Shadow enhancers flanking the HoxB cluster direct dynamic Hox expression in early heart and endoderm development

The products of Hox genes function in assigning positional identity along the anterior–posterior body axis during animal development. In mouse embryos, Hox genes located at the 3′ end of HoxA and HoxB complexes are expressed in nested patterns in the progenitors of the secondary heart field during early cardiogenesis and the combined activities of both of these clusters are required for proper looping of the heart. Using Hox bacterial artificial chromosomes (BACs), transposon reporters, and transgenic analyses in mice, we present the identification of several novel enhancers flanking the HoxB complex which can work over a long range to mediate dynamic reporter expression in the endoderm and embryonic heart during development. These enhancers respond to exogenously added retinoic acid and we have identified two retinoic acid response elements (RAREs) within these control modules that play a role in potentiating their regulatory activity. Deletion analysis in HoxB BAC reporters reveals that these control modules, spread throughout the flanking intergenic region, have regulatory activities that overlap with other local enhancers. This suggests that they function as shadow enhancers to modulate the expression of genes from the HoxB complex during cardiac development. Regulatory analysis of the HoxA complex reveals that it also has enhancers in the 3′ flanking region which contain RAREs and have the potential to modulate expression in endoderm and heart tissues. Together, the similarities in their location, enhancer output, and dependence on retinoid signaling suggest that a conserved cis-regulatory cassette located in the 3′ proximal regions adjacent to the HoxA and HoxB complexes evolved to modulate Hox gene expression during mammalian cardiac and endoderm development. This suggests a common regulatory mechanism, whereby the conserved control modules act over a long range on multiple Hox genes to generate nested patterns of HoxA and HoxB expression during cardiogenesis.     

Combined function of HoxA and HoxB clusters in neural crest cells

The evolution of chordates was accompanied by critical anatomical innovations in craniofacial development, along with the emergence of neural crest cells. The potential of these cells to implement a craniofacial program in part depends upon the (non-)expression of Hox genes. For instance, the development of jaws requires the inhibition of Hox genes function in the first pharyngeal arch. In contrast, Hox gene products induce craniofacial structures in more caudal territories. To further investigate which Hox gene clusters are involved in this latter role, we generated HoxA;HoxB cluster double mutant animals in cranial neural crest cells. We observed the appearance of a supernumerary dentary-like bone with an endochondral ossification around a neo-Meckel's cartilage matrix and an attachment of neo-muscle demonstrating that HoxB genes enhance the phenotype induced by the deletion of the HoxA cluster alone. In addition, a cervical and hypertrophic thymus was associated with the supernumerary dentary-like bone, which may reflect its ancestral position near the filtrating system. Altogether these results show that the HoxA and HoxB clusters cooperated during evolution to lead to present craniofacial diversity.     

Homeobox b5 (Hoxb5) regulates the expression of Forkhead box D3 gene (Foxd3) in neural crest

Patterning of neural crest (NC) for the formation of specific structures along the anterio-posterior (A-P) body axis is governed by a combinatorial action of Hox genes, which are expressed in the neuroepithelium at the time of NC induction. Hoxb5 was expressed in NC at both induction and migratory stages, and our previous data suggested that Hoxb5 played a role in the NC development. However, the underlying mechanisms by which Hoxb5 regulates the early NC development are largely unknown. Current study showed that both the human and mouse Foxd3 promoters were bound and trans-activated by Hoxb5 in NC-derived neuroblastoma cells. The binding of Hoxb5 to Foxd3 promoter in vivo was further confirmed in the brain and neural tube of mouse embryos. Moreover, Wnt1-Cre mediated perturbation of Hoxb5 signaling at the dorsal neural tube in mouse embryos resulted in Foxd3 down-regulation. In ovo, Foxd3 alleviated the apoptosis of neural cells induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Foxd3 expression in the chick neural tube. This study demonstrated that Hoxb5 (an A-P patterning gene) regulated the NC development by directly inducing Foxd3 (a NC specifier and survival gene).     

Positioning of the mouse Hox gene clusters in the nuclei of developing embryos and differentiating embryoid bodies

Expression of Hox genes located on different chromosomes is precisely regulated and synchronized during development. In order to test the hypothesis that the Hox loci might cluster in nuclear space in order to share regulatory components, we performed 3D FISH on cryosections of developing mouse embryos and differentiating embryoid bodies. We did not observe any instances of co-localization of 4 different Hox alleles. Instances of 2 different alleles touching each other were found in 20-47% of nuclei depending on the tissue. The frequency of such “kissing” events was not significantly different in cells expressing a high proportion of the Hox clusters when compared to cells expressing none or only a few Hox genes. We found that the HoxB and HoxC clusters, which are located in gene-rich regions, were involved more frequently in gene kissing in embryonic nuclei. In the case of HoxB, this observation correlated with the positioning of the corresponding chromosome towards the interior of the nucleus. Our results indicate that co-regulation of the different Hox clusters is not associated with co-localization of the loci at a single regulatory compartment and that the chromosomal context may influence the extent to which they contact each other in the nucleus.     

