2003).

Hox genes require homothorax and extradenticle for body wall identity specification but not for appendage identity specification during metamorphosis of Tribolium castaneum

The establishment of segment identity is a key developmental process that allows for divergence along the anteroposterior body axis in arthropods. In Drosophila, the identity of a segment is determined by the complement of Hox genes it expresses. In many contexts, Hox transcription factors require the protein products of extradenticle (exd) and homothorax (hth) as cofactors to perform their identity specification functions. In holometabolous insects, segment identity may be specified twice, during embryogenesis and metamorphosis. To glean insight into the relationship between embryonic and metamorphic segmental identity specification, these processes were compared in the flour beetle Tribolium castaneum, which develop ventral appendages during embryogenesis that later metamorphose into adult appendages with distinct morphologies. At metamorphosis, comparisons of RNAi phenotypes indicate that Hox genes function jointly with Tc-hth and Tc-exd to specify several region-specific aspects of the adult body wall. In contrast, Hox genes specify appendage identities along the anteroposterior axis independently of Tc-hth/Tc-exd and Tc-hth/Tc-exd specify proximal vs. distal identity within appendages independently of Hox genes during this stage. During embryogenesis, Tc-hth and Tc-exd play a broad role in the segmentation process and are required for specification of body wall identities in the thorax; however, contrasting with results from other species, no homeotic transformations of embryonic appendages were obtained in response to Tc-hth or Tc-exd RNAi. In general, the homeotic effects of interference with the function of Hox genes and Tc-hth/Tc-exd during metamorphosis did not match predictions based on embryonic roles of these genes. Comparing metamorphic patterning in T. castaneum to embryonic and post-embryonic development in hemimetabolous insects suggests that holometabolous metamorphosis combines patterning processes of both late embryogenesis and metamorphosis of the hemimetabolous life cycle (Smith, 2014).

extradenticle homologs in other invertebrates

Co-factor homeodomain proteins such as Drosophila Homothorax (Hth) and Extradenticle (Exd) and their respective vertebrate homologs, the Meis/Prep and Pbx proteins, can increase the DNA-binding specificity of Hox protein transcription factors and appear to be required for many of their developmental functions. unc-62 gene encodes the C. elegans ortholog of Hth, and maternal-effect unc-62 mutations can cause severe posterior disorganization during embryogenesis (Nob phenotype), superficially similar to that seen in embryos lacking function of either the two posterior-group Hox genes nob-1 and php-3 or the caudal homolog pal-1. Other zygotically acting unc-62 alleles cause earlier embryonic arrest or incompletely penetrant larval lethality with variable morphogenetic defects among the survivors, suggesting that unc-62 functions are required at several stages of development. The differential accumulation of four unc-62 transcripts is consistent with multiple functions. The C. elegans exd homologs ceh-20 and ceh-40 interact genetically with unc-62 and may have overlapping roles in embryogenesis: neither CEH-20 nor CEH-40 appears to be required when the other is present, but loss of both functions causes incompletely penetrant embryonic lethality in the presence of unc-62(+) and complete embryonic lethality in the presence of an unc-62 hypomorphic allele (Van Auken, 2002).

In Drosophila, similar phenotypes result from loss of function mutations in the Meis-family homolog hth and the PBC-family homolog exd, consistent with other evidence for strong interactions between Meis- and PBC-family proteins. Although inactivation of the only previously studied C. elegans exd homolog, ceh-20, causes only larval and adult defects and no embryonic lethality, this result may be at least partially due to overlapping functions of two C. elegans PBC-family genes, ceh-20 and ceh-40. These two genes interact genetically: ceh-40(RNAi) causes no significant embryonic lethality, while ceh-20(RNAi); ceh-40(RNAi) causes a substantial decrease in larval lethality with a corresponding increase in embryonic lethality compared with ceh-20(RNAi) alone. In addition, whereas unc-62(RNAi) embryos arrest with the Nob phenotype, ceh-20(RNAi); ceh-40(RNAi) embryos arrest earlier, prior to morphogenesis. Recent phylogenetic comparisons with similar genes in other nematodes support the view that ceh-40 may be a C. elegans exd ortholog. The RNAi results suggest that ceh-20 and ceh-40 functions may overlap, with ceh-40 normally functioning earlier than ceh-20. The incomplete penetrance of the ceh-20(RNAi); ceh-40(RNAi) lethality compared with the complete penetrance of unc-62(s472) or unc-62(RNAi) lethality could indicate either that silencing by RNAi was incomplete, or that the functions of ceh-20 and ceh-40 are not essential for unc-62 function since exd is essential for hth function in Drosophila (Van Auken, 2002).

In the background of the hypomorphic unc-62(e644) allele, which is predicted to reduce or eliminate production of a functional protein from 7b-containing transcripts (one of the four splice varients), ceh-20 and ceh-40 show a pattern of interaction with each other similar to that described above. Individually, their RNAi phenotypes are roughly additive to the unc-62(e644) phenotype. However, the triple combination of ceh-20(RNAi); unc-62(e644); ceh-40(RNAi) results in complete embryonic inviability, with terminal phenotypes similar to those caused by the null allele unc-62(s472). One interpretation of these results is that when levels of the putatively interacting UNC-62 and CEH-20/CEH-40 proteins both fall below some threshold level, the result is a strong Unc-62 phenotype. A more intriguing possibility is that there is some overlap in the functions of CEH-20/CEH-40 and the UNC-62 proteins encoded by 7b-containing transcripts. Perhaps, for example, the latter could act directly as a Hox transcription cofactor (Van Auken, 2002).

There are several differences between C. elegans and Drosophila that may somehow be related to the finding that phenotypes caused by unc-62, ceh-20 and ceh-40 mutations are not as expected from observations on the homologous Drosophila genes. Whereas in Drosophila only one PBC-family and one MEIS-family gene have been reported, C. elegans has at least two PBC-family genes. Furthermore, the unc-62 gene generates four different transcripts by alternative splicing, while Drosophila hth transcripts are not alternatively spliced. Therefore, C. elegans, with several PBC-family and Meis-family proteins, resembles vertebrates more closely than Drosophila with regard to the repertoire and possible interactions of these cofactors. A second possibly relevant difference between C. elegans and Drosophila could be that C. elegans requires only anterior-group and posterior-group Hox gene functions for embryonic survival. Since PBC- and Meis-family proteins can act as cofactors for Hox proteins, the fact that medial-group Hox proteins are essential for completion of embryogenesis in Drosophila but not C. elegans might account for different phenotypes resulting from loss of co-factor functions (Van Auken, 2002).

