Homothorax genes in C. elegans

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).

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).

Joint actions of diverse transcription factor families establish neuron-type identities and promote enhancer selectivity

To systematically investigate the complexity of neuron specification regulatory networks, this study carried out an an RNA interference (RNAi) screen against all 875 transcription factors (TFs) encoded in Caenorhabditis elegans genome and searched for defects in nine different neuron types of the monoaminergic (MA) superclass and two cholinergic motoneurons. 91 TF candidates to be required for correct generation of these neuron types, of which 28 were confirmed by mutant analysis. Correct reporter expression in each individual neuron type requires at least nine different TFs. Individual neuron types do not usually share TFs involved in their specification but share a common pattern of TFs belonging to the five most common TF families: homeodomain (HD), basic helix loop helix (bHLH), zinc finger (ZF), basic leucine zipper domain (bZIP), and nuclear hormone receptors (NHR). HD TF members are overrepresented, supporting a key role for this family in the establishment of neuronal identities. These five TF families are also prevalent when considering mutant alleles with previously reported neuronal phenotypes in C. elegans, Drosophila, and mouse. In addition, terminal differentiation complexity was studied focusing on the dopaminergic terminal regulatory program. Two HD TFs [UNC-62 (Homothorax paralog) and VAB-3 (Toy paralog)] were found that work together with known dopaminergic terminal selectors [AST-1 (Ets65A paralog), CEH-43 (Dll paralog), CEH-20 (Exd paralog)]. Combined TF binding sites for these five TFs constitute a cis-regulatory signature enriched in the regulatory regions of dopaminergic effector genes. These results provide new insights on neuron-type regulatory programs in C. elegans that could help better understand neuron specification and evolution of neuron types (Jimeno-Martin, 2022).

Homothorax homologs in other insects

The expression patterns of Gryllus (cricket) homothorax (Gbhth) and dachshund (Gbdac) are described, together with localization of Distal-less or Extradenticle protein during leg development. Their expression patterns have been correlated with the morphological segmentation of the leg bud. The boundary of Gbhth/GbDll subdivision is correlated with the segment boundary of the future trochanter/femur at early stages. Gbdac expression subdivides the leg bud into the presumptive femur and more distal region. During the leg proximodistal formation, although the early expression patterns of GbDll, Gbdac, and Gbhth significantly differ from those of Drosophila imaginal disc, their expression patterns in the fully segmented Gryllus leg are similar to those in the Drosophila late third instar disc (Inoue, 2002).

Although the legs of adult flies and crickets are very similar in their segmental compositions, the developmental processes producing their morphologies are quite distinct. In the Drosophila imaginal disc, leg formation occurs through the concentric folding and the subsequent segmentation of monolayered epithelia in the leg disc during later larval stages, although the early leg disc remains as a flattened two-dimensional structure. In contrast, the cricket leg bud is formed directly from the body wall and segmented during the subsequent outward growth in the early embryogenesis period. The leg segmentation in Gryllus occurs intercalatively step by step until stage 11 (6 days after EL). The changes have been classified into the five following stages: (1) formation of the leg bud at stages 6-7 (2.5 days after EL); (2) the first morphological segmentation at the future trochanter/femur boundary at stage 8 (3 days after EL); (3) the second segmentation in the femur/distal telopodite boundary at stage 9 (4 days after EL); (4) the third segmentation in the tibia/tarsus boundary at stage 10 (5 days after EL); (5) the fourth segmentation at the coxa/trochanter boundary. Then, the elongation of each segment takes place to form the legs of the nymph until hatching (14 days after EL) (Inoue, 2002).

Before the onset of the leg bud formation (stage 5), Gbhth is expressed uniformly throughout the embryos. Just before the onset of leg bud formation (stage 5, 40 h after egg laying), GbDll starts to be expressed in the presumptive thoracic leg region. Subsequently, Gbhth expression was downregulated in the same regions (stage 6, 48 h after egg laying). Thus, the Gbhth/GbDll antagonistic subdomains seem to be established in the early cricket embryos, as observed in Drosophila. In the early stages of leg bud formation (stages 7-9), Gbhth is expressed in the proximal region of the leg bud. In embryos at stages 8-10, when the leg segments are visible, the distal boundary of the Gbhth expression domain is localized at the proximal region of the femur segment (Inoue, 2002).

To compare expression patterns of Gbhth and Gbdac with localization of GbDll or GbExd, double staining was performed, using the corresponding RNA probes and an antibody against Dll or Exd, which are used as distal and proximal markers, respectively, in Drosophila, Acheta (Orthoptera, cricket), and Schistocerca (Orthoptera, grasshopper).Prior to the leg bud formation (stage 5), due to weak expression of GbDll, no double stainings could be observed. Prior to the first leg segmentation, Gbhth is expressed in the proximal region of the leg bud, while GbDll is detected in the distal region of the leg bud (stages 6-7). A merged panel reveals that the leg bud is stained with red and green without significant overlap, indicating that the bud is divided into two domains: a distal GbDll-localized domain and a proximal Gbhth-expressing domain. These domains are complementary at this stage, but by the end of stage 7 they partially overlap. At the same stage, GbExd accumulation, caused by nuclear localization of GbExd, is strongly detected in the proximal region of the leg bud, though low levels of GbExd expression can be seen in the distal region. The GbExd accumulation domain in the proximal region overlaps the Gbhth expression domain, as is observed in Drosophila (Inoue, 2002).

The subdivision of the leg bud into proximal and distal domains becomes morphologically discernible as a circumferential constriction, which is the boundary between the trochanter and femur (stage 8). The proximal limit of the GbDll domain corresponds to this boundary at this stage. However, the GbDll domain includes the distal region of the trochanter in later stages (Inoue, 2002).

At stages 8-9, Gbhth is expressed continuously in the proximal domain of the leg bud, overlapping the GbExd accumulation domain. By early stage 8, GbDll is localized throughout the distal tip of the leg bud, including the telopodite. Gbdac is expressed in the middle of the leg bud as a narrow ring, overlapping the GbDll expression domain. GbDll expression starts to become downregulated in the central region at the end of stage 8, in a region where Gbdac is expressed. The domain of GbDll and Gbhth co-expression continues to be observed at stage 9. The proximal ring of GbDll expression, corresponding to the proximal domain of the femur segment, remains unchanged from stage 9 to stage 11 (Inoue, 2002).

By stage 9, the Gbdac expression domain observed at stage 8 has divided into two domains: the proximal and distal domains. Meanwhile GbDll expression becomes undetectable in the middle region. The distal Gbdac domain therefore overlaps a proximal region of the distal GbDll domain, while no such co-expression is observed in the proximal domain. A narrow domain showing no expression of Gbhth, Gbdac, or GbDll can be observed between the two Gbdac domains. Consequently, six proximodistal expression domains appear in the Gryllus leg (Inoue, 2002).

At stage 10, the third morphological segmentation is observed in the distal leg bud, which divides into the tibia and tarsus. Subsequently, the fourth segmentation is discernible in the proximal leg bud, which divides into the coxa and trochanter. It has not been possible to find any of the boundaries of expression of the known appendage genes corresponding of the tibia/tarsus and coxa/trochanter boundaries (Inoue, 2002).

At stages 11-12, following the establishment of the leg segments, structures that connect adjacent segments such as articulates, muscle patterns, etc., are constructed at the segment boundaries. These stages are therefore designated here as the articulation phases. In Gryllus, since the major leg segments can be identified morphologically, the leg segments and the six domains determined by expression patterns of Gbhth, Gbdac, and GbDll can be easily correlated. The proximal-most domain in which only Gbhth is expressed, corresponds to the body wall, coxa segment, and proximal trochanter. The proximal boundary of the GbDll/Gbhth domain lies in the trochanter, and the overlapping GbDll/Gbhth domain extends into the proximal femur. The Gbdac domain corresponds to the middle of the femur segment, including the articulation between the femur and tibia. At stages 11-12, a narrow domain can also be found in which neither GbDll nor Gbdac is expressed, corresponding to the proximal tibia. The Gbdac/GbDll domain includes the articulate between the tibia and tarsus, and extends to the presumptive boundary between tarsal segments 1 and 2. The distal-most GbDll domain corresponds to presumptive tarsal segments 2 and 3 and the pretarsus. The intermediate three expression domains, i.e. the GbDll/Gbhth, Gbdac, and GbDll/Gbdac domains, include the segmental boundaries of the trochanter/femur, the femur/tibia, and the tibia/tarsus, respectively, but do not correspond to the segments (Inoue, 2002).

Expression patterns of Gryllus hth, dac, and Dll in the leg bud with those in the Drosophila leg imaginal disc were compared. The results reveal that these expression domains resolve into similar patterns. In contrast, some differences in the elaborating processes of the expression patterns of the three genes can be seen between Gryllus and Drosophila (Inoue, 2002).

The position of the proximal limit of the GbDll domain has a very sharp boundary between stages 6-8, corresponding to the morphological segmental boundary between the femur and trochanter. Thus, the most proximal segment of the telopodite, i.e. the trochanter, is not included in the GbDll domain in early stages. In contrast, in Drosophila, genetic evidence has demonstrated the subdivision of the leg disc by Dmexd and DmDll into the coxopodite and telopodite. In addition, it has been reported that Dll is detected throughout the telopodite during early development in Acheta (Orthoptera, cricket). Despite these discrepancies, the observations of the expression patterns of GbDll and Gbhth or GbExd in later stages are basically consistent with the results obtained for Acheta, Schistocerca (Orthoptera, grasshopper, and Drosophila (Inoue, 2002).

Following the primary subdivision by GbDll and Gbhth, their expression domains overlap at the boundary at later stages. The domain of Gbhth expression does not strictly correspond to the coxopodite, but expands distally into the femur. This is the same for Drosophila leg, in which the limit of expression of Dmhth expands into the proximal femur. In Drosophila, the first intercalated region between the Dmhth and DmDll domains is the intermediate region expressing Dmdac, and as a result, three discrete domains are established in the leg imaginal disc. In contrast, in Gryllus, three discrete expression domains of the three genes are not observed during leg development (Inoue, 2002).

