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.     

What are the roles of retinoids,other morphogens,and Hox genes in setting up the vertebrate body axis?

This article is concerned with the roles of retinoids and other known anterior–posterior morphogens in setting up the embryonic vertebrate anterior–posterior axis. The discussion is restricted to the very earliest events in setting up the anterior–posterior axis (from blastula to tailbud stages in Xenopus embryos). In these earliest developmental stages, morphogen concentration gradients are not relevant for setting up this axis. It emerges that at these stages, the core patterning mechanism is timing: BMP‐anti BMP mediated time space translation that regulates Hox temporal and spatial collinearities and Hox‐Hox auto‐ and cross‐ regulation. The known anterior–posterior morphogens and signaling pathways––retinoids, FGF's, Cdx, Wnts, Gdf11 and others––interact with this core mechanism at and after space–time defined “decision points,” leading to the separation of distinct axial domains. There are also other roles for signaling pathways. Besides the Hox regulated hindbrain/trunk part of the axis, there is a rostral part (including the anterior part of the head and the extreme anterior domain [EAD]) that appears to be regulated by additional mechanisms. Key aspects of anterior–posterior axial patterning, including: the nature of different phases in early patterning and in the whole process; the specificities of Hox action and of intercellular signaling; and the mechanisms of Hox temporal and spatial collinearities, are discussed in relation to the facts and hypotheses proposed above.     

Hox Genes and Axial Specification in Vertebrates

The colinear, anterior to posterior expression domains of theHox genes in vertebrate embryos is strongly correlated withregional changes in vertebral morphology. The limbs of tetrapodsare consistently aligned with specific areas of the vertebralcolumn. However, control of limb development is apparently situatedin the lateral plate mesoderm, and has been experimentally shownto be independent of an axial Hox code (Cohn et al., 1997, Nature387:97–101). We have used experimental manipulation ofchick embryos to test the causal role of Hox genes in patterningderivatives of the paraxial mesoderm. Hox expression in heterotopicallytransplanted segmental plate responds in a manner consistentwith a patterning role for these genes in the morphologicalbehavior of the transplants. Expression is maintained in dorsalparaxial regions where patterning is also intrinsic to the donorsite of the graft. However, expression is apparently lost insomite cells that migrate into the host lateral plate environmentand form appropriate host-level muscles. This arrangement couldenable increased plasticity in the evolution of transpositionalvariation in the vertebrate body plan.     

Conserved genetic programs in insect and mammalian brain development

In recent years it has become evident that the developmental regulatory genes involved in patterning the embryonic body plan are conserved throughout the animal kingdom. Striking examples are the orthodenticle (otd/Otx) gene family and the Hox gene family, both of which act in the specification of anteroposterior polarity along the embryonic body axis. Studies carried out in Drosophila and mouse now demonstrate that these genes are also involved in the formation of the insect and mammalian brain; the otd/Otx genes are involved in rostral brain development and the Hox genes are involved in caudal brain development. These studies also show that the genes of the otd/Otx family can functionally replace each other in cross-phylum rescue experiments and indicate that the genetic mechanisms underlying pattern formation in insect and mammalian brain development are evolutionarily conserved. BioEssays 21:677–684, 1999. © 1999 John Wiley & Sons, Inc.     

Molecular mechanisms of segmental patterning in the vertebrate hindbrain and neural crest

Recent work has shown that segmentation underlies the patterning of the vertebrate hindbrain and its neural crest derivatives. Several genes have been identified with segment-restricted expression, and evidence is now emerging regarding their function and regulatory relationships. The expression patterns of Hox genes and the phenotype of null mutants indicate roles in specifying segment identity. A zinc finger gene Krox-20 is a segment-specific regulator of Hox expression, and it seems probable that retinoic acid receptors also regulate Hox genes in the hindbrain. The receptor tyrosine kinase gene Sek may mediate cell-cell interactions that lead to segmentation. These studies provide a starting point for understanding the molecular basis of segmental patterning in the hindbrain.     

miR-196 regulates axial patterning and pectoral appendage initiation

Vertebrate Hox clusters contain protein-coding genes that regulate body axis development and microRNA (miRNA) genes whose functions are not yet well understood. We overexpressed the Hox cluster microRNA miR-196 in zebrafish embryos and found four specific, viable phenotypes: failure of pectoral fin bud initiation, deletion of the 6th pharyngeal arch, homeotic aberration and loss of rostral vertebrae, and reduced number of ribs and somites. Reciprocally, miR-196 knockdown evoked an extra pharyngeal arch, extra ribs, and extra somites, confirming endogenous roles of miR-196. miR-196 injection altered expression of hox genes and the signaling of retinoic acid through the retinoic acid receptor gene rarab. Knocking down rarab mimicked the pectoral fin phenotype of miR-196 overexpression, and reporter constructs tested in tissue culture and in embryos showed that the rarab 3′UTR is a miR-196 target for pectoral fin bud initiation. These results show that a Hox cluster microRNA modulates development of axial patterning similar to nearby protein-coding Hox genes, and acts on appendicular patterning at least in part by modulating retinoic acid signaling.     