C. elegans contains a set of six cluster-type homeobox (Hox) genes that are required during larval development. Some of them, (but unlike in flies, not all of them) are also required during embryogenesis. It has been suggested that the control of the embryonic expression of the worm Hox genes might differ from that of other species by being regulated in a lineal rather than a regional mode. Here, a trans-species analysis of the cis-regulatory region of ceh-13, the worm ortholog of the Drosophila labial and the vertebrate Hox1 genes has been performed; the molecular mechanisms that regulate its expression may be similar to what has been found in species that follow a regulative, non-cell-autonomous mode of development. Two enhancer fragments have been identified that are involved in different aspects of the embryonic ceh-13 expression pattern. Important features of comma-stage expression depend on an autoregulatory input that requires ceh-13 and ceh-20 functions. The data show that the molecular nature of Hox1 class gene autoregulation has been conserved among worms, flies, and vertebrates. The second regulatory sequence is sufficient to drive correct early embryonic expression of ceh-13. Interestingly, this enhancer fragment acts as a response element of the Wnt/WG signaling pathway in Drosophila embryos (Streit, 2002).

pMF1. containing 8.1 kb of upstream sequences as well as the first exon, the first intron, and most of the second exon, mimics endogenous ceh-13 expression in transgenic animals. Comparative sequence analysis of pMF1 led to the identification of a 10-bp-long sequence motif (TGATGGATGG) in enh450 (starting at nucleotide position 26483 relative to the ATG start codon of ceh-13) that is identical to that of the HOXB1/PBX autoregulatory element of the mouse gene Hoxb1 and differs by only one base pair substitution from that of the LAB/EXD autoregulatory element of the Drosophila gene labial. In order to assess the importance of the 10-bp element and to test whether this sequence represents an autoregulatory element similar to the one described for the mouse and the fly, a mutational analysis was performed in the context of the enh450 (a fragment able to drive most aspects of the normal ceh-13 in the expression pattern of comma-stage embryos). In Drosophila and in mice, it has been shown that the activity of the element is sensitive to point mutations in the core binding sites of the LAB(HOXB1) and EXD(PBX) proteins. In analogy to these findings, two separate 1-bp substitutions were intoduced in the 10-bp worm element that were expected to disrupt the potential binding sites for CEH-13 and for a putative worm ortholog of EXD/PBX. In the double-mutant construct mutE/L, the point mutations in the two half sites of the element were combined. The results of this single point mutation analysis confirmed that the 10-bp sequence element is absolutely required for the function of enh450 and strongly suggest that it functions as a direct autoregulatory element of ceh-13 (Streit, 2002).

Anteroposterior cell migration and patterning in C. elegans are governed by multiple, interacting signaling pathways and transcription factors. In this study, the roles of ceh-20, the C. elegans ortholog of the HOX co-factor Extradenticle (Exd/Pbx), and unc-62, the C. elegans ortholog of Homothorax (Hth/Meis/Prep), were investigated in two processes that are regulated by Hox gene lin-39: cell migration and vulva formation. As in lin-39 mutants, the anterior migrations of neuroblasts in the Q lineage are truncated in Hox co-factor mutants. Surprisingly, though, the findings suggested that the roles of ceh-20 and unc-62 are different from that of lin-39; specifically, ceh-20 and unc-62 but not lin-39 are required for the transmembrane protein MIG-13 to promote anterior migration. ceh-20 and unc-62 are the only genes that have been implicated in the mig-13 pathway. ceh-20 and unc-62 are also required for several steps in vulva development. Surprisingly, ceh-20 and unc-62 mutants have phenotypes that are starkly different from those of lin-39 mutants. Thus, in this process, too, ceh-20 and unc-62 are likely to have functions that are independent of lin-39 (Yang, 2005).

Do ceh-20 and unc-62 function in similar ways as their homologs? First, previous studies in Drosophila and vertebrates have demonstrated that the phenotypes produced by mutations in the homologs of ceh-20 and unc-62 resemble one another. For example, in Drosophila, exd and hth mutants both exhibit posterior transformations of embryonic segments. In zebrafish, mutations in either lazarus or meis (the exd and hth homologs, respectively) cause defects in hindbrain segmentation. This property of these genes is conserved: mutations in C. elegans ceh-20 and unc-62 both disrupt, in similar ways, the migration of Q descendants, Pn.p fusion, vulval morphogenesis and the patterning of V cell descendants (Yang, 2005).

Second, in many situations, mutations in Hox co-factor genes produce phenotypes that mimic those of Hox mutants. In fact, Drosophila exd and hth were identified based on their sharing embryonic segmentation defects with Hox mutants. In C. elegans, although there are some similarities between the Q migration defects of unc-62, ceh-20 and Hox gene mutants, the details of their phenotypes are very different. The same holds true for vulva development. Thus, ceh-20 and unc-62 may have functions independent of lin-39 in anteriorwards migration and vulva development. In Drosophila, Hox-independent functions of exd and hth have been demonstrated for antennal formation. However, in contrast to the C. elegans situation in which lin-39 is required and expressed in the cells with defects in migration, fusion or division, Drosophila Hox genes are neither required nor expressed in the eye-antennal disc from which the antenna forms (Yang, 2005).

Third, Hox gene expression is regulated, in part, by Hox co-factors. In Drosophila, exd and hth are required to maintain Sex-combs reduced expression in the salivary gland. In zebrafish, Hox genes interact with their co-factors to cross-regulate the expression of other Hox genes. As a result, reducing the function or altering the intracellular localization of zebrafish lazarus or meis/prep genes results in decreased expression of various Hox genes. In C. elegans, although ceh-20 does not activate mab-5 or lin-39 expression in the mesoderm, it is required for the autoregulatory expression of ceh-13, the C. elegans ortholog of Drosophila Hox gene labial. lin-39 expression is upregulated, instead of downregulated, in the Q cells of some animals with reduced ceh-20 or unc-62 function. Although this is somewhat unexpected, it is possible that ceh-20 and unc-62 downregulate Hox gene expression in conjunction with non-HOX proteins downstream of signaling pathways. Although the wingless and the TGFß pathways have not been shown to inhibit Hox gene expression, they do influence Hox gene expression in C. elegans. Because lin-39 upregulation is not restricted to the Q cells in ceh-20 or unc-62 mutants, the possibility is raised that ceh-20 and unc-62 could function with repressors of lin-39 expression such as SAM domain proteins (Yang, 2005).

Taken together, these comparisons suggest that C. elegans ceh-20 and unc-62 share many properties with their homologs, but the degree to which the Hox-independent and Hox-dependent roles of these genes and their homologs dominate may differ in different organisms and even between different tissues within the same organism (Yang, 2005).

The Notch signaling pathway controls growth, differentiation and patterning in divergent animal phyla; in humans, defective Notch signaling has been implicated in cancer, stroke and neurodegenerative disorders. Despite its developmental and medical significance, little is known about the factors that render cells to become competent for Notch signaling. This study shows that during vulval development in the nematode C. elegans the HOX protein LIN-39 and its EXD/PBX-like cofactor CEH-20 are required for LIN-12/Notch-mediated lateral signaling that specifies the 2° vulval cell fate. Inactivation of either lin-39 or ceh-20 resulted in the misspecification of 2° vulval cells and suppresses the multivulva phenotype of lin-12(n137) gain-of-function mutant animals. Furthermore, both LIN-39 and CEH-20 are required for the expression of basal levels of the genes encoding the LIN-12/Notch receptor and one of its ligands in the vulval precursor cells, LAG-2/Delta/Serrate, rendering them competent for the subsequent lin-12/Notch induction events. These results suggest that the transcription factors LIN-39 and CEH-20, which function at the bottom of the RTK/Ras and Wnt pathways in vulval induction, serve as major integration sites in coordinating and transmitting signals to the LIN-12/Notch cascade to regulate vulval cell fates (Takács-Vellai, 2007).

