Homeotic Complex (Hox) gene regulation and homeosis in the mesoderm of the Drosophila melanogaster embryo: the roles of signal transduction and cell autonomous regulation

In this paper we evaluate homeosis and Homeotic Complex (Hox) regulatory hierarchies in the somatic and visceral mesoderm. We demonstrate that both Hox control of signal transduction and cell autonomous regulation are critical for establishing normal Hox expression patterns and the specification of segmental identity and morphology. We present data identifying novel regulatory interactions associated with the segmental register shift in Hox expression domains between the epidermis/somatic mesoderm and visceral mesoderm. A proposed mechanism for the gap between the expression domains of Sex combs reduced (Scr) and Antennapedia (Antp) in the visceral mesoderm is provided. Previously, Hox gene interactions have been shown to occur on multiple levels: direct cross-regulation, competition for binding sites at downstream targets and through indirect feedback involving signal transduction. We find that extrinsic specification of cell fate by signaling can be overridden by Hox protein expression in mesodermal cells and propose the term autonomic dominance for this phenomenon.     

Overlapping roles of two Hox genes and the exd ortholog ceh-20 in diversification of the C. elegans postembryonic mesoderm

Members of the Hox family of homeoproteins and their cofactors play a central role in pattern formation of all germ layers. During postembryonic development of C. elegans, non-gonadal mesoderm arises from a single mesoblast cell M. Starting in the first larval stage, M divides to produce 14 striated muscles, 16 non-striated muscles, and two non-muscle cells (coelomocytes). We investigated the role of the C. elegans Hox cluster and of the exd ortholog ceh-20 in patterning of the postembryonic mesoderm. By examining the M lineage and its differentiation products in different Hox mutant combinations, we found an essential but overlapping role for two of the Hox cluster genes, lin-39 and mab-5, in diversification of the postembryonic mesoderm. This role of the two Hox gene products required the CEH-20 cofactor. One target of these two Hox genes is the C. elegans twist ortholog hlh-8. Using both in vitro and in vivo assays, we demonstrated that twist is a direct target of Hox activation. We present evidence from mutant phenotypes that twist is not the only target for Hox genes in the M lineage: in particular we show that lin-39 mab-5 double mutants exhibit a more severe M lineage defect than the hlh-8 null mutant.     

Plasticity within the lateral somatic mesoderm of Drosophila embryos

Each of 30 Drosophila larval somatic muscles has its individual shape, insertion sites and innervation. From the very beginning, the formation of individual muscles is controlled by a set of muscle identity genes. The four lateral transverse muscles (LT1-LT4) are thought to be specified by the combinatorial activity of Krüppel (Kr), apterous (ap) and muscle specific homeobox (msh) genes whilst the activity of the ladybird (lb) genes is required for proper formation of the neighbouring segmental border muscle (SBM). We have recently shown that ectopic expression of lb changes the identity of Kr-expressing lateral muscle precursors and recruits them to form enlarged or duplicated SBMs. Here we report that loss of msh function leads to a similar transformation resulting in the overproduction of SBMs. Inversely, in msh gain of function embryos, the prospective SBM myoblasts change their identity resulting in the formation of enlarged lateral transverse muscles. These data indicate a key role for the msh and lb genes in the specification and diversification of myoblast lineages from the lateral domain, and reveal a plasticity of cell fate within the somatic mesoderm of Drosophila.     

Anterior boundaries of Hox gene expression in mesoderm-derived structures correlate with the linear gene order along the chromosome

The developmental expression patterns of four genes, Hox 1.1, Hox 1.2, Hox 1.3 and Hox 3.1, were examined by in situ hybridization to serial embryonic sections. The three genes of the Hox 1 cluster, used in this study, map to adjacent positions along chromosome 6, whereas the Hox 3.1 gene maps to the Hox 3 cluster on chromosome 15. The anterior expression limits in segmented mesoderm varied among the four genes examined. Interestingly, a linear correlation exists between the position of the gene along the chromosome and the extent of anterior expression. Genes that are expressed more posterior are also more restricted in their expression in other mesoderm-derived tissues. The order of expression anterior to posterior was determined as: Hox 1.3, Hox 1.2, Hox 1.1 and Hox 3.1. Similarly, genes of the Drosophila Antennapedia and Bithorax complex specifying segment identity also exhibit anterior expression boundaries that correlate with gene position. The data suggest that Hox genes may specify positional information along the anterior-posterior axis during the formation of the body plan.     