PI3K/Akt pathway regulates retinoic acid‐induced Hox gene expression in F9 cells

Retinoic acid (RA), the most potent natural form of vitamin A, is a key morphogen in vertebrate development and a potent regulator of both adult and embryonic cell differentiation. Specifically, RA regulates clustered Hox gene expression during embryogenesis and is required to establish the anteroposterior body plan. The PI3K/Akt pathway was also reported to play an essential role in the process of RA‐induced cell differentiation. Therefore, we tested whether the PI3K/Akt pathway is involved in RA‐induced Hox gene expression in a F9 murine embryonic teratocarcinoma cells. To examine the effect of PI3K/Akt signaling on RA‐induced initiation of collinear expression of Hox genes, F9 cells were treated with RA in the presence or absence of PI3K inhibitor LY294002, and time‐course gene expression profiles for all 39 Hox genes located in four different clusters—Hoxa, Hoxb, Hoxc, and Hoxd—were analyzed. Collinear expression of Hoxa and ‐b cluster genes was initiated earlier than that of the ‐c and ‐d clusters upon RA treatment. When LY294002 was applied along with RA, collinear expression induced by RA was delayed, suggesting that the PI3K/Akt signaling pathway somehow regulates RA‐induced collinear expression of Hox genes in F9 cells. The initiation of Hox collinear expression by RA and the delayed expression following LY294002 in F9 cells would provide a good model system to decipher the yet to be answered de novo collinear expression of Hox genes during gastrulation, which make the gastrulating cells to remember their positional address along the AP body axis in the developing embryo.     

Secondary DNA structure formation for Hoxb9 promoter and identification of its specific binding protein

Hox genes determine anterior–posterior specificity of an animal body. In mammals, these genes map onto four chromosomal loci in a clustered manner, and their expression is regulated in a coordinated manner according to their chromosomal structure. In the present study, we analysed the Hoxb9 promoter and found that promoter activity in cultured cells is linked to secondary structure formation of promoter DNA. In nuclear extracts, we also detected binding activity specific for secondary-structured DNA. We successfully isolated a candidate gene encoding this specific DNA-binding protein, FBXL10, and demonstrated the effects of the gene product on Hoxb9 promoter activity. Our results suggest that DNA can regulate gene expression by other, non-sequence-specific modes of genetic coding.     

Hox gene expression patterns in Lethenteron japonicum embryos--insights into the evolution of the vertebrate Hox code

The Hox code of jawed vertebrates is characterized by the colinear and rostrocaudally nested expression of Hox genes in pharyngeal arches, hindbrain, somites, and limb/fin buds. To gain insights into the evolutionary path leading to the gnathostome Hox code, we have systematically analyzed the expression pattern of the Hox gene complement in an agnathan species, Lethenteron japonicum (Lj). We have isolated 15 LjHox genes and assigned them to paralogue groups (PG) 1-11, based on their deduced amino acid sequences. LjHox expression during development displayed gnathostome-like spatial patterns with respect to the PG numbers. Specifically, lamprey PG1-3 showed homologous expression patterns in the rostral hindbrain and pharyngeal arches to their gnathostome counterparts. Moreover, PG9-11 genes were expressed specifically in the tailbud, implying its posteriorizing activity as those in gnathostomes. We conclude that these gnathostome-like colinear spatial patterns of LjHox gene expression can be regarded as one of the features already established in the common ancestor of living vertebrates. In contrast, we did not find evidence for temporal colinearity in the onset of LjHox expression. The genomic and developmental characteristics of Hox genes from different chordate species are also compared, focusing on evolution of the complex body plan of vertebrates.     