The expression domain of Gbdac transiently overlaps with that of GbDll at early stages. In Drosophila, Dmdac expression is asymmetrically turned on in dorsal cells that still express DmDll, in the early third instar leg disc. Thus, in both insects, the overlapping Dll/dac domain is observed transiently and subsequently resolves into two domains; proximally Gbdac and distally Gbdac/GbDll in Gryllus, and proximally Dmdac and distally DmDll in Drosophila. At stages 8-9, the GbDll expression fades in the intermediate portion of the leg, and this leads to a new proximal boundary of GbDll. This boundary is correlated with the second segmentation of the femur/distal telopodite. No corresponding expression boundary has been reported in the Drosophila leg imaginal disc. In Drosophila, the expression boundary between the DmDll/Dmdac domain and distal DmDll domain corresponds to that between tarsal segments 1 and 2. Future tarsal segment 1 may be generated in the distal-most region of the Dmdac expression domain. In Gryllus, the distal limit of the Gbdac domain is likely to correspond to the boundary between tarsal segments 1 and 2 (Inoue, 2002).

From these results it was found that the expression patterns of the three genes are essentially conserved between Drosophila and Gryllus, although the time course of the pattern varies according to the developmental mode. The following conclusions were reached: (1) at early stages, expressions of Gbhth and GbDll do not correspond to the future coxopodite and telopodite, but rather to the presumptive trochanter/femur boundary; (2) Gbdac expression subdivides the leg bud into the presumptive femur and more distal region; (3) the expression patterns of GbDll, Gbdac, and Gbhth in the fully segmented Gryllus leg are similar to those in the Drosophila late third instar disc (Inoue, 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).

Homothorax homologs in amphibians

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).

Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins

The mechanisms by which cells obtain instructions to precisely re-create the missing parts of an organ remain an unresolved question in regenerative biology. Urodele limb regeneration is a powerful model in which to study these mechanisms. Following limb amputation, blastema cells interpret the proximal-most positional identity in the stump to reproduce missing parts faithfully. Classical experiments showed the ability of retinoic acid (RA) to proximalize blastema positional values. Meis homeobox genes are involved in RA-dependent specification of proximal cell identity during limb development. To understand the molecular basis for specifying proximal positional identities during regeneration, the axolotl Meis homeobox family was isolated. Axolotl Meis genes are RA-regulated during both regeneration and embryonic limb development. During limb regeneration, Meis overexpression relocates distal blastema cells to more proximal locations, whereas Meis knockdown inhibits RA proximalization of limb blastemas. Meis genes are thus crucial targets of RA proximalizing activity on blastema cells (Mercader, 2005).

During vertebrate limb development, Meis1 and Meis2 act downstream of RA in a proximalizing pathway. The results of this study show that the same pathway used during limb development is re-used in regeneration to specify proximal identity. In contrast to its effect on urodele limb regeneration, RA does not induce obvious PD duplications in the developing chick limb. A possible explanation is that during normal limb development, reprogrammed cells have the chance to migrate and integrate into proximal compartments, whereas during regeneration, they are 'trapped' in a distal region and are thus forced to generate an ectopic proximal compartment (Mercader, 2005).

During amniote limb development, the limit of Meis gene expression in the patterned chicken limb corresponds to the boundary between the stylopod and the zeugopod. Localized inhibition of RA signaling in the chick disrupts skeletal elements proximal to the elbow/knee, but does not affect regions distal to this point. Conversely, localized RA excess disrupts elements distal to the elbow/knee but spares elements proximal to this point. These results suggested a major subdivision of the limb bud into an RA-Meis domain that generates regions proximal to the stylopod/zeugopod boundary, and an RA-Meis-negative area that generates regions beyond this boundary. Meis gene expression and regulation through RA is conserved between amniote and amphibian limb development. Nonetheless, in the chick, RA exposure or Meis overexpression does not impose a stylopod character, but proximalizes cells at any distal position in a graded manner. Similar results are obtained after RA exposure during limb regeneration; duplications induced by decreasing amounts of RA result in increasing distalization of the proximal-most identity of the regenerate. The alterations in PD identity promoted by changes in Meis activity seem to follow similar rules; Meis overexpression proximalizes cell affinity at any position of the regenerating PD axis, and Meis knockdown distalizes the proximal-most identity of the regenerate, irrespective of the initial specification status of the blastema. These observations suggest that positional information is encoded uniformly, and is recognized continuously along the PD axis during both limb development and regeneration. In addition, the components of the positional code appear to remain sensitive to the RA-Meis pathway at any PD position (Mercader, 2005).

Meis regulation is mainly transcriptional in amniotes, whereas in the axolotl it appears to be at least partially regulated by translational and/or by subcellular protein localization. Mouse Meis proteins and their Drosophila ortholog Hth are translated and imported into the nucleus constitutively, whereas mouse Pbx1 and its Drosophila ortholog Exd require Meis/Hth expression to be imported into the nucleus and become functional. Data from Xenopus, zebrafish and spiders provide examples of the opposite situation; Pbx/Exd proteins are expressed constitutively in the nucleus whereas Meis/Hth proteins remain in the cytoplasm by default, and become nuclear only after co-expression with Pbx/Exd. These observations suggest that the mechanisms for the regulation of Meis and Pbx activity have remained labile during evolution. This flexibility would only be possible if, as suggested by most Meis/Pbx functional experiments, Meis and Pbx proteins always work together, with no functional independence for each partner. Any change in the ability of a partner to enter the nucleus on its own would have no functional consequence, and would thus be unrestricted during evolution. In the current experiments, however, Pbx neither induced alterations in cell behavior nor substantially changed the phenotype induced by Meis protein overexpression. These results might indicate that Meis alone can reprogram limb cell PD identity, although it is most likely to interact with Pbx proteins constitutively expressed in limb blastemas (Mercader, 2005).

Another interesting aspect of limb PD specification mechanisms is the regulation of cell affinity. PD axial level-specific affinity is a hallmark of PD identity, and is modulated by RA during development and regeneration. The fact that Meis rapidly induces cell relocation in the early blastema suggests that PD-specific affinity is already established at this stage. Cell lineage tracing and transplantation experiments have also suggested an early PD sub-division of the limb blastema. Alterations of Meis activity at later stages similarly induced cell relocation, but within a restricted subregion of the regenerate, suggesting progressive PD compartmentalization of the blastema, or progressive limitation in cell migratory ability (Mercader, 2005).

The GPI-anchored Prod1 molecule is downstream of RA in the proximalizing pathway, at least as part of the PD affinity code. Prod1 appears to form part of the positional memory system that enables blastema cells to 'know' which limb parts are missing. By contrast, no evidence was found of Meis genes as part of the positional memory system, suggesting that the Meis pathway belongs exclusively to the patterning network re-activated after amputation. In this case, after proximal amputation, the Meis pathway would be activated by the positional memory system, so that the initial Prod1 status may determine the level of Meis activation. The answers to these questions must await isolation of the axolotl Prod1 counterpart (Mercader, 2005).

Meis1 related genes: Patterns of expression

Knotted (Kn) genes are expressed within restricted domains of the plant meristems and play a key role in the control of plant morphogenesis. The recently isolated Kn-related murine gene Meis2 labels the lateral somitic compartment and its derivatives during early mouse embryogenesis and later becomes a marker for the dorso-ectodermal region, overlying cells of the paraxial mesoderm. Meis2 is also highly expressed in specific areas of the developing central nervous system from embryonic day 9 (e9) onward. In later developmental stages, a strong expression is detectable in differentiating nuclei and regions of the forebrain, midbrain, hindbrain, and spinal cord. This temporal and spatial expression pattern suggests that Meis2 may play an important role in the cascade of induction leading to somitic differentiation as well as in brain regionalization and histogenesis (Cecconi, 1997).

Meis1 related genes: Homothorax homologs involved in oncogenesis

Leukemia results from the accumulation of multiple genetic alterations that disrupt the control mechanisms of normal growth and differentiation. The use of inbred mouse strains that develop leukemia has greatly facilitated the identification of genes that contribute to the neoplastic transformation of hematopoietic cells. BXH-2 mice develop myeloid leukemia as a result of the expression of an ecotropic murine leukemia virus that acts as an insertional mutagen to alter the expression of cellular proto-oncogenes. A new locus, Meis1, serves as a site of viral integration in 15% of the tumors arising in BXH-2 mice. Meis1 was mapped to a distinct location on proximal mouse chromosome 11, suggesting that it represents a novel locus. Analysis of somatic cell hybrids that segregate human chromosomes pinpoints the localization of MEIS1 to human chromosome 2p23-p12, in a region known to contain translocations found in human leukemias. Northern (RNA) blot analysis demonstrates that a Meis1 probe detects a 3.8-kb mRNA present in all BXH-2 tumors, whereas tumors containing integrations at the Meis1 locus express an additional truncated transcript. A Meis1 cDNA clone that encodes a novel member of the homeobox gene family has been identified. The homeodomain of Meis1 is most closely related to those of the PBX/exd family of homeobox protein-encoding genes, suggesting that Meis1 functions in a similar fashion by cooperative binding to a distinct subset of HOX proteins. Collectively, these results indicate that altered expression of the homeobox gene Meis1 may be one of the events that lead to tumor formation in BXH-2 mice (Moskow, 1997).

Stra10 is a novel retinoic acid-inducible gene in P19 embryonal carcinoma cells. Four murine cDNA isoforms have been isolated that are likely to result from alternative splicing. The predicted protein sequences exhibit approximately 85% identity with the Pbx-related Meis1 homeobox gene products, which are involved in myeloid leukemia in BXH-2 mice, and one of the Stra10 isoforms corresponds to the recently published Meis2 sequence. The Meis2 homeodomain is identical to that of Meis1, and is most closely related to homeodomains of the Pbx/TGIF homeobox gene products. The Meis2 gene displays spatially restricted expression patterns in the developing nervous system, limbs, face, and in various viscera. In adult mice, Meis2 is mainly expressed in the brain and female genital tract, with a different distribution of the alternative splice forms in these organs (Oulad-Abdelghani, 1997).