Convergent intercellular signals must be precisely coordinated in order to elicit specific biological responses. The C. elegans vulva provides an excellent experimental microcosm for studying how cell fate is specified according to the combined effects of different signaling pathways. This paper has studied the role of the Hox gene lin-39 and the Exd ortholog ceh-20 in vulval development. Genetic and molecular evidence is presented that the HOX protein LIN-39 and its putative cofactor CEH-20 are required for basal expression levels of lin-12 and lag-2 in the VPCs prior to vulval induction; this regulation may be important to render the VPCs competent for the subsequent lin-12/Notch induction events at the L3 larval stage. Identifying transcriptional regulators of lateral signaling in C. elegans vulval development will be essential for understanding how the Notch signaling pathway specifies cell fate in divergent animal species, and how compromised Notch signaling leads to human diseases (Takács-Vellai, 2007).

LIN-39 and CEH-20 are both required at the first larval stage to prevent fusion of the VPCs to the surrounding hypodermis. The data lead to the attractive possibility that LIN-39 and its putative cofactor CEH-20 regulate the competence of the VPCs to respond to any of the patterning signals during vulval formation. Along this line, it is challenging to speculate that, besides regulating lin-12 and lag-2 expression, they might also promote the expression of components of the inductive pathway (such as let-23) or other Notch pathway genes in the VPCs (Takács-Vellai, 2007).

It has been shown that CEH-20 binds in vitro, together with LIN-39, to the promoter of the twist transcription factor ortholog hlh-8 to regulate its expression in postembryonic mesodermal cells. ChIP experiments demonstrate that LIN-39 associates with the lag-2promoter, suggesting that the regulation of lag-2 expression by LIN-39 may be direct. It is proposed that LIN-39 forms a heterodimer with CEH-20 to promote the basal transcription of lag-2 and lin-12 in the VPCs. Based on their different expression pattern in the Pn.p lineages, ceh-20 is assumed to have some functions that are independent of lin-39. Indeed, mab-5 has been shown to be expressed in the descendants of the posterior VPCs, P7.p and P8.p, and to prevent them from adopting an induced vulval fate. Thus, it is possible that CEH-20 also interacts and functions with MAB-5 in controlling certain aspects of vulval fate specification. Furthermore, it is noted that ceh-20(ay9) mutant animals sometimes displayed a dual AC phenotype, whereas lin-39 mutants never did. RNAi-mediated depletion of mab-5 sometimes resulted in 2 ACs, suggesting that the correct AC specification requires the combined activity of mab-5 and ceh-20 (Takács-Vellai, 2007).

Finally, CEH-20 has been shown to be required as a cofactor for autoregulatory expression of the anterior Hox paralog (labial-like) ceh-13 in embryonic cells. Because ceh-13 is expressed all along the anteroposterior body axis in the ventral mid-line during the L1–L4 larval stages and a few percent of the ceh-13(sw1) mutant animals that are able to develop into fertile adults exhibit various defects in vulval formation, it is possible that CEH-13 acts with CEH-20 to control cell fate in the anterior VPC lineages. The future analysis of a potential role of ceh-13 in vulval development would help to establish the role of all of the major body Hox genes in this important process (Takács-Vellai, 2007).

Interaction of Extradenticle homologs and homothorax homologs

The Pbx1 and Meis1 (Drosophila homolog: homothorax) proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively. These oncogenes implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate Hox protein DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. It is concluded that Meis proteins are the preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1 (Chang, 1997).

E2a-Pbx1 is a chimeric transcription factor oncoprotein produced by the t(1;19) translocation found in human pre-B cell leukemia. Class I Hox proteins bind DNA cooperatively with both Pbx proteins and oncoprotein E2a-Pbx1, suggesting that leukemogenesis by E2a-Pbx1 and Hox proteins may alter transcription of cellular genes regulated by Pbx-Hox motifs. Likewise, in murine myeloid leukemia, transcriptional coactivation of Meis1 with HoxA7/A9 suggests that Meis1-HoxA7/9 heterodimers may evoke aberrant gene transcription. Both Meis1 and its relative, pKnox1, dimerize with Pbx1 on the same TGATTGAC motif selected by dimers of Pbx proteins and unidentified partner(s) in nuclear extracts, including those from t(1;19) pre-B cells. Outside their homeodomains, Meis1 and pKnox1 are highly conserved in only two motifs required for cooperativity with Pbx1. Like the unidentified endogenous partner(s), both Meis1 and pKnox1 fail to dimerize significantly with E2a-Pbx1. The Meis1/pKnox1-interaction domain in Pbx1 resides predominantly in a conserved N-terminal Pbx domain deleted in E2a-Pbx1. Thus, the leukemic potential of E2a-Pbx1 may require abrogation of its interaction with members of the Meis and pKnox families of transcription factors, permitting selective targeting of genes regulated by Pbx-Hox complexes. Because most motifs bound by Pbx-Meis1/pKnox1 are not bound by Pbx1-Hox complexes, the leukemic potential of Meis1 in myeloid leukemias may involve shifting Pbx proteins from promoters containing Pbx-Hox motifs to those containing Pbx-Meis motifs (Knoepfler, 1997b).

The human transcription factor, UEF3, is important in regulating the activity of the urokinase plasminogen activator (uPA) gene enhancer. The UEF3 DNA target site is a regulatory element in the promoters of several growth factor and protease genes. Purified UEF3 is a complex of several subunits. One of the subunits encodes a novel human homeodomain protein, which has been termed Prep1. The Prep1 homeodomain belongs to the TALE class of homeodomains; is most closely related to those of the TGIF and Meis1 proteins, and like these, recognizes a TGACAG motif. The other UEF3 subunit has been identified as a member of the Pbx protein family. Unlike other proteins known to interact with Pbx, Prep1 forms a stable complex with Pbx independent of DNA binding. Heterodimerization of Prep1 and Pbx results in a strong DNA binding affinity toward the TGACAG target site of the uPA promoter. Overall, these data indicate that Prep1 is a stable intracellular partner of Pbx in vivo (Berthelsen, 1998a).

The products of the mammalian Pbx and Drosophila extradenticle genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. Exist as stable heterodimers with a novel homeodomain protein, Prep1. The highest homeodomain homology of Prep1 is found with members of human, murine and Xenopus Meis proto-oncogene family, and with human TGIF. In addition, Prep1 contains two short regions, HR1 and HR2, which display strong homology with all members of the Meis protein family . Prep1-Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N-terminal portions of both proteins. The Prep1-Pbx protein-protein interaction is essential for DNA-binding activity. Prep1-Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, the formation of a ternary Prep1-Pbx1-HOXB1 complex on a HOXB1-responsive target is observed in vitro. Interaction with Prep1 enhances the ability of the HOXB1-Pbx1 complex to activate transcription in a cooperative fashion from the same target. These data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins (Berthelsen, 1998b).

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, which mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which is required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. These data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element (Jacobs, 1999).

