Molecular cloning and expression of stromalin protein from Drosophila melanogaster: homologous to mammalian stromalin family of nuclear proteins.

We report the cloning of a new cDNA from Drosophila melanogaster that encodes an open reading frame of 1116 amino acid residues. It is the insect homolog of the previously reported stromalin (SA) family of nuclear proteins in mammals (Carramolino et al. [1997]. Gene 195, 151-159). Taking into account the identical domain present in all the SA family members characterized to date, we have carried out polymerase chain reaction (PCR) using degenerate oligonucleotides from the 5' and 3' ends of one of those regions of the molecule and cDNA from D. melanogaster embryos. We isolated the homologous domain of the putative Drosophila SA molecule (DSA). This cDNA fragment was used as a radiolabeled probe for screening a cDNA library from Drosophila embryos, and we have cloned a full-length cDNA for the SA homolog from an insect. The protein shows a good degree of identity with the mammalian stromalins SA-1 and SA-2, with the N and C ends being the most divergent regions of the molecule. The mRNA coding for this protein shows a molecular size of about 3.7 kb by Northern blot analysis and is essentially expressed in embryonic stage. The in situ hybridization experiments indicate that the DSA messenger is expressed mainly in neurogenic territories in the embryonic development of Drosophila. The DSA protein has been cloned and expressed in a baculovirus system, and polyclonal antibodies were generated against the recombinant molecule. Western blot analysis using these antibodies detected a main band corresponding to about 120 kDa, principally in embryos.

Antagonism between extradenticle function and Hedgehog signalling in the developing limb.

The Drosophila homeobox gene extradenticle (exd) encodes a highly conserved cofactor of Hox proteins. exd activity is regulated post-translationally by a mechanism involving nuclear translocation; only nuclear Exd protein is functional. The exd gene is required for patterning of the proximal region of the leg, whereas patterning of the distal region requires signalling by the Wingless (Wg) and Decapentaplegic (Dpp) proteins, which are in turn activated by Hedgehog (Hh). Here we show that exd function and Dpp/Wg signalling are antagonistic and divide the leg into two mutually exclusive domains. In the proximal domain, exd activity prevents cells from responding to Dpp and Wg. Conversely, in the distal domain, exd function is suppressed by the Dpp/Wg response gene Distal-less (Dll), which prevents the nuclear transport of Exd. We also found that the product of a murine homologue of exd (Pbx1) is regulated at the subcellular level, and that its pattern of nuclear localization in the mouse limb resembles that of Exd in the Drosophila leg. These findings suggest that the division of the limb into two antagonistic domains, as defined by exd (Pbx1) function and Hh signalling, may be a general feature of limb development.

Differential splicing of the growth hormone-releasing hormone gene in rat placenta generates a novel pre-proGHRH mRNA that encodes a different C-terminal flanking peptide.

We have isolated and characterized a novel rat placental pre-proGHRH mRNA (pre-proGHRH-2 mRNA). This mRNA is generated by an alternative splicing process which results in the presence of an additional exon of 156 bp (designated exon 4.5) located between exons 4 and 5 of the previously reported hypothalamic and placental pre-proGHRH mRNA (pre-proGHRH-1 mRNA). Since the sequences encoding mature GHRH are included within exons 3 and 4, the processing of pre-proGHRH-2 would not affect the synthesis of mature GHRH but would generate a C-terminal peptide (designated GCTP-2) different from that previously reported in the hypothalamus and placenta (GCTP-1). The putative GCTP-2 has 64 amino acids, and the first 18 N-terminal residues are identical to those present in GCTP-1 (30 amino acids long). Pre-proGHRH-2 mRNA has not been detected in the hypothalamus.

Genetic evidence for the subdivision of the arthropod limb into coxopodite and telopodite.

Arthropod appendages are thought to have evolved as outgrowths from the body wall of a limbless ancestor. Snodgrass, in his Principles of Insect Morphology (1935), proposed that, during evolution, expansion of the body wall would originate the base of the appendages, or coxopodite, upon which the most distal elements that represent the true outer limb, or telopodite, would develop. The homeobox gene Distal-less (Dll), which is required in the Drosophila appendages for development of distal regions, has been proposed to promote formation of telopodite structures above the evolutionary ground-state of non-limb or body wall. Here, we present evidence that another homeobox gene, extradenticle (exd), which is required for appropriate development of the trunk and the proximal parts of the appendages, represents a coxopodite gene. We show that exd function is eliminated from the distal precursors in the developing limb and remains restricted to proximal precursors throughout development. This elimination is important because, when ectopically expressed, exd prevents distal development and gives rise to truncated appendages lacking distal elements. Moreover, the maintenance of exd expression during larval stages, contrary to Dll, does not require the hedgehog (hh) signaling pathway, suggesting that the proximal regions of the appendages develop independently of hh function. Finally, we show that in the crustacean Artemia, exd and Dll are expressed in comparable patterns as in Drosophila, suggesting a conserved genetic mechanism subdividing the arthropod limb.