The Meis1 locus was isolated and found to be a common site of viral integration, involved in myeloid leukemia in BXH-2 mice. Meis1 encodes a novel homeobox protein belonging to the TALE (three amino acid loop extension) family of homeodomain-containing proteins. The homeodomain of Meis1 is the only known motif within the entire 390-amino-acid protein. Southern blot analyses using the Meis1 homeodomain as a probe revealed the existence of a family of Meis1-related genes (Mrgs) in several diverged species. The 3' untranslated region (UTR) of Meis1 is remarkably conserved in evolution. To gain a further understanding of the role Meis1 plays in leukemia and development, as well as to identify conserved regions of the protein that might elucidate function, Mrgs from the mouse and human genomes were cloned and characterized. The sequence of Mrg1 and MRG2 as well as their chromosomal locations in murine and human genomes is now known. Both Mrgs share a high degree of sequence identity with the protein coding region of Meis1. The Xenopus laevis ortholog (XMeis1) has also been cloned. Sequence comparison of the murine and Xenopus clones reveals that Meis1 is highly conserved throughout its coding sequence as well as the 3' UTR. Comparison of Meis1 and the closely related Mrgs to known homeoproteins suggests that Meis1 represents a new subfamily of TALE homeobox genes (Steelman, 1997).

Hoxa9, Meis1 and Pbx1 encode homeodomain containing proteins implicated in leukemic transformation in both mice and humans. Hoxa9, Meis1 and Pbx1 proteins have been shown to physically interact with each other, as Hoxa9 cooperatively binds consensus DNA sequences with Meis1 and with Pbx1, while Meis1 and Pbx1 form heterodimers in both the presence and absence of DNA. Could Hoxa9 transform hemopoietic cells in collaboration with either Pbx1 or Meis1? Primary bone marrow cells, retrovirally engineered to overexpress Hoxa9 and Meis1a simultaneously, induce growth factor-dependent oligoclonal acute myeloid leukemia in 3 months when transplanted into syngenic mice. In contrast, overexpression of Hoxa9, Meis1a or Pbx1b alone, or the combination of Hoxa9 and Pbx1b fail to transform these cells acutely within 6 months post-transplantation. Similar results were obtained when FDC-P1 cells, engineered to overexpress these genes, are transplanted to syngenic recipients. Thus, these studies demonstrate a selective collaboration between a member of the Hox family and one of its DNA-binding partners in transformation of hemopoietic cells (Kroon, 1998).

Meis1 related proteins: copartners of Hox proteins

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).

Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and has been identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. The Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. The Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs (Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13) also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets (Shen, 1997).

The Pbx1 and Meis1 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).

The expression of Meis1 (a novel Pbx-related homeobox gene) and either Hoxa7 or Hoxa9 are coactivated by retroviral integration in BXH2 murine myeloid leukemias. Since Pbx proteins are Hox cofactors, cooperatively binding DNA with Hox proteins to modulate the otherwise similar DNA binding specificities of Hox proteins, these results suggested that Meis1 may function as a cofactor for Hoxa7 and Hoxa9 in the induction of murine myeloid leukemias. Using DNA cross-hybridization, two Meis1-related genes, Meis2 and Meis3, have been identified. Sequence analysis reveals extensive amino acid similarity among the three Meis proteins, both within and outside of the homeodomain. Phylogenetic analysis showsthat the Meis genes belong to a distinct family of Pbx-related genes. Chromosome mapping studies indicate that Meis2 and Meis3 are unlinked to Meis1 and map to mouse chromosome 2 and 7, respectively. The Meis genes display distinct but overlapping patterns of expression in normal tissues and are expressed in some cases of murine myeloid leukemia. The identification of two additional Meis genes identifies a new family of potential Hox cofactors as well as two new potential disease genes (Nakamura, 1996b).

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, 1997).

Retroviruses induce myeloid leukemia in BXH-2 mice by the insertional mutation of cellular proto-oncogenes or tumor suppressor genes. Disease genes can thus be identified by proviral tagging through the identification of common viral integration sites in BXH-2 leukemia. This paper describes a new approach for proviral tagging that greatly facilitates the identification of BXH-2 leukemia genes. Using this approach, three genes have been identified whose expression is activated by proviral integration in BXH-2 leukemias: Hoxa7, Hoxa9, and a Pbx1-related homeobox gene, Meis1. Proviral activation of Hoxa7 or Hoxa9 is strongly correlated with proviral activation of Meis1, implying that Hoxa7 and Hoxa9 cooperate with Meis1 in leukemia formation. These studies provide the first genetic evidence that Pbx1-related genes cooperate with Hox genes in leukemia formation and identify a number of new murine myeloid leukemia genes (Nakamura, 1996a).

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 (see Drosophila PKA), 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 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, 1998).

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, 1998).

HOX proteins and some orphan homeodomain proteins form complexes with either PBX or MEIS subclasses of homeodomain proteins. This interaction can increase the binding specificity and transcriptional effectiveness of the HOX partner. Specific members of both PBX and MEIS subclasses are shown to form a multimeric complex with the pancreatic homeodomain protein PDX1 and switch the nature of PDX1's transcriptional activity. The two activities of PDX1 are exhibited through the 10-bp B element of the transcriptional enhancer of the pancreatic elastase I gene (ELA1). In pancreatic acinar cells the activity of the B element requires other elements of the ELA1 enhancer; in beta-cells the B element can activate a promoter in the absence of other enhancer elements. In acinar cell lines the activity is mediated by a complex comprising PDX1, PBX1b, and MEIS2. In contrast, beta-cell lines are devoid of PBX1b and MEIS2, so that a trimeric complex does not form, and the beta-cell-type activity is mediated by PDX1 without PBX1b and MEIS2. The presence of specific nuclear isoforms of PBX and MEIS is precisely regulated in a cell-type-specific manner. The beta-cell-type activity can be detected in acinar cells if the B element is altered to retain binding of PDX1 but prevent binding of the PDX1-PBX1b-MEIS2 complex. These observations suggest that association with PBX and MEIS partners controls the nature of the transcriptional activity of the organ-specific PDX1 transcription factor in exocrine versus endocrine cells (Swift, 1998).

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).

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

AbdB-like HOX proteins form DNA-binding complexes with the TALE superclass proteins MEIS1A and MEIS1B, and trimeric complexes have been identified in nuclear extracts that include a second TALE protein, PBX. Thus, soluble DNA-independent protein-protein complexes exist in mammals. The extent of HOX/TALE superclass interactions, protein structural requirements, and sites of in vivo cooperative interaction have not been fully explored. Hoxa13 and Hoxd13 expression has been shown to not overlap with that of Meis1-3 in the developing limb; however, coexpression occurs in the developing male and female reproductive tracts (FRTs). Both HOXA13 and HOXD13 associate with MEIS1B in mammalian and yeast cells, and HOXA13 can interact with all MEIS proteins but not more diverged TALE superclass members. In addition, the C-terminal domains (CTDs) of MEIS1A (18 amino acids) and MEIS1B (93 amino acids) are necessary for HOXA13 interaction; for MEIS1B, this domain is also sufficient. Yeast two hybrid studies reveal that MEIS proteins can interact with anterior HOX proteins, but for some, additional N-terminal MEIS sequences are required for interaction. Using deletion mutants of HOXA13 and HOXD13, evidence is provided for multiple HOX peptide domains interacting with MEIS proteins. These data suggest that HOX:MEIS interactions may extend to non-AbdB-like HOX proteins in solution and that differences may exist in the MEIS peptide domains utilized by different HOX groups. Finally, the capability of multiple HOX domains to interact with MEIS C-terminal sequences implies greater complexity of the HOX:MEIS protein-protein interactions and a larger role for variation of HOX amino-terminal sequences in specificity of function (Williams, 2005).

Polycomb potentiates meis2 activation in midbrain by mediating interaction of the promoter with a tissue-specific enhancer

Polycomb-group (PcG) proteins mediate repression of developmental regulators in a reversible manner, contributing to their spatiotemporally regulated expression. However, it is poorly understood how PcG-repressed genes are activated by developmental cues. This study used the mouse Meis2 gene as a model to identify a role of a tissue-specific enhancer in removing PcG from the promoter. Meis2 repression in early development depends on binding of RING1B, an essential E3 component of PcG, to its promoter, coupled with its association with another RING1B-binding site (RBS) at the 3' end of the Meis2 gene. During early midbrain development, a midbrain-specific enhancer (MBE) transiently associates with the promoter-RBS, forming a promoter-MBE-RBS tripartite interaction in a RING1-dependent manner. Subsequently, RING1B-bound RBS dissociates from the tripartite complex, leaving promoter-MBE engagement to activate Meis2 expression. This study therefore demonstrates that PcG and/or related factors play a role in Meis2 activation by regulating the topological transition of cis-regulatory elements (Kondo, 2014).

Meis1 related proteins: transcriptional targets

Three-amino acid extension loop (TALE) homeobox proteins are highly conserved transcription regulators. Two members of this family, Meis2 and TGIF, which frequently have overlapping consensus binding sites on complementary DNA strands in opposite orientations, can function competitively. For example, in the D(1A) gene, which encodes the predominant dopamine receptor in the striatum, Meis2 and TGIF bind to the activator sequence ACT (-1174 to -1154) and regulate transcription differentially in a cell type-specific manner. Among the five cloned splice variants of Meis2, isoforms Meis2a-d activate the D(1A) promoter in most cell types tested, whereas TGIF competes with Meis2 binding to DNA and represses Meis2-induced transcription activation. Consequently, Meis2 cannot activate the D(1A) promoter in a cell that has abundant TGIF expression. The Meis2 message is highly co-localized with the D(1A) message in adult striatal neurons, whereas TGIF is barely detectable in the adult brain. These observations provide in vitro and in vivo evidence that Meis2 and TGIF differentially regulate their target genes. Thus, the delicate ratio between Meis2 and TGIF expression in a given cell type determines the cell-specific expression of the D(1A) gene. Splice variant Meis2e, which has a truncated homeodomain, cannot bind to the D(1A) ACT sequence or activate transcription. However, Meis2e is an effective dominant negative regulator by blocking Meis2d-induced transcription activation. Thus, truncated homeoproteins with no DNA binding domains can have important regulatory functions (Yang, 2000).