The observation that Meis contributes to the DNA binding requirements of ternary complexes differs from previous observations and raises the possibility that its contributions may vary with different enhancers or under different cellular conditions. The most compelling evidence that DNA binding by a Meis-related protein is required for the in vivo function of ternary complexes is provided by the analysis of the requirements for function of the Hoxb2 r4 enhancer in rhombomere 4 of the developing hindbrain. Elegant genetic studies have demonstrated that this enhancer directs the appropriate expression of the Hoxb2 gene in response to Hoxb1 cross-regulation in rhombomere 4 at approximately 8.5 to 10 days of hindbrain development. Extensive mapping has shown that a consensus Pbx-Hox site is essential for r4 enhancer-mediated expression in rhombomere 4, but not in rhombomeres 3 and 5. These earlier studies also indicate that the Pbx-Hox site is sufficient for r4-directed expression, a conclusion that conflicts with the current findings that mutation of the flanking Meis site phenocopies Pbx-Hox site mutation. This disparity may be accounted for by the fact that the previous studies employed synthetic elements that were not in a natural configuration and that contained iterated copies of Pbx-Hox sites. Since Meis crossbinds to Pbx-Hox consensus sites, synthetic elements containing them in tandem resemble the natural tripartite Meis-Pbx-Hox elements identified here and, in fact, weakly support DNA binding by ternary Hoxb1 complexes in vitro. This may also account for the Meis-mediated enhancement of the expression of reporter genes containing similar multimerized configurations of the ARE r3 site (Jacobs, 1999 and references).

The current studies demonstrate a consistent requirement for the Meis site in vitro and in vivo, but it is not yet clear which of the various Meis-Prep1 family members may be directly responsible for r4 enhancer function in the developing hindbrain. Western blot analyses show that both Meis and Prep1 proteins are expressed in the hindbrain at embryonic day 9.5. Since Meis genes display dynamic expression profiles during embryonic development, a more precise determination of the in vivo roles of individual Meis-related proteins in r4 functions will require studies with mice that are nullizygous for one or more of the Meis genes. In summary, these studies provide support at the molecular level for previous observations that each component of the TALE heterodimer interacts and functions on common genetic pathways with a subset of Hox proteins. Although its generality for Hox function remains to be determined, a trimeric model invoking a higher-order assembly of Hox and TALE proteins provides a molecular framework for integrating the functions of these developmentally important proteins (Jacobs, 1999).

Human PREP1, a novel homeodomain protein of the TALE superfamily, forms a stable DNA-binding complex with PBX proteins in solution, a ternary complex with PBX and HOXB1 on DNA, and is able to act as a co-activator in the transcription of PBX-HOXB1 activated promoters. DNA-binding PREP1-PBX complexes are present in murine cells. In vivo, PREP1 is a predominant partner of PBX proteins in various murine tissues. However, the choice of PBX family member associated with PREP1 is largely tissue-type specific. The cloning and expression domain of the murine Prep1 gene is reported. Murine PREP1 shares 100% identity with human PREP1 in the homeodomain and 95% similarity throughout the whole protein. In the adult mouse, PREP1 is expressed ubiquitously, with peaks in testis and thymus. Murine Prep1 mRNA and protein and multiple DNA-binding PREP1-PBX complexes are present in mouse embryos from at least 9.5 days p.c. PREP1 is present in all embryonic tissues from at least 7.5-17.5 days of development, with a predominantly nuclear staining. PREP1 is able to superactivate the PBX-HOXB-1 autoregulated Hoxb-1 promoter, and all three proteins, PREP1, PBX and HOXB-1, are present together in the mouse rhombomere 4 domain in vivo, compatible with a role for PREP1 as a regulator of PBX and HOXB-1 proteins activity during development (Ferretti, 1999).

The Meis and Pbx genes encode for homeodomain proteins of the TALE class and have been shown to act as co-factors for other homeodomain transcription factors. The expression of these genes in the mouse telencephalon has been studied, and Meis1 and Meis2 have been found to display region-specific patterns of expression from embryonic day (E)10.5 until birth, defining distinct subterritories in the developing telencephalon. The expression of the Meis genes and their proteins is highest in the subventricular zone (SVZ) and mantle regions of the ventral telencephalon. Compared to the Meis genes, Pbx genes show a broader expression within the telencephalon. However, as is the case in Drosophila, nuclear localized PBX proteins correlate highly with Meis expression. In addition, DLX proteins co-localize with nuclear PBX in distinct regions of the ventral telencephalon (Toresson, 2000).

The Meis gene family in vertebrates consists of Meis1-3 and the related Prep1/Pknox. High level expression of two of these genes, Meis1 and Meis2, has been detected within the telencephalon. Telencephalic Meis1 and Meis2 expression is first detected around E10.5 in the ventrolateral telencephalon. At this time, Meis1 transcripts are expressed at low levels in the ventricular zone (VZ) while Meis2 is expressed at high levels in cells underlying the VZ, lateral to the medial ganglionic emeinence (MGE) [i.e. the presumptive lateral ganglionic eminence (LGE)]. From E11.5, when the MGE and LGE are morphologically distinct, Meis1 is expressed at high levels in the caudal ganglionic eminence (CGE) and developing amygdala and weaker in the LGE and MGE. At E12.5, Meis1 transcripts are found in the lateral edges of both the MGE and LGE. Meis1 continues to be expressed in the ventro-lateral regions of the striatum and cortex with higher expression in the ventral pallidum and medial septum at later stages. Unlike Meis1, Meis2 is found at moderate levels throughout the VZ of the entire telencephalon with the exception of the ventro- and dorso-medial regions. The highest expression is detected in the SVZ of the LGE and developing striatum. By E16.5, Meis2 transcripts are also found in the cortical plate. As was the case for Meis1, the CGE and amygdala also express high levels of Meis2. Meis gene expression in the telencephalon remains unchanged at birth and even into adulthood. At all stages examined, MEIS protein is found where the respective gene is expressed (Toresson, 2000).

The Drosophila homolog of the Meis genes, homothorax, is required for the nuclear localization of the PBX homolog, Extradenticle. Interestingly, in the mouse there is a strong correlation between Meis gene expression and nuclear localized PBX proteins (including PBX1, 2 and 3). In fact, double in situ hybridization-immunohistochemistry reveals co-localization of PBX proteins and Meis2 transcripts. This protein localization does not completely parallel that of Pbx gene expression. While Pbx1 expression is rather broad, Pbx3 transcripts show a similar localization to Meis1 and Meis2. Thus, interactions between MEIS and PBX molecules may have been conserved from Drosophila to mouse. MEIS and PBX proteins form DNA binding complexes with a number of homeodomain transcription factors, including one another. The Dlx family of homeobox genes is interesting in this respect, since they are expressed in both the LGE and MGE, both of which are known to give rise to distinct neuronal phenotypes in the ventral telencephalon. The LGE represents the principal source of striatal projection neurons. In fact, both Dlx1 and Dlx2 are required for normal striatal differentation. DLX protein expression overlaps extensively with the domains of MEIS and PBX expression in the telencephalon. Indeed, within the SVZ of the LGE but not the MGE, cells expressing DLX proteins were also found to co-express nuclear PBX proteins (Toresson, 2000).