Conserved Anterior Boundaries of Hox Gene Expression in the Central Nervous System of the LeechHelobdella

Molecular developmental studies of fly and mouse embryos have shown that the identity of individual body segments is controlled by a suite of homeobox-containing genes called the Hox cluster. To examine the conservation of this patterning mechanism in other segmented phyla, we here describe four Hox gene homologs isolated from glossiphoniid leeches of the genusHelobdella.Based on sequence similarity and phylogenetic analysis, the leech genesLox7, Lox6, Lox20,andLox5are deemed to be orthologs of theDrosophilageneslab, Dfd, Scr,andAntp,respectively. Sequence similarities betweenLox5andAntpoutside the homeodomain and phylogenetic reconstructions suggest that the Antennapedia family of Hox genes (as defined by Bürglin, 1994) had already expanded to include at least two discreteAntpandUbx/abdAprecursors prior to the annelid/arthropod divergence.In situhybridization reveals that the fourLoxgenes described in this study are all expressed at high levels within the segmented portion of the central nervous system (CNS), with variable levels of expression in the segmental mesoderm. Little or no expression was seen in peripheral ectoderm or endoderm, or in the unsegmented head region (prostomium). EachLoxgene has a distinct anterior expression boundary within one of the four rostral segments, and the anterior-posterior (AP) order of these expression boundaries is identical to that reported for the orthologous Hox gene products in fly and mouse. This finding supports the idea that the process of AP axis differentiation is conserved among the higher metazoan phyla with respect to the regional expression of individual Hox genes along that axis. One unusual feature of leech Hox genes is the observation that some genes are only expressed during later development -- beginning at the time of terminal cell differentiation -- whereas others begin expression at a much earlier stage, and their RNA ceases to be detectable shortly after the onset of expression of the ‘late’ Hox genes. The functional significance of this temporal disparity is unknown, but it is noteworthy that only the two ‘early’ Hox genes display high levels of mesodermal expression.     

Specification and maintenance of the spinal cord stem zone

Epiblast cells adjacent to the regressing primitive streak behave as a stem zone that progressively generates the entire spinal cord and also contributes to paraxial mesoderm. Despite this fundamental task, this cell population is poorly characterised, and the tissue interactions and signalling pathways that specify this unique region are unknown. Fibroblast growth factor (FGF) is implicated but it is unclear whether it is sufficient and/or directly required for stem zone specification. It is also not understood how establishment of the stem zone relates to the acquisition of spinal cord identity as indicated by expression of caudal Hox genes. Here, we show that many cells in the chick stem zone express both early neural and mesodermal genes; however, stem zone-specific gene expression can be induced by signals from underlying paraxial mesoderm without concomitant induction of an ambivalent neural/mesodermal cell state. The stem zone is a site of FGF/MAPK signalling and we show that although FGF alone does not mimic paraxial mesoderm signals, it is directly required in epiblast cells for stem zone specification and maintenance. We further demonstrate that caudal Hox gene expression in the stem zone also depends on FGF and that neither stem zone specification nor caudal Hox gene onset requires retinoid signalling. These findings thus support a two step model for spinal cord generation - FGF-dependent establishment of the stem zone in which progressively more caudal Hox genes are expressed, followed by the retinoid-dependent assignment of spinal cord identity.     