Pax6 is a pivotal regulator of eye development throughout Metazoa, but the direct upstream regulators of vertebrate Pax6 expression are unknown. In vertebrates, Pax6 is required for formation of the lens placode, an ectodermal thickening that precedes lens development. The Meis1 and Meis2 homeoproteins are direct regulators of Pax6 expression in prospective lens ectoderm. In mice, Meis1 and Meis2 are developmentally expressed in a pattern remarkably similar to Pax6 and their expression is Pax6-independent. Biochemical and transgenic experiments reveal that Meis1 and Meis2 bind a specific sequence in the Pax6 lens placode enhancer that is required for its activity. Furthermore, Pax6 and Meis2 exhibit a strong genetic interaction in lens development, and Pax6 expression is elevated in lenses of Meis2-overexpressing transgenic mice. When expressed in embryonic lens ectoderm, dominant-negative forms of Meis down-regulate endogenous Pax6. These results contrast with those in Drosophila, where the single Meis homolog, Homothorax, has been shown to negatively regulate eye formation. Therefore, despite the striking evolutionary conservation of Pax6 function, Pax6 expression in the vertebrate lens is uniquely regulated (Zhang, 2002).

Despite the striking evolutionary conservation of the Pax6 pathway, it is interesting to contrast the current result with Drosophila, where the sole Meis homolog, Homothorax (Hth), suppresses eye formation in the ventral half of the eye and has been proposed to delimit the eye field. Meis/Homothorax function in controlling anterior-posterior embryonic patterning and proximo-distal limb development is conserved between Drosophila and vertebrates. Nevertheless, the fact that Meis1 and Meis2 are expressed throughout the developing lens, retina, and cornea further indicates that Meis1 and Meis2 cannot play exclusively repressive roles in vertebrate eye development. In addition, although the Meis binding site in the Pax6 lens enhancer is conserved from fish to human, it does not appear to be present in the eye imaginal disc enhancer of the Drosophila Pax6 homolog eyeless. In fact, when the eyeless enhancer was introduced into transgenic mice, it reproduced endogenous Pax6 expression in retina and spinal cord, but not in lens. Therefore, the mechanism of Pax6 regulation in the vertebrate lens placode differs in at least one respect from that in the Drosophila eye imaginal disc. Although Pax6 function is evolutionarily conserved, potentially indicating a monophyletic origin for the eye, it is attractive to suggest that divergent mechanisms regulating Pax6 expression also exist. These enhancer-specific differences may underlie the unique aspects of oculogenesis in vertebrates and invertebrates (Zhang, 2002).

The patterning of the cardiovascular system into systemic and pulmonic circulations is a complex morphogenetic process, the failure of which results in clinically important congenital defects. This process involves extensive vascular remodeling and coordinated division of the cardiac outflow tract (OFT). The homeodomain transcription factor Pbx1 orchestrates separate transcriptional pathways to control great-artery patterning and cardiac OFT septation in mice. Pbx1-null embryos display anomalous great arteries owing to a failure to establish the initial complement of branchial arch arteries in the caudal pharyngeal region. Pbx1 deficiency also results in the failure of cardiac OFT septation. Pbx1-null embryos lose a transient burst of Pax3 expression in premigratory cardiac neural crest cells (NCCs) that ultimately specifies cardiac NCC function for OFT development, but does not regulate NCC migration to the heart. Pbx1 directly activates Pax3, leading to repression of its target gene Msx2 in NCCs. Compound Msx2/Pbx1-null embryos display significant rescue of cardiac septation, demonstrating that disruption of this Pbx1-Pax3-Msx2 regulatory pathway partially underlies the OFT defects in Pbx1-null mice. Conversely, the great-artery anomalies of compound Msx2/Pbx1-null embryos remain within the same spectrum as those of Pbx1-null embryos. Thus, Pbx1 makes a crucial contribution to distinct regulatory pathways in cardiovascular development (Chang, 2008).

In vitro studies were conducted to further assess the potential role of Pbx1 in the transcriptional regulation of Pax3, which contains Pbx1 binding sites in its promoter. Electrophoretic mobility shift assays (EMSA) confirmed that Site A, which contains a consensus Pbx1/Meis1 binding sequence (5'-TGACAGTT-3'), supported robust cooperative binding by Pbx1 and Meis1, but not binding by either protein alone. By contrast, Pbx1 did not form binding complexes with several representative Hox proteins (HoxB2, HoxB4 or HoxB7) on Site A. Site B, which is located 1.1 kb upstream of the Pax3 transcriptional start site, was bound robustly by HoxB4 or Meis1 in the presence of Pbx1. DNA binding by HoxB2 and HoxB7 on Site B was also dependent on Pbx1. Pbx1-Meis1-Hox trimeric complexes did not form on either isolated Site A or Site B. The requirement for Pbx1 in regulating Pax3 promoter activity was assessed using a reporter gene containing the 1.6 kb Pax3 promoter fragment in PC12 pheochromocytoma cells, which are derivatives of NCCs. Whereas HoxB4 alone produced a modest increase in Pax3 promoter activity, co-transfection of Pbx1 and HoxB4 strongly activated transcription. Consistent with the binding studies, adding Meis1 to the transfection mixture did not further enhance the Pax3 transcriptional response. Thus, Pbx1 partners with Meis and Hox proteins to directly activate expression of Pax3 through its proximal promoter elements. Taken together, these results show that Pbx1 is essential for Pax3 proximal promoter activity and for transient premigratory cardiac NCC expression of Pax3. It is proposed that activity of the Pax3 1.6 kb proximal promoter in vivo partially reflects Pbx1-dependent Pax3 expression in premigratory cardiac NCCs. The broader and sustained dorsal neural tube expression of Pax3 is likely to require additional regulatory elements outside the 1.6 kb region that are not under Pbx1 control (Chang, 2008).

How transcription factors interpret the cis-regulatory logic encoded within enhancers to mediate quantitative changes in spatiotemporally restricted expression patterns during animal development is not well understood. Pax6 is a dosage-sensitive gene essential for eye development. This study identified the Prep1 (pKnox1) transcription factor, a homeobox gene of the TALE superfamily that was identified along with closely related Meis TFs as a regulator of the Pax6 pancreatic enhancer, as a critical dose-dependent upstream regulator of Pax6 expression during lens formation. Prep1 activates the Pax6 lens enhancer by binding to two phylogenetically conserved lower-affinity DNA-binding sites. Finally, a mechanism is described whereby Pax6 levels are determined by transcriptional synergy of Prep1 bound to the two sites, while timing of enhancer activation is determined by binding site affinity (Rowan, 2010).

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).

Proliferative and transcriptional identity of distinct classes of neural precursors in the mammalian olfactory epithelium

Neural precursors in the developing olfactory epithelium (OE) give rise to three major neuronal classes - olfactory receptor (ORNs), vomeronasal (VRNs) and gonadotropin releasing hormone (GnRH) neurons. Nevertheless, the molecular and proliferative identities of these precursors are largely unknown. Two precursor classes were characterized in the olfactory epithelium (OE) shortly after it becomes a distinct tissue at midgestation in the mouse: slowly dividing self-renewing precursors that express Meis1/2 at high levels, and rapidly dividing neurogenic precursors that express high levels of Sox2 and Ascl1. Precursors expressing high levels of Meis genes primarily reside in the lateral OE, whereas precursors expressing high levels of Sox2 and Ascl1 primarily reside in the medial OE. Fgf8 maintains these expression signatures and proliferative identities. Using electroporation in the wild-type embryonic OE in vitro as well as Fgf8, Sox2 and Ascl1 mutant mice in vivo, it was found that Sox2 dose and Meis1 -- independent of Pbx co-factors -- regulate Ascl1 expression and the transition from lateral to medial precursor state. Thus, proliferative characteristics and a dose-dependent transcriptional network were characterized that define distinct OE precursors: medial precursors that are most probably transit amplifying neurogenic progenitors for ORNs, VRNs and GnRH neurons, and lateral precursors that include multi-potent self-renewing OE neural stem cells (Tucker, 2010).

Subcellular localization of multiple PREP2 isoforms is regulated by actin, tubulin, and nuclear export

The PREP, MEIS, and PBX families are mammalian members of the TALE (three amino acid loop extension) class of homeodomain-containing transcription factors. These factors have been implicated in cooperative DNA binding with the HOX class of homeoproteins, but PREP and MEIS interact with PBX in apparently non-HOX-dependent cooperative DNA binding as well. PREP, MEIS, and PBX have all been reported to reside in the cytoplasm in one or more tissues of the developing vertebrate embryo. In the case of PBX, cytoplasmic localization is due to the modulation of nuclear localization signals, nuclear export sequences, and interaction with a cytoplasmic anchoring factor, non-muscle myosin heavy chain II B. Murine PREP2 exists in multiple isoforms distinguished by interaction with affinity-purified antibodies raised to N- and C-terminal epitopes and by nuclear versus cytoplasmic localization. Alternative splicing gives rise to some of these PREP2 isoforms, including a 25-kDa variant lacking the C-terminal half of the protein and homeodomain and having the potential to act as dominant-negative. Cytoplasmic localization is due to the concerted action of nuclear export, as evidenced by sensitivity to leptomycin B, and cytoplasmic retention by the actin and microtubule cytoskeletons. Cytoplasmic PREP2 colocalizes with both the actin and microtubule cytoskeletons and coimmunoprecipitates with actin and tubulin. Importantly, disruption of either cytoskeletal system redirects cytoplasmic PREP2 to the nucleus. It is suggested that transcriptional regulation by PREP2 is modulated through the subcellular distribution of multiple isoforms and by interaction with two distinct cytoskeletal systems (Haller, 2004: full text of article).