Pbx1, in partnership with Meis1b, can regulate posterior neural markers and neural crest marker genes during Xenopus development. A Xenopus homolog of the Pbx1b homeodomain protein was isolated and shown to be expressed throughout embryogenesis. Xpbx1b expression overlaps with Xmeis1 in several areas, including the lateral neural folds, caudal branchial arch, hindbrain, and optic cup. When ectopically expressed, Xpbx1b can synergize with Xmeis1b to promote posterior neural and neural crest gene expression in ectodermal explants. Further, a physical interaction between these two homeodomain proteins is necessary for induction of these genes in embryonic tissue. In addition, coexpression of Xmeis1b and Xpbx1b leads to a prominent shift in the localization of Xmeis1b from the cytoplasm to the nucleus, suggesting that nuclear transport or retention of Xmeis1b may depend upon Xpbx1b. Finally, expression of a mutant construct in which Xpbx1b protein is fused to the repressor domain from Drosophila Engrailed inhibits posterior neural and neural crest gene expression. These data indicate that Xpbx1b and its partner, Xmeis1b, function in a transcriptional activation complex during hindbrain and neural crest development (Maeda, 2002).

The epidermal barrier varies over the body surface to accommodate regional environmental stresses. Regional skin barrier variation is produced by site-dependent epidermal differentiation from common keratinocyte precursors and often manifests as site-specific skin disease or irritation. There is strong evidence for body-site-dependent dermal programming of epidermal differentiation in which the epidermis responds by altering expression of key barrier proteins, but the underlying mechanisms have not been defined. The LCE (Late cornified envelope) multigene cluster encodes barrier proteins that are differentially expressed over the body surface, and perturbation of LCE cluster expression is linked to the common regional skin disease psoriasis. LCE subclusters comprise genes expressed variably in either external barrier-forming epithelia (e.g. skin) or in internal epithelia with less stringent barriers (e.g. tongue). A complex of TALE homeobox transcription factors PBX1, PBX2 and Pknox (homologues of Drosophila Extradenticle and Homothorax) preferentially regulate external rather than internal LCE gene expression, competitively binding with SP1 and SP3. Perturbation of TALE protein expression in stratified squamous epithelia in mice produces external but not internal barrier abnormalities. It is concluded that epidermal barrier genes, such as the LCE multigene cluster, are regulated by TALE homeodomain transcription factors to produce regional epidermal barriers (Jackson, 2011).

PBX1 nuclear export is regulated by PKA phosphorylation of the PBC-B domain

The PBC subfamily of TALE (Three-Amino-acid-Loop-Extension) homeodomain proteins includes the products of the vertebrate Pbx1, Pbx2, Pbx3 and Pbx4 genes, and the Drosophila extradenticle (exd) gene. PBC proteins form stable heterodimers with MEINOX proteins, which belong to a different subfamily of TALE homeodomain proteins and include the products of the vertebrate Meis and Prep genes and the Drosophila homothorax (hth) gene. The regulation of PBC protein function through subcellular distribution is a crucial evolutionarily conserved mechanism for appendage patterning. The processes controlling PBX1 nuclear export was investigated. In the absence of MEINOX proteins nuclear export is not a default pathway for PBX1 subcellular localization. In different cell backgrounds, PBX1 can be imported or exported from the nucleus independently of its capacity to interact with MEINOX proteins. The cell context-specific balance between nuclear export and import of PBX1 is controlled by the PBC-B domain, which contains several conserved serine residues corresponding to phosphorylation sites for Ser/Thr kinases. PBX1 subcellular localization correlates with the phosphorylation state of these residues whose dephosphorylation induces nuclear export. Protein kinase A (PKA) specifically phosphorylates PBX1 at these serines, and stimulation of endogenous PKA activity in vivo blocks PBX1 nuclear export in distal limb mesenchymal cells. These results reveal a novel mechanism for the control of PBX1 nuclear export in addition to the absence of MEINOX protein, which involves the inhibition of PKA-mediated phosphorylation at specific sites within the PBC-B domain (Kilstrup-Nielsen, 2003).

Cooperation between PBX proteins and their Hox partners

The vertebrate hindbrain is divided into rhombomeres (r); serially homologous segments. Pbx and Hox proteins are hypothesized to form heterodimeric, DNA binding transcription complexes which specify rhombomere identities. Eliminating zebrafish Lazarus (Lzr/Pbx4) and Pbx2 function prevents hindbrain segmentation and causes a wholesale anterior homeotic transformation of r2-r6, to r1 identity. Pbx proteins interact with Hox paralog group 1 proteins to specify segment identities broadly within the hindbrain, and this process involves the Pbx:Hox-1-dependent induction of Fgf signals in r4. It is proposed that in the absence of Pbx function, r2-r6 acquire a homogeneous ground state identity, that of r1, and that Pbx proteins, functioning primarily with their Hox partners, function to modify this ground state identity during normal hindbrain development (Waskiewicz, 2002).

Interactions between Pbx and Hox-1 paralogs (hoxa1 and hoxb1 in the mouse, hoxb1a and hoxb1b in the zebrafish) directly specify r4 identity via essential Hox-1/Pbx binding sites in the regulatory regions of target genes. In mouse Hoxa1-/-;b1-/- double mutants, r4 identity is not specified. In the zebrafish, morpholino knockdown of hoxb1a and hoxb1b do not prevent r4 specification; however, this is likely due to the failure of the morpholinos to completely eliminate gene function rather than due to a basic difference in the mechanism of hox-1 function in mouse and zebrafish, since reducing hoxb1a and hoxb1b function in a zygotic lzr/pbx4 background prevents r4 specification. Furthermore, reducing pbx activity uncovers a role for hox-1 genes in specifying pattern more broadly in the hindbrain, since krox20 expression in r3 and r5 and val/mafB expression in r5 and r6 are also virtually eliminated in Zlzr; hox-1 MO-injected embryos (Waskiewicz, 2002).

These observations suggest that Hox-1 proteins, together with their Pbx partners, function at the top of the hindbrain patterning hierarchy, possibly specifying rhombomere identities through the regulation of signals derived from r4. Indeed, previous work in the mouse hasdemonstrated that hoxa1, which is never expressed in r3, nevertheless contributes non-cell autonomously to its specification. fgf3 and fgf8 are expressed in r4 in zebrafish and are required for the specification of r5 and r6. In zebrafish embryos lacking pbx2 and pbx4 function, upregulation of Fgf signals (Fgf3 and Fgf8) in r4 fails to occur. hox-1 genes are both required and sufficient to drive fgf3 expression in r4. These observations, together with previous work in the zebrafish and mouse, led to a model for hindbrain patterning in the zebrafish. In this model, Pbx interacts with Hox-1 genes to specify r4 identity including the upregulation of hoxb1a, hoxa2, fgf3, and fgf8. Fgf3 and Fgf8 in turn signal to the surrounding rhombomeres, contributing to the establishment of r3, r5, and r6 identity (Waskiewicz, 2002).