Knockdown of spalt function by RNAi causes de-repression of Hox genes and homeotic transformations in the crustacean Artemia franciscana

Hox genes play a central role in the specification of distinct segmental identities in the body of arthropods. The specificity of Hox genes depends on their restricted expression domains, their interaction with specific cofactors and selectivity for particular target genes. spalt genes are associated with the function of Hox genes in diverse species, but the nature of this association varies: in some cases, spalt collaborates with Hox genes to specify segmental identities, in others, it regulates Hox gene expression or acts as their target. Here we study the role of spalt in the branchiopod crustacean Artemia franciscana. We find that Artemia spalt is expressed in the pre-segmental 'growth zone' and in stripes in each of the trunk (thoracic, genital and post-genital) segments that emerge from this zone. Using RNA interference (RNAi), we show that knocking down the expression of spalt has pleiotropic effects, which include thoracic to genital (T-->G), genital to thoracic (G-->T) and post-genital to thoracic (PG-->T) homeotic transformations. These transformations are associated with a stochastic de-repression of Hox genes in the corresponding segments of RNAi-treated animals (AbdB for T-->G and Ubx/AbdA for G-->T and PG-->T transformations). We discuss a possible role of spalt in the maintenance of Hox gene repression in Artemia and in other animals.     

Patterning in time and space: HoxB cluster gene expression in the developing chick embryo

The developing embryo is a paradigmatic model to study molecular mechanisms of time control in Biology. Hox genes are key players in the specification of tissue identity during embryo development and their expression is under strict temporal regulation. However, the molecular mechanisms underlying timely Hox activation in the early embryo remain unknown. This is hindered by the lack of a rigorous temporal framework of sequential Hox expression within a single cluster. Herein, a thorough characterization of HoxB cluster gene expression was performed over time and space in the early chick embryo. Clear temporal collinearity of HoxB cluster gene expression activation was observed. Spatial collinearity of HoxB expression was evidenced in different stages of development and in multiple tissues. Using embryo explant cultures we showed that HoxB2 is cyclically expressed in the rostral presomitic mesoderm with the same periodicity as somite formation, suggesting a link between timely tissue specification and somite formation. We foresee that the molecular framework herein provided will facilitate experimental approaches aimed at identifying the regulatory mechanisms underlying Hox expression in Time and Space.     

The Drosophila Hox gene deformed sculpts head morphology via direct regulation of the apoptosis activator reaper

Hox proteins control morphological diversity along the anterior-posterior body axis of animals, but the cellular processes they directly regulate are poorly understood. We show that during early Drosophila development, the Hox protein Deformed (Dfd) maintains the boundary between the maxillary and mandibular head lobes by activating localized apoptosis. Dfd accomplishes this by directly activating the cell death promoting gene reaper (rpr). One other Hox gene, Abdominal-B (Abd-B), also regulates segment boundaries through the regional activation of apoptosis. Thus, one mechanism used by Drosophila Hox genes to modulate segmental morphology is to regulate programmed cell death, which literally sculpts segments into distinct shapes. This and other emerging evidence suggests that Hox proteins may often regulate the maintenance of segment boundaries.     

A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis

The decapentaplegic (dpp) gene product, a member of the transforming growth factor-beta family, is required in Drosophila embryos for normal gastrulation and the establishment of dorsal-ventral polarity in the embryo. dpp is also expressed at specific positions in the visceral mesoderm along the developing midgut. We find that mutations that eliminate the visceral mesoderm expression of dpp lead to defects in midgut morphogenesis and alter the spatially localized expression of the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), and Antennapedia (Antp) in the visceral mesoderm. The extracellular dpp protein migrates from the visceral mesoderm across the apposing endodermal cell layer in a region of the endoderm that expresses the homeotic gene labial (lab). Mesodermal expression of dpp is required for the expression of lab in these endodermal cells indicating that dpp mediates an inductive interaction between the two germ layers. We propose that extracellular dpp protein regulates gut morphogenesis, in part, by regulating homeotic gene expression in the visceral mesoderm and endoderm of the developing midgut.