Transformations in null mutants of hox genes: Do they represent intercalary regenerates?

In the minds of many, Hox gene null mutant phenotypes have confirmed the direct role that these genes play in specifying the pattern of vertebrate embryos. The genes are envisaged as defining discrete spatial domains and, subsequently, conferring specific segmental identities on cells undergoing differentiation along the antero-posterior axis. However, several aspects of the observed mutant phenotypes are inconsistent with this view. These include: the appearance of other, unexpected transformations along the dorsal axis; the occurrence of mirror-image duplications; and the development of anomalies outside the established domains of normal Hox gene expression. In this paper, Hox gene disruptions are shown to elicit regeneration-like responses in tissues confronted with discontinuities in axial identity. The polarities and orientations of transformed segments which emerge as a consequence of this response obey the rules of distal transformation and intercalary regeneration. In addition, the incidence of periodic anomalies suggests that the initial steps of Hox-mediated patterning occurs in Hensen's node. As gastrulation proceeds, mesoderm cell cycle kinetics impose constraints upon subsequent cellular differentiation. This results in the delayed manifestation of transformations along the antero-posterior axis. Finally, a paradigm is sketched in which temporal, rather than spatial axial determinants direct differentiation. Specific, testable predictions are made about the role of Hox genes in the establishment of segmental identity.     

Fin duplications and deletions induced by disruption of retinoic acid signaling

 Retinoic acid (RA), a derivative of vitamin A, plays a critical role as a signaling molecule in axial patterning of vertebrates. Here we report that RA exposure of zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus) embryos during gastrulation results in homeotic duplications of the pectoral fins in up to 94% of fish. We have observed three to four pairs of fins in an individual fish. Although some duplications are partial, many represent complete axial duplications of the pectoral girdle and fin and include coracoscapulae, proximal radials, and dermal fin elements. Fin duplications are observed only at a defined dose of RA. Inhibition of RA synthesis by exposure to citral during a narrow developmental window leads to fish which lack pectoral fins but can be rescued by addition of exogenous RA, suggesting that RA signaling is critical to fin specification during early development. The ability to consistently induce multiple fins in a large number of vertebrate embryos should contribute to the understanding of genetic regulation of the normal positioning of limbs during embryogenesis. Received: 30 August 1997 / Accepted: 6 December 1997     

Combinatorial roles for zebrafish retinoic acid receptors in the hindbrain, limbs and pharyngeal arches

Retinoic acid (RA) signaling regulates multiple aspects of vertebrate embryonic development and tissue patterning, in part through the local availability of nuclear hormone receptors called retinoic acid receptors (RARs) and retinoid receptors (RXRs). RAR/RXR heterodimers transduce the RA signal, and loss-of-function studies in mice have demonstrated requirements for distinct receptor combinations at different stages of embryogenesis. However, the tissue-specific functions of each receptor and their individual contributions to RA signaling in vivo are only partially understood. Here we use morpholino oligonucleotides to deplete the four known zebrafish RARs (raraa, rarab, rarga, and rargb). We show that while all four are required for anterior-posterior patterning of rhombomeres in the hindbrain, there are unique requirements for rarga in the cranial mesoderm for hindbrain patterning, and rarab in lateral plate mesoderm for specification of the pectoral fins. In addition, the alpha subclass (raraa, rarab) is RA inducible, and of these only raraa expression is RA-dependent, suggesting that these receptors establish a region of particularly high RA signaling through positive-feedback. These studies reveal novel tissue-specific roles for RARs in controlling the competence and sensitivity of cells to respond to RA.     

The <Emphasis Type="Italic">C. elegans Hox</Emphasis> gene <Emphasis Type="Italic">ceh-13</Emphasis> regulates cell migration and fusion in a non-colinear way. Implications for the early evolution of <Emphasis Type="Italic">Hox</Emphasis>clusters

Background  

Hox genes play a central role in axial patterning during animal development. They are clustered in the genome and specify cell fate in sequential domains along the anteroposterior (A-P) body axis in a conserved order that is co-linear with their relative genomic position. In the soil worm Caenorhabditis elegans, this striking rule of co-linearity is broken by the anterior Hox gene ceh-13, which is located between the two middle Hox paralogs, lin-39 and mab-5, within the loosely organized nematode Hox cluster. Despite its evolutionary and developmental significance, the functional consequence of this unusual genomic organization remains unresolved.     

