Drosophila endoderm development requires a novel homeobox gene which is a target of Wingless and Dpp signalling.

We have identified and cloned a novel type of homeobox gene that is composed of two homeodomains and is expressed in the Drosophila endoderm. Mutant analysis reveals that its activity is required at the foregut/midgut boundary for the development of the proventriculus. This organ regulates food passage from the foregut into the midgut and forms by the infolding of ectoderm and endoderm-derived tissues. The endodermal outer wall structure of the proventriculus is collapsed in the mutants leading to a failure of the ectodermal part to invaginate and build a functional multilayered organ. Lack-of-function and gain-of-function experiments show that the expression of this homeobox gene in the proventriculus endoderm is induced in response to Wingless activity emanating from the ectoderm/endoderm boundary whereas its expression in the central midgut is controlled by Dpp and Wingless signalling emanating from the overlying visceral mesoderm.     

An ascidian homologue of the gonadotropin-releasing hormone receptor is a retinoic acid target gene

Transdifferentiation of the multipotent atrial epithelium is a key event during budding of the ascidian Polyandrocarpa misakiensis. The transdifferentiation is induced by mesenchyme cells that were stimulated by retinoic acid. The fluorescent differential display identified a few cDNA fragments for retinoic acid-inducible genes. One of the cDNA clones, named Pm-GnRHR, encoded a seven-pass transmembrane receptor similar to gonadotropin-releasing hormone receptors. Putative amino acid sequence showed high similarity to Ciona intestinalis GnRHRs and formed a cluster with other GnRHR proteins in a phylogenetic tree. The level of expression of the Pm-GnRHR mRNA increased during the early stage of bud development, suggesting that the Pm-GnRHR function is involved in some aspects of bud development.     

A rat homeobox gene, rNKx-2.5, is a homologue of the tinman gene in Drosophila and is mainly expressed during heart development

 We report the cloning of a rat homeobox-containing gene, rNkx-2.5, and investigation of its expression in adult tissues and during embryonic development. The rNkx-2.5 gene is a homologue of the tinman gene in Drosophila. Both genes share an identical TN domain (tinman-like amino-terminal decapeptide) and about 66.7% sequence identity within their homeodomain sequences. In vertebrates, the rNKx-2.5 gene is most closely related to the mouse NKx-2.5 (mNKx-2.5) gene. Coding sequences for both NKx-2.5 genes have 94.1% sequence identity, including three identical conserved domains, the TN, homeo and NK-2 domains (NK-2 specific domain, carboxy-terminal to the homeodomain in vertebrate tinman homologues). The rat NKx-2.5 gene is encoded by a 1.4-kb mRNA and in adult tissues is mainly expressed in heart, with weaker expression in testis, spleen and lung. During embryonic development, expression of rNKx-2.5 is strongly observed in developing heart, specifically in the myocardium of both atrial and ventricular chambers. In addition, rNKx-2.5 expression marks other developing tissues, including tongue, thyroid, stomach, spleen and pulmonary veins. These data show that rNKx-2.5 is expressed in a pattern similar but not identical to that previously observed for mNKx-2.5. Received: 24 February 1997 / Accepted: 23 June 1997     

The RING domain of Siaz, the zebrafish homologue of Drosophila seven in absentia, is essential for cellular growth arrest

Siah is a mammalian homologue of Drosophila seven in absentia (sina) that is required for R7 photoreceptor development. Both the SINA and Siah family interact with ubiquitin-conjugating enzymes via an N-terminal RING domain and the C-terminal domain of SINA/ Siahs interacts with proteins targeted for degradation. Siah induces cell growth arrest by promoting beta-catenin degradation in a phosphorylation-independent manner as a result of indirect binding to beta-catenin. We previously cloned a zebrafish homologue (Siaz) of Siah. Siaz shares high sequence homology with vertebrate Siah-2. We have now examined the role of Siaz in growth regulation using the trypan blue exclusion assay and flow cytometry and found that Siaz induces cellular growth arrest by inhibiting the G2/M transition. The C-terminal domain of Siaz that interacts with target proteins is not required for growth inhibition. We conclude that the N-terminal RING and central domain of Siaz are sufficient to block the G2/M phase transition.     