It is important to note, however, that Pbx function in hindbrain development is not limited to driving the expression of r4-derived patterning signals. If this were the case, mutant cells would be able to acquire r2-r6 identities in a wild-type host. However, mosaic analysis demonstrates unambiguously that Pbx is required cell autonomously in r2-r6 since mutant cells cannot contribute to, and as a result are excluded from, these rhombomeres in a wild-type host. Thus it is proposed that Pbx is required at multiple levels of the hindbrain patterning hierarchy, first with Hox-1 proteins in r4 but then subsequently with other Hox or as yet unidentified non-Hox partners to specify aspects of r2-r6 identity in a cell-autonomous manner. Consistent with this, Hox-3 expression in r5 and r6 has been shown to be maintained by a Pbx-dependent autoregulatory loop, and Hoxa2, which requires Pbx function in zebrafish has been shown to contribute to r2 and r3 specification in the mouse (Waskiewicz, 2002).

In contrast to the strong effects seen on patterning in r2-r6 in MZlzr; pbx2 MO embryos, patterning of the posterior-most vagal region of the hindbrain is relatively mildly affected. The anterior limit of hoxb4 expression is diffuse; however, levels of hoxb4 expression are normal. Vagal motor neurons characteristic of the posterior hindbrain are also present, although reduced in number. Most strikingly, cells lacking Pbx function are able to contribute normally to the hindbrain posterior to the r6/r7 boundary, suggesting that they are able to acquire the cell surface characteristics of the posterior-most hindbrain. The observation that posterior hindbrain identities are less strongly affected by loss of Pbx function is unexpected, given that the Hox genes that are expressed in this region are strongly auto- and cross-regulated, and the required autoregulatory elements contain essential Pbx:Hox binding sites. However, it is noted that in a cross-species reporter assay designed to test the Pbx dependence of such a Hoxb4 autoregulatory element, reporter expression was only partially suppressed in Exd mutant flies, suggesting that Hox-4 may be partially Pbx independent in vertebrates. Other critical Hox-4 targets may be similarly Pbx independent. Another explanation is that aspects of posterior hindbrain patterning may not be strictly dependent on Hox function. The vagal hindbrain is outside of the meristic series, forming a nonsegmented transition zone between the segmental region of the hindbrain and the spinal cord. The robust ability of MZlzr; pbx2 MO cells to contribute to this region in genetic mosaics may reflect a reduced importance of cell sorting for the maintenance of identity in this region of the hindbrain (Waskiewicz, 2002).

In this work, an important distinction was made between r1 and r0, with r1 being the narrow, ephA4a, fgfr3-expressing domain immediately anterior to r2, and r0 being the larger engrailed-expressing domain between r1 and the mid-hindbrain junction. A similar distinction has been hypothesized based on morphological criteria in the chick, and molecular analysis has suggested that the r1-r0 distinction may indeed be a common feature of vertebrate embryos since fgfr3 is expressed in a similar domain in the chick. Importantly, r1 is the posterior-most region of the neural tube which does not express any hox genes; however, it is competent to respond to Hox-encoded patterning information. Taken together with the strong evidence presented (physical and genetic interactions between Pbx and Hox proteins) it is proposed that the primary function of Pbx genes during hindbrain patterning is to facilitate Hox function, and that r1 is a hindbrain ground state: the default fate established in the absence of Pbx:Hox activity (Waskiewicz, 2002).

Individual vertebrate Hox genes specify aspects of segment identity along the anterior-posterior axis. The exquisite in vivo specificity of Hox proteins is thought to result from their interactions with members of the Pbx/Exd family of homeodomain proteins. A zebrafish gene, lazarus, which is required globally for segmental patterning in the hindbrain and anterior trunk, has been identified and cloned. lazarus is a novel pbx gene and evidence is provided that it is the primary pbx gene required for the functions of multiple hox genes during zebrafish development. lazarus plays a critical role in orchestrating the corresponding segmentation of the hindbrain and the pharyngeal arches, a key step in the development of the vertebrate body plan (Pöpperl, 2000).

lzr plays an evolutionarily conserved role in broadly mediating hox gene function. This is based on three lines of evidence: (1) lzr mutants mimic Hox loss of function phenotypes in the mouse; (2) those aspects of Hox gene expression that have been shown to be dependent on auto-, para-, and cross-regulatory interactions in the mouse are disrupted in lzr- embryos; (3) lzr is required for hoxb2 function, since the effects of ectopic hoxb2 misexpression are not observed in lzr- embryos (Pöpperl, 2000).

If lzr is required as a DNA binding partner for zebrafish Hox proteins, then lzr- embryos are expected to mimic hox loss of function phenotypes. Indeed, with respect to the primary reticulospinal neurons, lzr mutants exhibit an anterior homeotic transformation toward an r2 identity. Furthermore, where it is possible to compare lzr to Hox mutants in the mouse many specific similarities are seen. The absence of rhombomere boundaries anterior to r4 is a characteristic of the Hoxa2;b2 double mutant, while the failure to downregulate ZDK1 in r4, and fusions of first and second pharyngeal arch cartilages are characteristics of the Hoxa2 mutant. The failure of facial (nVII) motor neurons to migrate posteriorly from r4 is a characteristic of the mouse Hoxb1 mutant, while hypoplastic first- and second-arch-derived cartilages characterize the Hoxa1 mutant. Finally, the absence of thymic primordia is a characteristic of the mouse Hoxa3 mutant (Pöpperl, 2000 and references therein).

Mice with mutations in the more posterior Hox genes (paralog groups 4-13) frequently exhibit homeotic vertebral transformations. In zebrafish, the ability to assay for Hox loss of function phenotypes in the trunk is limited by the fact that vertebrae form relatively late in development, after the point when lzr- embryos die. However, the observation that pectoral fins fail to be specified in lzr- embryos suggests that lzr does cooperate with hox genes in the anterior trunk. A correlation between the anterior limits of Hox-5 and Hox-6 paralog expression and the limits of the forelimb or pectoral fin buds in several vertebrate species has implied Hox genes in the specification of these appendages. The observation that lzr- embryos lack pectoral fins thus supports a key role for Hox genes, together with their pbx partners, in the specification of the vertebrate paired appendages (Pöpperl, 2000).

If lzr is required as a DNA binding partner for zebrafish Hox proteins, the expression of hox transcriptional targets is expected to be disrupted in lzr- embryos. Among the few targets of vertebrate Hox genes that have been identified are the Hox genes themselves, which experience a complex set of auto- and cross-regulatory interactions during the refinement phase of their expression in mouse embryos. These interactions have been suggested to involve Pbx proteins, and indeed, it is the in the context of Hox autoregulation that vertebrate Hox:Pbx interactions are understood at the structural level. In lzr- embryos, reduced hoxb1a and hoxb2 expression in r4 is seen, consistent with the existence of regulatory input on mouse Hoxb1 by both Hoxa1 and Hoxb1 and on Hoxb2 by Hoxb1. The failure to generate a sharp anterior limit of hoxb4 expression at the r6/7 boundary is consistent with the existence of regulatory input on Hoxb4 by other Hox4 paralogs. Taken together, these data demonstrate that lzr function is required for those aspects of hox gene expression that have been shown to be dependent on other Hox genes in the mouse and suggest that lzr is a required partner in those regulatory interactions (Pöpperl, 2000).