Homeobox Genes, Retinoic Acid and the Development and Evolution of Dual Body Plans in the Ascidian Herdmania curvata

Ascidians, along with other urochordates, are the most evolutionarydistant group from vertebrates to display definitive chordate-specificcharacters, such as a notochord, dorsal hollow nerve cord, pharynxand endostyle. Most solitary ascidians have a biphasic lifehistory that has partitioned the development of these charactersbetween a planktonic microscopic tadpole larva (notochord anddorsal nerve cord) and a larger sessile adult (pharynx and endostyle).Very little is known of the molecular axial patterning processesoperating during ascidian postlarval development. Two axialpatterning homeobox genes Otx and Cdx are expressed in a spatiallyrestricted manner along the ascidian anteroposterior axis duringembryogenesis and postlarval development (i.e., metamorphosis).Comparisons of these patterns with those of homologous cephalochordateand vertebrate genes suggest that the novel ascidian biphasicbody plan was not accompanied by a deployment of these genesinto new pathways but by a heterochronic shift in tissue-specificexpression. Studies examining the role of all-trans retinoicacid (RA) in axial patterning in chordates also contribute toour understanding of the role of homeobox genes in the developmentof larval and adult ascidian body plans. Our studies demonstratethat RA does not regulate axial patterning in the developingascidian larval neuroaxis in a manner homologous to that foundin vertebrates. Although RA may regulate the expression of someascidian homeobox genes, ectopic application of RA does notappear to alter the morphology of the larval CNS. However, treatmentwith similar or lower concentrations of RA, have a profoundeffect on postlarval development and the juvenile body plan.These changes are correlated to a dramatic reduction of Otxexpression. Through these RA-induced effects we infer that whileRA may regulate the expression of some homeobox genes duringembryogenesis it has a far more dramatic impact on postlarvaldevelopment where regulative processes predominate.     

Essential role of Bmp signaling and its positive feedback loop in the early cell fate evolution of chordates

In chordates, early separation of cell fate domains occurs prior to the final specification of ectoderm to neural and non-neural as well as mesoderm to dorsal and ventral during development. Maintaining such division with the establishment of an exact border between the domains is required for the formation of highly differentiated structures such as neural tube and notochord. We hypothesized that the key condition for efficient cell fate separation in a chordate embryo is the presence of a positive feedback loop for Bmp signaling within the gene regulatory network (GRN), underlying early axial patterning. Here, we therefore investigated the role of Bmp signaling in axial cell fate determination in amphioxus, the basal chordate possessing a centralized nervous system. Pharmacological inhibition of Bmp signaling induces dorsalization of amphioxus embryos and expansion of neural plate markers, which is consistent with an ancestral role of Bmp signaling in chordate axial patterning and neural plate formation. Furthermore, we provided evidence for the presence of the positive feedback loop within the Bmp signaling network of amphioxus. Using mRNA microinjections we found that, in contrast to vertebrate Vent genes, which promote the expression of Bmp4, amphioxus Vent1 is likely not responsible for activation of cephalochordate ortholog Bmp2/4. Cis-regulatory analysis of amphioxus Bmp2/4, Admp and Chordin promoters in medaka embryos revealed remarkable conservation of the gene regulatory information between vertebrates and basal chordates. Our data suggest that emergence of a positive feedback loop within the Bmp signaling network may represent a key molecular event in the evolutionary history of the chordate cell fate determination.     

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.     

Delta-Notch signaling and lateral inhibition in zebrafish spinal cord development

Background  

Vertebrate neural development requires precise coordination of cell proliferation and cell specification to guide orderly transition of mitotically active precursor cells into different types of post-mitotic neurons and glia. Lateral inhibition, mediated by the Delta-Notch signaling pathway, may provide a mechanism to regulate proliferation and specification in the vertebrate nervous system. We examined delta and notch gene expression in zebrafish embryos and tested the role of lateral inhibition in spinal cord patterning by ablating cells and genetically disrupting Delta-Notch signaling.     

Axial and appendicular skeletal transformations, ligament alterations, and motor neuron loss in Hoxc10 mutants

Vertebrate Hox genes regulate many aspects of embryonic body plan development and patterning. In particular, Hox genes have been shown to regulate regional patterning of the axial and appendicular skeleton and of the central nervous system. We have identified patterning defects resulting from the targeted mutation of Hoxc10, a member of the Hox10 paralogous family. Hoxc10 mutant mice have skeletal transformations in thoracic, lumbar, and sacral vertebrae and in the pelvis, along with alterations in the bones and ligaments of the hindlimbs. These results suggest that Hoxc10, along with other members of the Hox10 paralogous gene family, regulates vertebral identity at the transition from thoracic to lumbar and lumbar to sacral regions. Our results also suggest a general role for Hoxc10 in regulating chondrogenesis and osteogenesis in the hindlimb, along with a specific role in shaping femoral architecture. In addition, mutant mice have a reduction in lumbar motor neurons and a change in locomotor behavior. These results suggest a role for Hoxc10 in generating or maintaining the normal complement of lumbar motor neurons.