Meis1 related proteins: roles in development

The mechanisms controlling growth and patterning along the proximal-distal axis of the vertebrate limb are yet to be understood. Restriction of expression of the homeobox gene Meis2 to proximal regions of the limb bud is essential for limb development, since ectopic Meis2 severely disrupts limb outgrowth. An antagonistic relationship between the secreted factors Gremlin and BMPs required to maintain the Shh/FGF loop that regulates distal outgrowth has been uncovered. These proximal and distal factors have coordinated activities: Meis2 can repress distal genes, and Bmps and Hoxd genes restrict Meis2 expression to the proximal limb bud. Moreover, combinations of BMPs and AER factors are sufficient to distalize proximal limb cells. These results unveil a novel set of proximal-distal regulatory interactions that establish and maintain outgrowth of the vertebrate limb (Capdevila, 1999).

Vertebrate limbs develop in a temporal proximodistal sequence, with proximal regions specified and generated earlier than distal ones. Whereas considerable information is available on the mechanisms promoting limb growth, those involved in determining the proximodistal identity of limb parts remain largely unknown. Retinoic acid (RA) is an upstream activator of the proximal determinant genes Meis1 and Meis2. RA promotes proximalization of limb cells and endogenous RA signaling is required to maintain the proximal Meis domain in the limb. RA synthesis and signaling range, which initially span the entire lateral plate mesoderm, become restricted to proximal limb domains by the apical ectodermal ridge (AER) activity following limb initiation. Fibroblast growth factor (FGF) has been identified as the main molecule responsible for this AER activity and a model is proposed integrating the role of FGF in limb cell proliferation, with a specific function in promoting distalization through inhibition of RA production and signaling (Mercader, 2000).

The progress zone (PZ) model currently explains how limb cells acquire their PD identity in the PZ and become increasingly distalized with time. The first requirement for distalization would be sufficient cell divisions in the PZ. FGFs, as factors essential for PZ cell proliferation, are required for limb cell distalization, and different FGFs can induce the development of a complete limb from embryo flanks triggering the whole limb developmental program, including its distalization. A specific molecular mechanism by which FGFs could regulate limb distalization has not, however, been demonstrated until now. It is suggested that FGFs promote limb distalization by counteracting the RA pathway, which is essential to maintain the proximalizing Meis activity in the limb. This FGF activity is achieved by at least two different effects on the RA pathway: inhibition of RA synthesis by repressing Raldh2, and parallel direct inhibition of RA signaling, resulting in inactivation of Meis and other RA targets. As long as AER function is not affected, neither Meis activation, nor RA, inhibit limb growth during the PZ-dependent phase. The role of FGF in repressing RA/Meis pathway therefore does not appear to be related to the promotion of cell proliferation, but rather to a specific function in promoting distalization. In contrast, strong Meis activation in the AER or high RA doses applied directly beneath it, destroy the AER and lead to limb truncations. The AER thus appears to be a structure especially sensitive to RA/Meis activity. The strong expression of the RA-degrading enzyme Cyp26 in the cells delimiting the AER may preserve it from RA/Meis pathway activation during early stages of limb development. Whereas FGF signaling might be the principal and primary signal involved in Meis restriction, other diffusible molecules such as BMP and Wnt, which can also inhibit Meis expression, are likely to cooperate in this role (Mercader, 2000).

Fibroblast growth factor (FGF) has been proposed to be involved in the specification and patterning of the developing vertebrate nervous system. There is conflicting evidence, however, concerning the requirement for FGF signaling in these processes. To provide insight into the signaling mechanisms that are important for neural induction and anterior-posterior neural patterning, the dominant negative Ras mutant, N17Ras, was employed, in addition to a truncated FGF receptor (XFD). Both N17Ras and XFD, when expressed in Xenopus laevis animal cap ectoderm, inhibit the ability of FGF to generate neural pattern. They also block induction of posterior neural tissue by XBF2 and XMeis3. However, neither XFD nor N17Ras inhibits noggin, neurogenin, or XBF2 induction of anterior neural markers. MAP kinase activation has been proposed to be necessary for neural induction, yet N17Ras inhibits the phosphorylation of MAP kinase that usually follows explantation of explants. In whole embryos, Ras-mediated FGF signaling is critical for the formation of posterior neural tissues but is dispensable for neural induction (Ribisi, 2000).

Posterior mesoderm tissue induces midbrain and hindbrain fates from prospective forebrain, an activity that is mimicked in explant culture by bFGF. Treatment of early gastrula age animal cap ectoderm with bFGF protein induces the expression of the spinal cord marker hoxB9. Late gastrula-age (stage 11) animal cap ectoderm treated with bFGF expresses the midbrain and hindbrain marker genes en2 and krox20, in addition to hoxB9, and the forebrain marker otx2 is not induced. The combination of somite tissue with animal caps of gastrula age (stage 10.5) induces the expression of hindbrain-specific genes and low levels of spinal cord-specific genes in the animal cap tissue and this induction is partially sensitive to XFD. Keller explants faithfully recapitulate the A-P distribution of neural markers observed in the whole embryo: blocking FGF signaling using XFD eliminates posterior neural development in Keller explants. The claim that FGF signaling is required for the formation of posterior neural tissue is supported by the results of explant assays. The induction of posterior neural markers requires FGF and Ras signaling. Animal caps do not express posterior neural markers in response to either XBF2 or XMeis3 when either FGF or Ras signaling is blocked. In addition, when MAPK activation is directly inhibited by MAP kinase phosphatase, the ability of XMeis3 to induce the expression of posterior neural markers is greatly curtailed (Ribisi, 2000 and references therein).

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).

Many Hox proteins are thought to require Pbx and Meis co-factors to specify cell identity during embryogenesis. Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish. Hoxb1b and Pbx4 act together to induce ectopic hoxb1a expression in rhombomere 2 of the hindbrain. In contrast, Hoxb1b and Pbx4 acting together with Meis3 induce hoxb1a, hoxb2, krox20 and valentino expression rostrally and cause extensive transformation of forebrain and midbrain fates to hindbrain fates, including differentiation of excess rhombomere 4-specific Mauthner neurons. This synergistic effect requires that Hoxb1b and Meis3 have intact Pbx-interaction domains, suggesting that their in vivo activity is dependent on binding to Pbx4. In the case of Meis3, binding to Pbx4 is also required for nuclear access. These results are consistent with Hoxb1b and Meis3 interacting with Pbx4 to form complexes that regulate hindbrain development during zebrafish embryogenesis (Vlachakis, 2001).

Notably, these experiments do not indicate the composition of Hoxb1b-, Pbx4- and Meis3-containing complexes, and several issues remain to be resolved. (1) It is not clear if all complexes contain a Meis family member. Ectopic expression of Hoxb1b by itself induces hoxb1a expression in r2. To perform this function, Hoxb1b needs to interact with an endogenous Pbx protein (most likely Pbx4 as this is the predominant Pbx protein at this stage), but does not require exogenous Meis3. While this is consistent with Hoxb1b and Pbx4 acting in the absence of a Meis protein, it leaves open the possibility that an endogenous Meis protein is involved. Indeed, the zebrafish prep1 gene appears to be ubiquitously expressed and endogenous Prep1 may interact with Hoxb1b and Pbx4 in these experiments. However, while the Pbx and Hox binding sites appear to be required for in vivo expression of most Hox-dependent genes, a Meis/Prep1 binding site is only required for some genes. Meis proteins may therefore not always be required (at least not as a DNA binding component) for Pbx and Hox proteins to function in vivo (Vlachakis, 2001).

(2) Different Hox proteins have different effects in vivo, but it is not clear if different Meis family members differ functionally. For instance, if Hoxb1b and Pbx4 require Prep1 to activate hoxb1a expression, the synergistic effect seen following co-expression of Meis3 could be due to Prep1 being limiting in vivo. In this scenario, Prep1 and Meis3 would be functionally equivalent. An alternative explanation to the synergistic effect is that exogenously supplied Meis3 provides a unique function, perhaps by replacing Prep1, thus playing an instructive role. The latter model is favored, primarily because published experiments suggest that Prep1 cannot substitute for Homothorax in Drosophila (Vlachakis, 2001 and references therein).

(3) Although both Hoxb1b and Meis3 appear to require Pbx4 interaction to be functional in vivo, it is not known whether Hoxb1b and Meis3 interact with the same Pbx4 molecule to form a trimeric complex, or whether they interact with separate Pbx4 molecules to form a pair of dimers. However, several pieces of data indicate the formation of trimeric complexes: (1) both the hoxb1 and hoxb2 r4 enhancers contain adjacent Hox/Pbx binding sites and a more distant Meis site, but there is no Pbx site near the Meis site; consistent with this, DNA fragments containing these sequences support formation of trimeric Hox/Pbx/Meis complexes, but not of a pair of dimers, in vitro; (2) a DNA-binding mutant Prep1 forms dimers with Pbx that bind DNA only very weakly. Therefore, if Meis3, Pbx4 and Hoxb1b act as a pair of dimers, a DNA-binding mutant of Meis3 (MutMeis3) should not be able to form a functional dimer with Pbx4 and should not have any in vivo activity. However, MutMeis3 still functions in vivo, as does a DNA-binding mutant Hth (Vlachakis, 2001).

(3) Misexpression of Xenopus Meis3 by itself has a minimal effect on krox20 and hoxb1 expression, but nevertheless mediates anterior deletions in Xenopus. This is in contrast with the analysis reported here, where zebrafish Meis3 requires Pbx4 and Hoxb1b for the transformation of anterior fates. Since lineage labeling was not utilized to analyze the deletions in Xenopus, it is not known if a distinct mechanism is at work, or if some Meis family members may be able to function independently of Pbx and Hox (Vlachakis, 2001 and references therein).

The effects mediated by Hoxb1b, Pbx4 and Meis3 co-expression are likely to be causally related and to occur in sequence. Since murine hoxb1 and hoxb2 have Pbx, Hox and Meis binding sites in their enhancers it is likely that zebrafish hoxb1a and hoxb2 are directly induced by Hoxb1b, Pbx4 and Meis3. Ectopic expression of hoxb2 induces krox20 and valentino expression in zebrafish, suggesting that these genes may be activated subsequent to hoxb2. Thus, expression of Hoxb1b, Pbx4 and Meis3 is sufficient to promote the differentiation of hindbrain fates, particularly r4 fates, and it is speculated that they normally perform this function within the caudal hindbrain during zebrafish embryogenesis (Vlachakis, 2001).