The Ketel gene encodes a Drosophila homologue of importin-beta

The Drosophila melanogaster Ketel gene was identified via the Ketel(D) dominant female sterile mutations and their ketel(r) revertant alleles that are recessive zygotic lethals. The maternally acting Ketel(D) mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1. 2-3 in four ketel(r) alleles. The Ketel(+) transgenes rescue ketel(r)-associated zygotic lethality and slightly reduce Ketel(D)-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-beta, the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm from stage 11 of oogenesis. In cleavage embryos the Ketel protein is cytoplasmic. The Ketel gene appears to be ubiquitously expressed in embryonic cells. Western blot analysis revealed that the Ketel gene is not expressed in several larval cell types of late third instar larvae.     

Cdx4 is a Cdx2 target gene

The products of the Cdx genes, Cdx1, Cdx2 and Cdx4, play multiple roles in early vertebrate development, and have been proposed to serve to relay signaling information from Wnt, RA and FGF pathways to orchestrate events related to anterior-posterior vertebral patterning and axial elongation. In addition, Cdx1 and Cdx2 have been reported to both autoregulate and to be subject to cross regulation by other family members. We have now found that Cdx4 expression is significantly down regulated in Cdx2(-/-) mutants suggesting previously unrecognized cross-regulatory interactions. Moreover, we have previously shown that Cdx4 is a direct target of the canonical Wnt signaling pathway, and that Cdx1 physically interacts with LEF/TCF members in an autoregulatory loop. We therefore investigated the means by which Cdx2 impacted on Cdx4 expression and assessed potential interaction between Cdx2 and canonical Wnt signaling on the Cdx4 promoter. We found that the Cdx4 promoter was regulated by Cdx2 in transient transfection assays. Electrophoretic mobility shift assays showed that Cdx2 bound to predicted Cdx response elements in the Cdx4 promoter which, when mutated, significantly reduced activity. Consistent with these data, chromatin immunoprecipitation assays from embryos demonstrated occupancy of the Cdx4 promoter by Cdx2 in vivo. However, we failed to observe an interaction between Cdx2 and components of the canonical Wnt signaling pathway. These findings suggest that, while both canonical Wnt and Cdx2 can regulate the activity of the Cdx4 promoter, they appear to operate through distinct mechanisms.     

Maternal almondex,a neurogenic gene,is required for proper subcellular Notch distribution in early Drosophila embryogenesis

Notch signaling plays crucial roles in the control of cell fate and physiology through local cell–cell interactions. The core processes of Notch signal transduction are well established, but the mechanisms that fine-tune the pathway in various developmental and post-developmental contexts are less clear. Drosophila almondex, which encodes an evolutionarily conserved double-pass transmembrane protein, was identified in the 1970s as a maternal-effect gene that regulates Notch signaling in certain contexts, but its mechanistic function remains obscure. In this study, we examined the role of almondex in Notch signaling during early Drosophila embryogenesis. We found that in addition to being required for lateral inhibition in the neuroectoderm, almondex is also partially required for Notch signaling-dependent single-minded expression in the mesectoderm. Furthermore, we found that almondex is required for proper subcellular Notch receptor distribution in the neuroectoderm, specifically during mid-stage 5 development. The absence of maternal almondex during this critical window of time caused Notch to accumulate abnormally in cells in a mesh-like pattern. This phenotype did not include any obvious change in subcellular Delta ligand distribution, suggesting that it does not result from a general vesicular-trafficking defect. Considering that dynamic Notch trafficking regulates signal output to fit the specific context, we speculate that almondex may facilitate Notch activation by regulating intracellular Notch receptor distribution during early embryogenesis.     

phoenixin(smim20), a gene coding for a novel reproductive ligand,is expressed in the brain of adult zebrafish

Gonadotropin-releasing hormone (GnRH) is a highly conserved neuroendocrine decapeptide that is essential for the onset of puberty and the maintenance of the reproductive state. In addition to its role as hypothalamic releasing hormone, GnRH has multiple functions including modulator of neural activity within the nervous system and of resulting behaviors. These multiple functions are reflected by the existence of multiple isoforms. Despite its importance as a critical hypothalamic releasing hormone, the gnrh1 gene has been lost in zebrafish, and its reproductive function is not compensated for by other GnRH isoforms (GnRH2 and GnRH3), suggesting that, surprisingly, zebrafish do not use any of the GnRH peptides to control reproduction and fertility. Previously we proposed that Phoenixin/SMIM20, a novel peptide identified in mammals and the ligand for the orphan GPR173, is a potential candidate to control the initiation of sexual development and fertility in the zebrafish. Here we confirm the sequence of the zebrafish phoenixin/smim20 gene and by RT-PCR show that it is expressed early in development through adulthood. Subsequently we show that phoenixin/smim20 is expressed in the adult brain including the regions of the hypothalamus important in the control of fertility and reproduction.