The striking similarities between zebrafish lzr and mouse Hox mutant phenotypes, the requirement for lzr in those aspects of hox regulation that in the mouse are Hox dependent, and the absolute requirement for lzr for the effects of ectopic hoxb2 expression all point to a critical role for lzr in mediating the functions of multiple hox genes in the developing zebrafish embryo. A total of five zebrafish pbx genes have been cloned, of which only lzr is expressed broadly and at high levels during the first 24 hr of development. lzr is not a clear ortholog of any of the three mammalian Pbx family members based on sequence similarity, and thus it defines a new pbx family member. However, Lzr protein forms heterodimers with Hox proteins in vitro on characterized Pbx/Hox binding sites, indicating that it has the in vitro DNA binding and dimerization properties of other Pbx proteins (Pöpperl, 2000).

Furthermore, other zebrafish pbx family members, which include pbx1 and pbx3 orthologs, can efficiently rescue the lzr- phenotype when ectopically expressed by mRNA injection. This indicates that differences in the in vivo functions of zebrafish pbx family members results from differences in their temporal and spatial expression patterns rather than differences in their biochemical functions. In the mouse, all three Pbx genes are expressed in the early embryo and thus may redundantly perform the roles of the single early expressed zebrafish gene. Consistent with this, lzr- embryos have a more severe mutant phenotype than mice with single targeted mutations in either Pbx1 or Pbx2 (Pöpperl, 2000).

In spite of the considerable similarities between the lzr- phenotype and Hox mutant phenotypes in the mouse, there are Hox loss of function phenotypes that are absent in lzr- embryos. Lzr is expressed maternally and zygotically, and the perdurance of maternal transcripts until 10 hpf may partially rescue the zygotic loss of lzr function since maternally encoded wild-type Lzr protein is available to interact with early-expressed zygotic Hox proteins to drive the initial expression of their targets in lzr- embryos. By generating germline clones of lzr-/- cells, the importance of maternal lzr in mediating early hox function in zebrafish could be addressed (Pöpperl, 2000).

As well as fulfilling the role of a required hox partner, lzr has functions that have not previously been attributed to Hox genes. Hox genes have not been shown to function upstream of krox20 expression in r3 nor have they been shown to be required for the specification of trigeminal motor neurons, yet lzr is required for both of these roles. While Hox genes are required for the specification of anterior-posterior identity in the cranial neural crest, they have not been shown to function in its segmentation or in the corresponding segmentation of the pharyngeal arches as lzr does. The mechanism of cranial neural crest segmentation, whether involving processes that occur within the hindbrain or involving interactions with the head periphery remains controversial. The observation that lzr functions in this process is intriguing in that it implicates Hox genes not only in the specification of regional identity of the cranial neural crest but also in the control of its migratory behavior. However the possibility that this and other novel lzr- phenotypes reflect hox-independent roles for lzr cannot be ruled out. The possibility that lzr may have functions that do not involve Hox genes has its precedent in Drosophila, where exd functions independently of Hox genes in antennal specification (Pöpperl, 2000 and references therein).

The formation of the pharyngeal arches is an essential step in the development of the vertebrate body plan. Indeed, the phylotypic stage for vertebrates is called the 'pharyngula' after these prominent structures. Nevertheless, the mechanisms that organize the corresponding segmentation of the cranial neural crest and the pharyngeal arches remain obscure. A recent report has shown that segmentation of the pharyngeal endoderm can occur independently of the presence of neural crest. Since lzr functions in the segmentation of both the crest and the pharyngeal endoderm, this raises the intriguing possibility that lzr functions throughout germ layers to establish a global segmental prepattern in the developing head. Given the apparent conservation of hox function between fish and mice as demonstrated by cross-species comparison of lzr and Hox mutant phenotypes, the combination of more Hox loss of function alleles in the mouse should allow future studies to determine whether lzr plays this global role in head segmentation through its interaction with hox genes or with other, as yet unidentified, partners (Pöpperl, 2000).

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).

Binding of PBX proteins and their HOX partners to DNA

Wild-type PBX proteins cooperatively bind promoter sequences from vertebrate homeotic proteins HOXB4, B6, and B7 of HOX/HOM proteins. A hexapeptide motif upstream of the Hox homeodomain is essential for HoxB6 and B7 to cooperatively bind DNA with PBX proteins. The PBX homeodomain is necessary but not sufficient for cooperativity, which requires conserved amino acids carboxy-terminal of the homeodomain. Interactions between HOX and PBX proteins modulate their DNA-binding properties, suggesting that PBX and HOX proteins act in parallel as heterotypic complexes to regulate expression of specific subordinate genes (Chang, 1995).

Heterodimers between the Pbx/Exd and Hox/HOM-C classes of homeodomain proteins bind regulatory elements in tissue-specific and developmentally regulated genes. The half-site bound by both Pbx1 and Hox proteins has been characterized on a prototypic element (TGATTAAT); also determined has been how the orientation of the Hox protein contributes to the DNA binding specificity of Pbx-Hox heterodimers. The Hox protein binds the 3' TAAT sequence as its recognition core and exhibits sequence-specific binding at positions 3' to the TAAT core. Unfavored sequences at this position, such as two cytosines, abrogate binding to the element. The upstream Pbx1 core sequence, TGAT, must immediately juxtapose the Hox core. This geometry maintains the preference of Hox/HOM-C proteins for a T base at position -1, as T represents the fourth position of the Pbx1 core, and suggests that this T base is bound by both Pbx1 and Hox proteins, Pbx1 binding in the major groove and the Hox protein binding in the minor groove. Pbx1 also exhibits base selectivity 5' to its TGAT recognition sequence (Knoepfler, 1996).

Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. The 2.35 A structure of a ternary complex containing a human HoxB1-Pbx1 heterodimer bound to DNA is reported. Cooperative binding of Hox proteins with Pbx1/exd is dependent on a conserved hexapeptide sequence located N-terminal to the Hox homeodomain. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three-amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional alpha helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. This C-terminal portion of Pbx1 is an integral part of the Pbx1 DNA-binding domain and does not contact either the DNA or HoxB1. Rather, the C-terminal residues form part of the enlarged, four-helix DNA-binding domain of Pbx1 and may play a role in maintaining the structure of the DNA recognition helix and the hexapeptide binding pocket. The observed DNA contacts mediated by both HoxB1 and Pbx1 with the DNA provide a basis for understanding differential DNA sequence discrimination by Hox/Pbx heterodimers (Piper, 1999).

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

A binding site selection strategy was used to determine the optimal binding sites for Pbx proteins by themselves and as heterodimeric partners with various Hox gene products. Among the Pbx proteins by themselves, only Pbx3 binds with high affinity, as a monomer or as a homodimer, to an optimal binding site: TGATTGATTTGAT. An inhibitory domain located N terminal to the Pbx1 homeodomain prevents intrinsic Pbx1 binding to this sequence. When complexed with Hoxc-6, each of the Pbx gene products binds the same consensus sequence, TGATTTAT, which differs from the site bound by Pbx3 alone. Three members of the Antennapedia family, Hoxc-6, Hoxb-7, and Hoxb-8, select the same binding site in conjunction with Pbx1. These proteins show similar affinities as heterodimeric partners with Pbx1 for the selected optimal binding site. However, the binding specificity of Hox proteins for optimal binding sites is increased, compared to nonspecific DNA, in the presence of Pbx proteins. Thus, while cooperative DNA binding involving heterodimers of Pbx and Hox gene products derived from members within the Antennapedia family does not increase binding site selectivity, DNA binding specificity of the Hox gene products is significantly enhanced in the presence of Pbx (Neuteboom, 1997).