Meis-family homeobox proteins have been shown to regulate cell fate specification in vertebrate and invertebrate embryos. Ectopic expression of RNA encoding the Xenopus Meis3 (XMeis3) protein causes anterior neural truncations with a concomitant expansion of hindbrain and spinal cord markers in Xenopus embryos. In naive animal cap explants, XMeis3 activates expression of posterior neural markers in the absence of pan-neural markers. Supporting its role as a neural caudalizer, XMeis3 is expressed in the hindbrain and spinal cord. XMeis3 acts like a transcriptional activator, and its caudalizing effects can be mimicked by injecting RNA encoding a VP16-XMeis3 fusion protein. To address the role of endogenous XMeis3 protein in neural patterning, XMeis3 activity was antagonized by injecting RNA encoding an Engrailed-XMeis3 antimorph fusion protein or XMeis3 antisense morpholino oligonucleotides. In these embryos, anterior neural structures are expanded and posterior neural tissues from the midbrain-hindbrain junction through the hindbrain are perturbed. In neuralized animal cap explants, XMeis3-antimorph protein modifies caudalization by basic fibroblast growth factor and Wnt3a. XMeis3-antimorph protein does not inhibit caudalization per se, but re-directs posterior neural marker expression to more anterior levels; it reduces expression of spinal cord and hindbrain markers, yet increases expression of the more rostral En2 marker. These results provide evidence that XMeis3 protein in the hindbrain is required to modify anterior neural-inducing activity, thus, enabling the transformation of these cells to posterior fates (Dibner, 2001).

Knockdown studies in Xenopus have demonstrated that the XMeis3 gene is required for proper hindbrain formation. An explant assay was developed to distinguish between autonomous and inductive activities of XMeis3 protein. Animal cap explants caudalized by XMeis3 were recombined with explants neuralized by the BMP dominant-negative receptor protein. XMeis3-expressing cells induce convergent extension cell elongations in juxtaposed neuralized explants. Elongated explants express hindbrain and primary neuron markers, and anterior neural marker expression is extinguished. Cell elongation is dependent on FGF/MAP-kinase and Wnt-PCP activities. XMeis3 activates FGF/MAP-kinase signaling, which then modulates the PCP pathway. In this manner, XMeis3 protein establishes a hindbrain-inducing center that determines anteroposterior patterning in the brain (Aamar, 2004).

Pbx1 is the product of a proto-oncogene originally discovered at the site of chromosomal translocations in acute leukemias. It binds DNA as a complex with a broad subset of homeodomain proteins, but its contributions to hematopoiesis have not been established. This paper reports that Pbx1 is expressed in hematopoietic progenitors during murine embryonic development and that its absence results in severe anemia and embryonic lethality at embryonic day 15 (E15) or E16. Definitive myeloerythroid lineages are present in Pbx1-/- fetal livers, but the total numbers of colony-forming cells are substantially reduced. Fetal liver hypoplasia reflects quantitative as well as qualitative defects in the most primitive multilineage progenitors and their lineage-restricted progeny. Hematopoietic stem cells from Pbx1-/- embryos have reduced colony-forming activity and are unable to establish multilineage hematopoiesis in competitive reconstitution experiments. Common myeloid progenitors (CMPs), the earliest known myeloerythroid-restricted progenitors, are markedly depleted in Pbx1-/- embryos at E14 and display clonogenic defects in erythroid colony formation. Comparative cell-cycle indexes suggest that these defects result largely from insufficient proliferation. Megakaryocyte- and erythrocyte-committed progenitors are also reduced in number and show decreased erythroid colony-forming potential. Taken together, these data indicate that Pbx1 is essential for the function of hematopoietic progenitors with erythropoietic potential and that its loss creates a proliferative constriction at the level of the CMP. Thus, Pbx1 is required for the maintenance, but not the initiation, of definitive hematopoiesis and contributes to the mitotic amplifications of progenitor subsets through which mature erythrocytes are generated (DiMartino, 2001).

Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. The zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. Meis functions in the same pathway as Pbx in zebrafish hindbrain development, since expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to those of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr- phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. These results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels (Waskiewicz, 2001).

Meis homeodomain proteins function as Hox-cofactors by binding Pbx and Hox proteins to form multimeric complexes that control transcription of genes involved in development and differentiation. It is not known what role Meis proteins play in these complexes, nor is it clear which Hox functions require Meis proteins in vivo. A divergent Meis family member, Prep1, acts as a Hox co-factor in zebrafish. This suggests that all Meis family members have at least one shared function and that this function must be carried out by a conserved domain. The Meinox domain, an N-terminal conserved domain shown to mediate Pbx binding, is sufficient to provide Meis activity to a Pbx/Hox complex. This activity is separable from Pbx binding and resides within the M1 subdomain. This finding also presents a rational strategy for interfering with Meis activity in vivo. This was accomplished by expressing the Pbx4/Lzr N-terminus, which sequesters Meis proteins in the cytoplasm away from the nuclear transcription complexes. Sequestering Meis proteins in the cytoplasm leads to extensive loss of rhombomere (r) 3- and r4-specific gene expression, as well as defective rhombomere boundary formation in this region. These changes in gene expression correlate with impaired neuronal differentiation in r3 and r4, e.g. the loss of r3-specific nV branchiomotor neurons and r4-specific Mauthner neurons. It is conclude that Meis family proteins are essential for the specification of r3 and r4 of the hindbrain (Choe, 2002).

The phenotype observed as a result of interfering with Meis activity is also qualitatively similar to that of the lazarus mutant (which carries a mutation in the pbx4 gene). Particularly, in both cases gene expression is affected primarily in r3 and r4 and less in r1, r2 or r5-r7. This suggests that Pbx and Meis function in the same pathway during hindbrain development. This is consistent with work in Drosophila, where the phenotypes of hth and exd mutants are largely indistinguishable and the genes are thought to act in the same pathway. An explanation for Meis and Pbx acting in the same pathway in the hindbrain probably comes from Meis proteins not interacting directly with Hox proteins expressed in the hindbrain (primarily paralog group 1-4), whereas Pbx proteins do interact directly with Hox proteins. Therefore, Meis proteins can only act as Hox cofactors in the hindbrain by binding to Pbx. The finding that Meis and Pbx loss-of-function give similar hindbrain phenotypes is therefore consistent with all hindbrain Hox functions that require Pbx also requiring Meis. However, although the meis loss-of-function and lazarus phenotypes are qualitatively similar, they differ quantitatively. Surprisingly, both a higher frequency and a more severe effect on hindbrain gene expression is observed in the absence of Meis function than reported for the lazarus mutant. It is speculated that this is unlikely to be a result of Pbx-independent effects of Meis proteins on Hox function, but may instead stem from the presence of maternal pbx4/lzr transcript, as well as additional pbx genes expressed in the lazarus mutant. If this is correct, complete removal of Pbx activity might be required to conclusively define the relative roles of Pbx and Meis in regulating Hox function (Choe, 2002).

In vitro studies have shown that Pbx1 regulates the activity of Ipf1 (also known as Pdx1), a ParaHox homeodomain transcription factor required for the development and function of the pancreas in mice and humans. To investigate in vivo roles of Pbx1 in pancreatic development and function, pancreatic Pbx1 expression, and morphogenesis, cell differentiation and function were examined in mice deficient for Pbx1. Pbx1-/- embryos have pancreatic hypoplasia and marked defects in exocrine and endocrine cell differentiation prior to death at embryonic day (E) 15 or E16. In these embryos, expression of Isl1 and Atoh5, essential regulators of pancreatic morphogenesis and differentiation, is severely reduced. Pbx1+/- adults have pancreatic islet malformations, impaired glucose tolerance and hypoinsulinemia. Thus, Pbx1 is essential for normal pancreatic development and function. Analysis of trans-heterozygous Pbx1+/- Ipf1+/- mice revealed in vivo genetic interactions between Pbx1 and Ipf1 that are essential for postnatal pancreatic function; these mice develop age-dependent overt diabetes mellitus, unlike Pbx1+/- or Ipf1+/- mice. Mutations affecting the Ipf1 protein may promote diabetes mellitus in mice and humans. This study suggests that perturbation of Pbx1 activity may also promote susceptibility to diabetes mellitus (Kim, 2002).

Pbx1 is required in the differentiation of urogenital organs, where Pbx1 is widely expressed in mesenchymal tissues. The complete lack of adrenal glands and formation of gonads displaying rudimentary sexual differentiation correlated with decreased cellular proliferation in Pbx1-/- genital ridges. Furthermore, expression of steroidogenic factor-1 (SF-1), a nuclear receptor essential for adrenal organogenesis, is reduced to minimal levels in Pbx1 mutants, indicating an upstream function for Pbx1 in adrenocortical development. Finally, loss of Pbx1 markedly reduces urogenital ridge outgrowth and results in impaired differentiation of the mesonephros and kidneys and the absence of Mullerian ducts. These findings establish a Pbx1-dependent pathway that regulates the expansion of SF-1 positive cells essential for adrenal formation and gonadal differentiation and demonstrate an early requirement for Pbx1 in urogenital development (Schnabel, 2003).

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

Pbx2 is one of four mammalian genes that encode closely related TALE homeodomain proteins, which serve as DNA binding partners for a subset of Hox transcription factors. The expression and contributions of Pbx2 to mammalian development remain undefined, in contrast to the essential roles recently established for family members Pbx1 and Pbx3. Pbx2 is widely expressed during embryonic development, particularly in neural and epithelial tissues during late gestation. Despite wide Pbx2 expression, mice homozygous mutant for Pbx2 are born at the expected Mendelian frequencies and exhibit no detectable abnormalities in development and organogenesis or reduction of long-term survival. The lack of an apparent phenotype in Pbx2-/- mice likely reflects functional redundancy, since the Pbx2 protein is present at considerably lower levels than comparable isoforms of Pbx1 and/or Pbx3 in embryonic tissues. In postnatal bone marrow and thymus, however, Pbx2 is the predominant high-molecular-weight isoform Pbx protein detectable by immunoblotting. Nevertheless, the absence of Pbx2 has no measurable effect on steady-state hematopoiesis or immune function in adult mice, suggesting possible compensation by low-MW-isoform Pbx proteins present in these tissues. It is concluded that the roles of Pbx2 in murine embryonic development, organogenesis, hematopoiesis, immune responses, and long-term survival are not essential (Selleri, 2004).