Perturbation of Hoxb5 signaling in vagal and trunk neural crest cells causes apoptosis and neurocristopathies in mice

Neural crest cells (NCCs) migrate from different regions along the anterior–posterior axis of the neural tube (NT) to form different structures. Defective NCC development causes congenital neurocristopathies affecting multiple NCC-derived tissues in human. Perturbed Hoxb5 signaling in vagal NCC causes enteric nervous system (ENS) defects. This study aims to further investigate if perturbed Hoxb5 signaling in trunk NCC contributes to defects of other NCC-derived tissues besides the ENS. We perturbed Hoxb5 signaling in NCC from the entire NT, and investigated its impact in the development of tissues derived from these cells in mice. Perturbation of Hoxb5 signaling in these NCC resulted in Sox9 downregulation, NCC apoptosis, hypoplastic sympathetic and dorsal root ganglia, hypopigmentation and ENS defects. Mutant mice with NCC-specific Sox9 deletion also displayed some of these phenotypes. In vitro and in vivo assays indicated that the Sox9 promoter was bound and trans-activated by Hoxb5. In ovo studies further revealed that Sox9 alleviated apoptosis induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Sox9 expression in chick NT. This study demonstrates that Hoxb5 regulates Sox9 expression in NCC and disruption of this signaling causes Sox9 downregulation, NCC apoptosis and multiple NCC-developmental defects. Phenotypes such as ENS deficiency, hypopigmentation and some of the neurological defects are reported in patients with Hirschsprung disease (HSCR). Whether dysregulation of Hoxb5 signaling and early depletion of NCC contribute to ENS defect and other neurocristopathies in HSCR patients deserves further investigation.     

Hox genes: Downstream “effectors” of retinoic acid signaling in vertebrate embryogenesis

One of the major regulatory challenges of animal development is to precisely coordinate in space and time the formation, specification, and patterning of cells that underlie elaboration of the basic body plan. How does the vertebrate plan for the nervous and hematopoietic systems, heart, limbs, digestive, and reproductive organs derive from seemingly similar population of cells? These systems are initially established and patterned along the anteroposterior axis (AP) by opposing signaling gradients that lead to the activation of gene regulatory networks involved in axial specification, including the Hox genes. The retinoid signaling pathway is one of the key signaling gradients coupled to the establishment of axial patterning. The nested domains of Hox gene expression, which provide a combinatorial code for axial patterning, arise in part through a differential response to retinoic acid (RA) diffusing from anabolic centers established within the embryo during development. Hence, Hox genes are important direct effectors of retinoid signaling in embryogenesis. This review focuses on describing current knowledge on the complex mechanisms and regulatory processes, which govern the response of Hox genes to RA in several tissue contexts including the nervous system during vertebrate development.     

Hoxc Gene Collinear Expression and Epigenetic Modifications Established during Embryogenesis Are Maintained until after Birth

The Hox genes, which are organized into clusters on different chromosomes, are key regulators of embryonic anterior-posterior (A-P) body pattern formation and are expressed at specific times and in specific positions in developing vertebrate embryos. Previously, we have shown that histone methylation patterns are closely correlated with collinear Hox gene expression patterns along the A-P axis of E14.5 mouse embryos. Since histone modification is thought to play a crucial mechanistic role in the highly coordinated pattern of collinear Hox gene expression, we examined the maintenance of the spatial collinear expression pattern of Hoxc genes and the corresponding histone modifications during embryogenesis and in early postnatal mice. Hox expression patterns and histone modifications were analyzed by semi-quantitative RT-PCR and chromatin immunoprecipitation (ChIP)-PCR analyses, respectively. The spatiotemporal expression patterns of Hoxc genes in a cluster were maintained until the early postnatal stage (from E8.5 through P5). Examination of histone modifications in E14.5 and P5 tissues revealed that level of H3K27me3 is only a weak correlation with collinear Hoxc gene expression in the trunk regions although diminished in general, however the enrichment of H3K4me3 is strongly correlated with the gene expression in both stages. In summary, the initial spatiotemporal collinear expression pattern of Hoxc genes and epigenetic modifications are maintained after birth, likely contributing to the establishment of the gene expression code for position in the anatomic body axis throughout the entire life of the organism.     

Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups

Initiation of Hox genes requires interactions between numerous factors and signaling pathways in order to establish their precise domain boundaries in the developing nervous system. There are distinct differences in the expression and regulation of members of Hox genes within a complex suggesting that multiple competing mechanisms are used to initiate their expression domains in early embryogenesis. In this study, by analyzing the response of HoxB genes to both RA and FGF signaling in neural tissue during early chick embryogenesis (HH stages 7-15), we have defined two distinct groups of Hox genes based on their reciprocal sensitivity to RA or FGF during this developmental period. We found that the expression domain of 5' members from the HoxB complex (Hoxb6-Hoxb9) can be expanded anteriorly in the chick neural tube up to the level of the otic vesicle following FGF treatment and that these same genes are refractory to RA treatment at these stages. Furthermore, we showed that the chick caudal-related genes, cdxA and cdxB, are also responsive to FGF signaling in neural tissue and that their anterior expansion is also limited to the level of the otic vesicle. Using a dominant negative form of a Xenopus Cdx gene (XcadEnR) we found that the effect of FGF treatment on 5' HoxB genes is mediated in part through the activation and function of CDX activity. Conversely, the 3' HoxB genes (Hoxb1 and Hoxb3-Hoxb5) are sensitive to RA but not FGF treatments at these stages. We demonstrated by in ovo electroporation of a dominant negative retinoid receptor construct (dnRAR) that retinoid signaling is required to initiate expression. Elevating CDX activity by ectopic expression of an activated form of a Xenopus Cdx gene (XcadVP16) in the hindbrain ectopically activates and anteriorly expands Hoxb4 expression. In a similar manner, when ectopic expression of XcadVP16 is combined with FGF treatment, we found that Hoxb9 expression expands anteriorly into the hindbrain region. Our findings suggest a model whereby, over the window of early development we examined, all HoxB genes are actually competent to interpret an FGF signal via a CDX-dependent pathway. However, mechanisms that axially restrict the Cdx domains of expression, serve to prevent 3' genes from responding to FGF signaling in the hindbrain. FGF may have a dual role in both modulating the accessibility of the HoxB complex along the axis and in activating the expression of Cdx genes. The position of the shift in RA or FGF responsiveness of Hox genes may be time dependent. Hence, the specific Hox genes in each of these complementary groups may vary in later stages of development or other tissues. These results highlight the key role of Cdx genes in integrating the input of multiple signaling pathways, such as FGFs and RA, in controlling initiation of Hox expression during development and the importance of understanding regulatory events/mechanisms that modulate Cdx expression.     

Adaptive Evolution of the Hox Gene Family for Development in Bats and Dolphins

Bats and cetaceans (i.e., whales, dolphins, porpoises) are two kinds of mammals with unique locomotive styles and occupy novel niches. Bats are the only mammals capable of sustained flight in the sky, while cetaceans have returned to the aquatic environment and are specialized for swimming. Associated with these novel adaptations to their environment, various development changes have occurred to their body plans and associated structures. Given the importance of Hox genes in many aspects of embryonic development, we conducted an analysis of the coding regions of all Hox gene family members from bats (represented by Pteropus vampyrus, Pteropus alecto, Myotis lucifugus and Myotis davidii) and cetaceans (represented by Tursiops truncatus) for adaptive evolution using the available draft genome sequences. Differences in the selective pressures acting on many Hox genes in bats and cetaceans were found compared to other mammals. Positive selection, however, was not found to act on any of the Hox genes in the common ancestor of bats and only upon Hoxb9 in cetaceans. PCR amplification data from additional bat and cetacean species, and application of the branch-site test 2, showed that the Hoxb2 gene within bats had significant evidence of positive selection. Thus, our study, with genomic and newly sequenced Hox genes, identifies two candidate Hox genes that may be closely linked with developmental changes in bats and cetaceans, such as those associated with the pancreatic, neuronal, thymus shape and forelimb. In addition, the difference in our results from the genome-wide scan and newly sequenced data reveals that great care must be taken in interpreting results from draft genome data from a limited number of species, and deep genetic sampling of a particular clade is a powerful tool for generating complementary data to address this limitation.     

Hox genes define distinct progenitor sub-domains within the second heart field

Much of the heart, including the atria, right ventricle and outflow tract (OFT) is derived from a progenitor cell population termed the second heart field (SHF) that contributes progressively to the embryonic heart during cardiac looping. Several studies have revealed anterior-posterior patterning of the SHF, since the anterior region (anterior heart field) contributes to right ventricular and OFT myocardium whereas the posterior region gives rise to the atria. We have previously shown that Retinoic Acid (RA) signal participates to this patterning. We now show that Hoxb1, Hoxa1, and Hoxa3, as downstream RA targets, are expressed in distinct sub-domains within the SHF. Our genetic lineage tracing analysis revealed that Hoxb1, Hoxa1 and Hoxa3-expressing cardiac progenitor cells contribute to both atria and the inferior wall of the OFT, which subsequently gives rise to myocardium at the base of pulmonary trunk. By contrast to Hoxb1^Cre, the contribution of Hoxa1-enhIII-Cre and Hoxa3^Cre-labeled cells is restricted to the distal regions of the OFT suggesting that proximo-distal patterning of the OFT is related to SHF sub-domains characterized by combinatorial Hox genes expression. Manipulation of RA signaling pathways showed that RA is required for the correct deployment of Hox-expressing SHF cells. This report provides new insights into the regulatory gene network in SHF cells contributing to the atria and sub-pulmonary myocardium.