The mammalian Pbx homeodomain proteins provide specificity and increased DNA binding affinity to other homeodomain proteins. A cAMP-responsive sequence (CRS1) from bovine CYP17 has previously been shown to be a binding site for Pbx1. A member of a second mammalian homeodomain family, Meis1, is now also demonstrated to be a CRS1-binding protein when purified using CRS1 affinity chromatography. CRS1 binding complexes from Y1 adrenal cell nuclear extract contain both Pbx1 and Meis1. This is the first transcriptional regulatory element reported as a binding site for members of the Meis1 homeodomain family. Pbx1 and Meis1 bind cooperatively to CRS1, whereas neither protein can bind this element alone. Mutagenesis of the CRS1 element indicates a binding site for Meis1 adjacent to the Pbx site. All previously identified Pbx binding partners have Pbx interacting motifs that contain a tryptophan residue amino-terminal to the homeodomain that is required for cooperative binding to DNA with Pbx. Members of the Meis1 family contain one tryptophan residue amino-terminal to the homeodomain, but site-directed mutagenesis indicates that this residue is not required for cooperative CRS1 binding with Pbx. Thus, the Pbx-Meis1 interaction is unique among Pbx complexes. Meis1 also cooperatively binds CRS1 with the Pbx homologs Extradenticle from Drosophila and ceh-20 from C. elegans, indicating that this interaction is evolutionarily conserved. Thus, CYP17 CRS1 is a transcriptional regulatory element containing both Pbx and Meis1 binding sites, which permits these two homeodomain proteins to bind and potentially regulate cAMP-dependent transcription through this sequence (Bischof, 1998).

The basis for this cAMP response is not yet well understood. Pbx1 enhances the cAMP activated transcriptional response, mediated by protein kinase A, of CRS1's regulation of a reporter gene. There are several possible mechanisms by which protein kinase A could regulate the activity of CRS1: (1) the levels of one or more of the CRS1-binding proteins could be regulated, for example, at the level of gene expression or translation. (2) Alternatively, Pbx1 or Meis1 could be posttranslationally modified by phosphorylation, which could affect DNA binding, dimerization ability, or transactivation function. For example, protein kinase A phosphorylation of the homeodomain protein thyroid transcription factor 1 is involved in the expression of the surfactant protein B gene promoter by this homeodomain protein. (3) An additional possibility is that protein kinase A modifies a non-DNA-bound protein, which may interact with the Pbx-Meis1 complex. (4) Concerning CRS1 activity, there may be other components of the CRS1 binding complex, such as the 60-kDa protein that was copurified. Preliminary data indicate that this protein may be the homeodomain protein, Pknox1, which is related to Meis1. The identification of this protein, as well as how protein kinase A may regulate transcription through this element, is being investigated (Bischof, 1998).

The HOX/HOM superfamily of homeodomain proteins controls cell fate and segmental embryonic patterning by a mechanism that is conserved in all metazoans. The linear arrangement of the Hox genes on the chromosome correlates with the spatial distribution of HOX protein expression along the anterior-posterior axis of the embryo. Most HOX proteins bind DNA cooperatively with members of the PBC family of TALE-type homeodomain proteins, which includes human Pbx1. Cooperative DNA binding between HOX and PBC proteins requires a residue N-terminal to the HOX homeodomain, termed the hexapeptide, which differs significantly in sequence between anterior- and posterior-regulating HOX proteins. The 1.9-Å-resolution structure of a posterior HOX protein, HoxA9, is reported to be complexed with Pbx1 and DNA; the structure reveals that the posterior Hox hexapeptide adopts an altered conformation as compared with that seen in previously determined anterior HOX/PBC structures. The additional nonspecific interactions and altered DNA conformation in this structure account for the stronger DNA-binding affinity and altered specificity observed for posterior HOX proteins when compared with anterior HOX proteins. DNA-binding studies of wild-type and mutant HoxA9 and HoxB1 show residues in the N-terminal arm of the homeodomains are critical for proper DNA sequence recognition despite lack of direct contact by these residues to the DNA bases. These results help shed light on the mechanism of transcriptional regulation by HOX proteins and show how DNA-binding proteins may use indirect contacts to determine sequence specificity (LaRonde-LeBlanc, 2003).

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).

Identification of a new type of PBX1 partner that contains zinc finger motifs and inhibits the binding of HOXA9-PBX1 to DNA

PBX1 belongs to the TALE-class of homeodomain protein and has a wide functional diversity during development. Indeed, PBX1 is required for haematopoiesis as well as for multiple developmental processes such as skeletal patterning and organogenesis. It has furthermore been shown that PBX1 functions as a HOX cofactor during development. More recent data suggest that PBX1 may act even more broadly by modulating the activity of non-homeodomain transcription factors. To better understand molecular mechanisms triggered by PBX1 during female genital tract development, additional PBX1 partners have been sought that might be involved in this process. Using a two hybrid screen, a new PBX1 interacting protein has been identified containing several zinc finger motifs; this protein has been called ZFPIP for Zinc Finger PBX1 Interacting Protein. ZFPIP is expressed in embryonic female genital tract but also in other PBX1 expression domains such as the developing head and the limb buds. ZFPIP is able to bind physically and in vivo to PBX1 and moreover, it prevents the binding of HOXA9/PBX complexes to their consensus DNA site. It is suggested that ZFPIP is a new type of PBX1 partner that could participate in PBX1 function during several developmental pathways (Laurent, 2007).

Degradation of Extradenticle homologs

Notch signals are important for lymphocyte development but downstream events that follow Notch signaling are not well understood. Signaling through Notch modulates the turnover of E2A proteins including E12 and E47, which are basic helix-loop-helix proteins crucial for B and T lymphocyte development. Notch-induced degradation requires phosphorylation of E47 by p42/p44 MAP kinases. Expression of the intracellular domain of Notch1 (N1-IC) enhances the association of E47 with the SCF(Skp2) E3 ubiquitin ligase and ubiquitination of E47, followed by proteasome-mediated degradation. Furthermore, N1-IC induces E2A degradation in B and T cells in the presence of activated MAP kinases. Activation of endogenous Notch receptors by treatment of splenocytes with anti-IgM or anti-CD3 plus anti-CD28 also leads to E2A degradation, which is blocked by the inhibitors of Notch activation or proteasome function. Notch-induced E2A degradation depends on the function of its downstream effector, RBP-Jkappa, probably to activate target genes involved in the ubiquitination of E2A proteins. Thus it is proposed that Notch regulates lymphocyte differentiation by controlling E2A protein turnover (Nie, 2003).

Role of the N-terminal arm of Hox genes in interacting with PBX proteins

Continued: extradenticle Evolutionary Homologs part 2/2

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

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