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).

Meis2 competes with the Groucho co-repressor Tle4 for binding to Otx2 and specifies tectal fate without induction of a secondary midbrain-hindbrain boundary organizer

The transcription factor Otx2 is expressed throughout the anterior neuroectoderm and is required for the formation of all forebrain- and midbrain-derived structures. The molecular determinants that cooperate with Otx2 to subdivide its expression domain into distinct functional units are, however, poorly understood at present. This study shows that the TALE-homeodomain protein Meis2 is expressed in the chick tectal anlage and is both necessary and sufficient for tectal development. Unlike known tectum-inducing genes, the ability of Meis2 to initiate tectal development does not involve the formation of a secondary midbrain-hindbrain boundary organizer, but instead requires direct interaction with Otx2. Using an Otx2-dependent reporter assay it was demonstrated that Meis2 competes with the Groucho co-repressor Tle4 (Grg4) for binding to Otx2 and thereby restores Otx2 transcriptional activator function. Together, these data suggest a model in which the balance between a co-repressor and a co-activator, which compete for binding to Otx2 in the mesencephalic vesicle, provides spatial and temporal control over tectal development. Controlled formation of Meis2-containing higher order protein complexes might thus serve as a general mechanism to achieve subdivision of the anterior neuroectoderm into distinct functional units during embryogenesis (Agoston, 2009).

Meis2 is a key regulator of tectal development. In contrast to other known genes involved in tectal development, Meis2 initiates tectal fate specification without inducing a secondary MHB organizer. Instead, Meis2 binds to Otx2 in the absence of DNA, competes with the co-repressor Tle4 for binding to Otx2 and thereby restores Otx2 transcriptional activator function. As discussed below, these results suggest a model in which the balance between a co-repressor and a co-activator, which compete for binding to Otx2 in the mesencephalic vesicle, provides spatial and temporal control over the onset of tectal development. These data thus argue for a novel, potentially DNA-independent function of TALE-homeodomain proteins: the controlled assembly and disassembly of transcription regulator complexes (Agoston, 2009).

Tectum development is induced when an ectopic Fgf8 source is generated in the prosencephalon through transplantation of an ectopic MHB organizer or implantation of Fgf8-releasing beads into the lateral wall of the diencephalon. In addition to Fgf8, several transcription factors can trigger tectal development upon misexpression, including Otx2, Pax2/5, En1/2 and Pax3/7. Unlike Meis2, expression of these proteins is not specific for the tectal anlage. Moreover, each of these proteins participates in the interdependent, positive maintenance loop at the MHB organizer and, consequently, induces ectopic expression of MHB marker genes, including Fgf8, when misexpressed. These molecules therefore evoke tectal development indirectly through formation of an ectopic MHB organizer. Meis2, by contrast, is unique as it can initiate tectal development without participating in MHB organizer function or maintenance. Endogenous Meis2 expression is repressed when metencephalic development is experimentally induced through activation of the Ras-MAP kinase pathway in the mesencephalon and is upregulated concomitantly to the metencephalic-to-mesencephalic fate change that occurs when Ras-MAP kinase signaling is blocked in rhombomere 1 (Vennemann, 2008; Agoston, 2009).

Meis2 expression must therefore be directly or indirectly under control of the MHB organizer. Notably, a single, transient transfection of Meis2 in the diencephalic alar plate at the 10- to 11-somite stage was sufficient to initiate long-term expression of endogenous Meis2 in transfected cells. Meis2, once induced, can therefore stabilize its own expression. Together, these results suggest a model in which regulation of tectal development by signals from the MHB is mediated via induction and subsequent auto-maintenance of Meis2 expression (Agoston, 2009).

Meis family proteins act as co-factors of other transcriptional regulators. To date, Meis-interacting proteins have been isolated from non-neuronal tissue and the posterior hindbrain, yet Meis co-factors in the developing anterior brain have remained elusive. GST pull-down experiments from tectal extracts or with in vitro translated proteins as well as co-immunoprecipitation experiments were performed with native tectal proteins to demonstrate direct interaction of Meis2 and Otx2 during early midbrain development. Using deletion constructs of Otx2, it was found that complex formation requires a short motif N-terminal of the Otx2 homeodomain. The region of the Otx2 polypeptide chain that contacts Meis2 thus differs from the tryptophan-containing hexapeptide that mediates cooperative DNA binding of Hox or myogenic bHLH proteins with TALE-homeodomain proteins. Meis family proteins can therefore interact with different protein motifs present in a variety of transcription factors (Agoston, 2009).

Employing an Otx2-dependent reporter assay, evidence was provided that Meis2 competes with the co-repressor Tle4 for binding to Otx2. Tle4 expression begins in the anterior primitive streak (thus preceding that of Meis2), is later strong in the anterior neural tube and decreases after the 20- to 25-somite stage. Tle4 binding to Otx2 was previously shown be required for the ability of Otx2 to repress Fgf8 anterior to the MHB, an important step in the formation and stabilization of the MHB organizer. Overexpression of Tle4 in the mesencephalic vesicle, in turn, disrupts normal development and lamination of the optic tecta. Together with the results presented in this study, these data might allow reconstruction of the probable temporal sequence of tectal fate specification in the embryo. In the anterior neural plate and anterior neural tube at early somite stages, Tle4 is co-expressed with Otx2 but Meis2 is missing. In the absence of Meis2, Otx2 and Tle4 can interact, prevent precocious tectal differentiation and inhibit Fgf8 expression anterior to the organizer, which stabilizes the MHB signaling center (Agoston, 2009).

Meis2 expression in the mesencephalic alar plate begins at HH11-12 (13-16 somites) and is strong from the 20- to 22-somite stage onwards, at which time the MHB organizer is established. Meis2 competes with Tle4 for binding to Otx2 in the tectal anlage, releases Otx2 from Groucho-mediated repression and thereby allows tectal development to commence (Agoston, 2009).

If correct, two predictions can be drawn from this model. First, loss of Tle4 from the diencephalic vesicle (where Tle4 is co-expressed with Otx2 at early somite stages) should lead to derepression of tectal genes. Second, precocious and ectopic expression of Meis2 in the MHB territory may destabilize the Fgf8 expression domain through premature restoration of Otx2 transcriptional activator function. Indeed, as previously demonstrated, transfection of a putative dominant-negative form of Tle4 - a truncation comprising only the first 203 amino acids of the protein - into the diencephalic vesicle causes widespread ectopic induction of En2 transcripts. In addition, when Meis2 was ectopically introduced into the MHB region at the 4- to 6-somite stage, small ectopic patches of Fgf8 transcripts anterior to the normal Fgf8 expression domain at the MHB were visible. These ectopic patches of Fgf8 expression might correspond to cells that have escaped Fgf8 downregulation by Otx2-Tle4 during the period of MHB organizer formation owing to the precocious inactivation of the Otx2-Tle4 complex by Meis2HA (Agoston, 2009).

Meis2 had to be transfected in excess to Tle4 in order to restore Otx2 transactivation in the Otx2-dependent reporter assays. This observation is consistent with the fact that between the 24- and 44-somite stages, Meis2 transcripts are abundant in the dorsal midbrain, whereas Tle4 expression is barely detectable. Tle4 thus appears to bind to Otx2 with higher affinity than does Meis2, which might allow for tight control over the tectum-inducing activity of Otx2. Recently, the spatial-temporal windows of Otx2 control over head, brain and body development were defined by Tamoxifen-induced deletion of Otx2. Interestingly, Otx2 deletion at E10.5-12.5 resulted in a mesencephalic-to-metencephalic fate change without shifting the molecular MHB. Hence, the interaction of Otx2 with Meis2 in the tectal anlage reported in this study occurs at a similar developmental stage to that at which Otx2 is required for mesencephalic fate determination but not MHB organizer positioning (Agoston, 2009).

Possible targets of Otx2/Meis2 include ephrin B1 (Efnb1) and Dbx1, both of which carry several potential consensus Bicoid- and Meis-binding sites upstream of their transcriptional start sites. Direct regulation of a midbrain-specific regulatory element of the EphA8 gene by Meis2 has also been demonstrated in mice. However, because Meis2 binding to Otx2 does not require either protein to be bound to DNA, Meis2-Otx2 interaction and restoration of Otx2 transcriptional activator function might in fact take place before both proteins have contacted the regulatory elements of downstream genes. Regulation of gene expression by putative transcription factors independent of DNA binding is not unprecedented. For instance, several Hox proteins can modulate gene expression by inhibiting the activity of CBP histone acetyltransferases (HATs) without forming DNA-binding complexes with CBP HAT. Meis family members might therefore affect gene expression by multiple, DNA-dependent and -independent mechanisms. This view is supported by the fact that despite the identification of Meis proteins as transcriptional co-factors, few direct target genes of these proteins have been reported to date (Agoston, 2009).

In summary, the results reported in this study strongly suggest that Tle4 and Meis2 compete for binding to Otx2 in the mesencephalic vesicle and that the balance between these proteins provides spatial and temporal control over the onset of tectal differentiation. Formation of spatially and temporally distinct higher order protein complexes involving Meis proteins and known regulators of neural patterning or fate determination might serve as a simple, yet versatile, mechanism to subdivide broad territories into smaller functional units during brain development (Agoston, 2009).

Meis2 is a Pax6 co-factor in neurogenesis and dopaminergic periglomerular fate specification in the adult olfactory bulb

Meis homeodomain transcription factors control cell proliferation, cell fate specification and differentiation in development and disease. Previous studies have largely focused on Meis contribution to the development of non-neuronal tissues. By contrast, Meis function in the brain is not well understood. This study provides evidence for a dual role of the Meis family protein Meis2 in adult olfactory bulb (OB) neurogenesis. Meis2 is strongly expressed in neuroblasts of the subventricular zone (SVZ) and rostral migratory stream (RMS) and in some of the OB interneurons that are continuously replaced during adult life. Targeted manipulations with retroviral vectors expressing function-blocking forms or with small interfering RNAs demonstrated that Meis activity is cell-autonomously required for the acquisition of a general neuronal fate by SVZ-derived progenitors in vivo and in vitro. Additionally, Meis2 activity in the RMS is important for the generation of dopaminergic periglomerular neurons in the OB. Chromatin immunoprecipitation identified doublecortin and tyrosine hydroxylase as direct Meis targets in newly generated neurons and the OB, respectively. Furthermore, biochemical analyses revealed a previously unrecognized complex of Meis2 with Pax6 and Dlx2, two transcription factors involved in OB neurogenesis. The full pro-neurogenic activity of Pax6 in SVZ derived neural stem and progenitor cells requires the presence of Meis. Collectively, these results show that Meis2 cooperates with Pax6 in generic neurogenesis and dopaminergic fate specification in the adult SVZ-OB system (Agoston, 2014).

Mesodermal Wnt signaling organizes the neural plate via Meis3

In vertebrates, canonical Wnt signaling controls posterior neural cell lineage specification. Although Wnt signaling to the neural plate is sufficient for posterior identity, the source and timing of this activity remain uncertain. Furthermore, crucial molecular targets of this activity have not been defined. This study identified the endogenous Wnt activity and its role in controlling an essential downstream transcription factor, Meis3. Wnt3a is expressed in a specialized mesodermal domain, the paraxial dorsolateral mesoderm, which signals to overlying neuroectoderm. Loss of zygotic Wnt3a in this region does not alter mesoderm cell fates, but blocks Meis3 expression in the neuroectoderm, triggering the loss of posterior neural fates. Ectopic Meis3 protein expression is sufficient to rescue this phenotype. Moreover, Wnt3a induction of the posterior nervous system requires functional Meis3 in the neural plate. Using ChIP and promoter analysis, this study shows that Meis3 is a direct target of Wnt/beta-catenin signaling. This suggests a new model for neural anteroposterior patterning, in which Wnt3a from the paraxial mesoderm induces posterior cell fates via direct activation of a crucial transcription factor in the overlying neural plate (Elkouby, 2010).

Is canonical Wnt activation of Meis gene expression a conserved phenomenon? In C. elegans, the PSA-3/Meis protein is required for daughter cell fates after asymmetric cell division, and it is a direct target of the Wnt pathway. POP-1/TCF protein binds the psa-3/Meis promoter and its binding site is required for expression. In Drosophila, the wingless (Wg/Wnt) protein activates expression of the sole Meis protein homolog, the homothorax (hth) gene, which is required for wing hinge development. In both beetles and spiders, hth expression seems to be regulated by Wg during appendage development. Thus, in diverged invertebrate systems, Wnt signaling controls Meis/hth family gene expression. This study shows the first evidence for Wnt regulation of Meis gene expression in vertebrates (Elkouby, 2010).

The canonical Wnt, FGF and RA signaling pathways are required for posterior CNS formation in Xenopus. In a unifying model, it is suggested that Wnt3a signaling from the Dorsal-lateral marginal zone (DLMZ) triggers Meis3 expression in the overlying neural plate. Meis3 protein directly activates FGF3 and FGF8 gene expression, and Meis3 protein cannot induce posterior cell fates in the absence of downstream FGF signaling. Supporting these data, in caudalized Xenopus explants, FGF acts downstream of canonical Wnt signaling. FGF3 and FGF8 expression in Wnt-caudalized explants is dependent on Meis3 protein activity. RA signaling also interacts with Meis3 protein to optimize Hox gene expression in the early neural plate to fine-tune the hindbrain pattern. Thus, in a regulatory network controlling posterior neural cell fates, Meis3 acts downstream to canonical Wnt, upstream to FGF, and in concert with RA signaling to activate gene expression (Elkouby, 2010).

What is the organizer's role in neural patterning? Clearly the DMZ is indispensable for neural induction, which is a prerequisite for neural patterning. In explants, BMP antagonism does not induce Meis3 expression but, in combination with Wnt signaling, BMP antagonism optimally induces Meis3 expression. BMP antagonism induces competent neuroectoderm that is responsive to caudalizing signals from the DLMZ. Optimal Meis3 expression in the embryo depends on Zic1 protein activity, and Zic gene family expression requires BMP antagonism. While the organizer does not caudalize neuroectoderm, it provides the competence for the Wnt pathway to activate Meis3 gene transcription (Elkouby, 2010).

There are still many open questions. In the presumptive hindbrain region of gastrula embryos, Meis3-expressing cells act as a hindbrain-inducing center by inducing posterior neural cell fates non-cell autonomously. At gastrula stages, when this center is active, the target cells are clustered in close proximity. How does Meis3 induce different cell fates in such proximal, but distinct, embryonic regions? How are the different posterior neural cell types, such as hindbrain, primary neuron and neural crest specified to such exact regions by similar signaling pathways and transcription factors during overlapping time windows? The future challenge is to determine how Meis3 protein acts with these signaling pathways and other transcription factors to generate multiple nervous system cell fates (Elkouby, 2010).

Meis1a and cancer

HOX genes have emerged as critical effectors of leukemogenesis, but the mechanisms that regulate their expression in leukemia are not well understood. Recent data suggest that the caudal homeobox transcription factors CDX1, CDX2, and CDX4, developmental regulators of HOX gene expression, may contribute to HOX gene dysregulation in leukemia. CDX4 is expressed normally in early hematopoietic progenitors and is expressed aberrantly in ~25% of acute myeloid leukemia (AML) patient samples. Cdx4 regulates Hox gene expression in the adult murine hematopoietic system and dysregulates Hox genes that are implicated in leukemogenesis. Furthermore, bone marrow progenitors that are retrovirally engineered to express Cdx4 serially replate in methylcellulose cultures, grow in liquid culture, and generate a partially penetrant, long-latency AML in bone marrow transplant recipients. Coexpression of the Hox cofactor Meis1a accelerates the Cdx4 AML phenotype and renders it fully penetrant. Structure-function analysis demonstrates that leukemic transformation requires intact Cdx4 transactivation and DNA-binding domains but not the putative Pbx cofactor interaction motif. Together, these data indicate that Cdx4 regulates Hox gene expression in adult hematopoiesis and may serve as an upstream regulator of Hox gene expression in the induction of acute leukemia. Inasmuch as many human leukemias show dysregulated expression of a spectrum of HOX family members, these collective findings also suggest a central role for CDX4 expression in the genesis of acute leukemia (Bansal, 2006).

Homeobox transcription factors Meis1 and Hoxa9 promote hematopoietic progenitor self-renewal and cooperate to cause acute myeloid leukemia (AML). While Hoxa9 alone blocks the differentiation of nonleukemogenic myeloid cell-committed progenitors, coexpression with Meis1 is required for the production of AML-initiating progenitors, which also transcribe a group of hematopoietic stem cell genes, including Cd34 and Flt3 (defined as Meis1-related leukemic signature genes). This study used dominant trans-activating (Vp16 fusion) or trans-repressing (engrailed fusion) forms of Meis1 to define its biochemical functions that contribute to leukemogenesis. Surprisingly, Vp16-Meis1 (but not engrailed-Meis1) functioned as an autonomous oncoprotein that mimicked combined activities of Meis1 plus Hoxa9, immortalizing early progenitors, inducing low-level expression of Meis1-related signature genes, and causing leukemia without coexpression of exogenous or endogenous Hox genes. Vp16-Meis1-mediated transformation required the Meis1 function of binding to Pbx and DNA but not its C-terminal domain (CTD). The absence of endogenous Hox gene expression in Vp16-Meis1-immortalized progenitors allowed investigation of how Hox alters gene expression and cell biology in early hematopoietic progenitors. Strikingly, expression of Hoxa9 or Hoxa7 stimulated both leukemic aggressiveness and transcription of Meis1-related signature genes in Vp16-Meis1 progenitors. Interestingly, while the Hoxa9 N-terminal domain (NTD) is essential for cooperative transformation with wild-type Meis1, it is dispensable in Vp16-Meis1 progenitors. The fact that a dominant transactivation domain fused to Meis1 replaces the essential functions of both the Meis1 CTD and Hoxa9 NTD suggests that Meis-Pbx and Hox-Pbx (or Hox-Pbx-Meis) complexes co-occupy cellular promoters that drive leukemogenesis and that Meis1 CTD and Hox NTD cooperate in gene activation. Chromatin immunoprecipitation confirmed co-occupancy of Hoxa9 and Meis1 on the Flt3 promoter (Wang, 2006 full test of article).

Oncogenic mutations of the MLL histone methyltransferase confer an unusual ability to transform non-self-renewing myeloid progenitors into leukemia stem cells (LSCs) by mechanisms that remain poorly defined. Misregulation of Hox genes is likely to be critical for LSC induction and maintenance but alone it does not recapitulate the phenotype and biology of MLL leukemias, which are clinically heterogeneous -- presumably reflecting differences in LSC biology and/or frequency. TALE (three-amino-acid loop extension) class homeodomain proteins of the Pbx and Meis families are also misexpressed in this context, and thus knockout, knockdown, and dominant-negative genetic techniques were employed to investigate the requirements and contributions of these factors in MLL oncoprotein-induced acute myeloid leukemia. The studies show that induction and maintenance of MLL transformation requires Meis1 and is codependent on the redundant contributions of Pbx2 and Pbx3. Meis1 in particular serves a major role in establishing LSC potential, and determines LSC frequency by quantitatively regulating the extent of self-renewal, differentiation arrest, and cycling, as well as the rate of in vivo LSC generation from myeloid progenitors. Thus, TALE proteins are critical downstream effectors within an essential homeoprotein network that serves a rate-limiting regulatory role in MLL leukemogenesis (Wong, 2007).

homothorax: Biological Overview | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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