Control of Drosophila adult pattern by extradenticle.

The homeobox gene extradenticle (exd) acts as a cofactor of the homeotic genes in the specification of larval patterns during embryogenesis. To study its role in adult patterns, we have generated clones of mutant exd- cells and examined their effect on the different body parts. In some regions, exd- clones exhibit homeotic transformations similar to those produced by known homeotic mutations such as Ultrabithorax (Ubx), labial (lab), spineless-aristapedia (ssa) or Antennapedia (Antp). In other regions, the lack of exd causes novel homeotic transformations producing ectopic eyes and legs. Moreover, exd is also required for functions normally not associated with homeosis, such as the maintenance of the dorsoventral pattern, the specification of subpatterns in adult appendages or the arrangement of bristles in the mesonotum and genitalia. Our findings indicate that exd is critically involved in adult morphogenesis, not only in the homeotic function but also in several other developmental processes.

Related target enhancers for dorsal and NF-kappa B signaling pathways.

Drosophila dorsoventral (DV) patterning and mammalian hematopoiesis are regulated by related signaling pathways (Toll, interleukin-1) and transcription factors (dorsal, nuclear factor-kappa B). These factors interact with related enhancers, such as the rhomboid NEE and kappa light chain enhancer, that contain similar arrangements of activator and repressor binding sites. It is shown that the kappa enhancer can generate lateral stripes of gene expression in transgenic Drosophila embryos in a pattern similar to that directed by the rhomboid NEE. Drosophila DV determinants direct these stripes through the corresponding mammalian cis regulatory elements in the kappa enhancer, including the kappa B site and kappa E boxes. These results suggest that enhancers can couple conserved signaling pathways to divergent gene functions.

Dif, a dorsal-related gene that mediates an immune response in Drosophila.

There are striking parallels between the regulation of gene expression along the dorsoventral (DV) axis of Drosophila embryos and lymphoid-restricted expression in the mammalian immune system. Both depend on regulatory factors containing rel domains (dorsal and NF-kappa B) that are controlled at the level of nuclear transport. A novel Rel-containing gene in Drosophila, Dif (dorsal-related immunity factor), provides a potential link between these seemingly disparate processes. Although Dif maps close to dorsal, it does not appear to participate in DV patterning, but instead mediates an immune response in Drosophila larvae. Dif is normally localized in the cytoplasm of the larval fat body, but quickly accumulates in the nucleus upon bacterial infection or injury. Evidence is presented that once in the nucleus, Dif binds to kappa B-like sequence motifs present in promoter regions of immunity genes. These results suggest that mammalian and insect immunity share a common evolutionary origin.

Interactions between dorsal and helix-loop-helix proteins initiate the differentiation of the embryonic mesoderm and neuroectoderm in Drosophila.

The dorsal (dl) morphogen has been implicated in the establishment of the embryonic mesoderm, neuroectoderm, and dorsal ectoderm in Drosophila. Here we show that the simultaneous reduction in the levels of dl and any one of several helix-loop-helix (HLH) proteins results in severe disruptions in the formation of mesoderm and neuroectoderm. Certain triple heterozygous combinations essentially lack mesoderm as a result of a block in ventral furrow formation during gastrulation. HLH proteins that have been implicated previously in sex determination and neurogenesis (daughterless, achaete, and scute) are shown to be required for the formation of these embryonic tissues. Evidence is also presented that dl-HLH interactions involve the direct physical association of these proteins in solution mediated by the rel and HLH domains. We discuss the striking parallels in mesoderm formation and sex determination.

Expression of the rat growth hormone-releasing hormone gene in placenta is directed by an alternative promoter.

Growth hormone-releasing hormone (GHRH) is a hypothalamic peptide that plays a critical role in controlling the synthesis and secretion of growth hormone by the anterior pituitary. GHRH has also been detected in other nonneural extrahypothalamic tissues, including rat placenta, although its role in the hormonal control of pregnancy and/or fetal development has not yet been defined. Here we present the isolation and characterization of cDNA clones corresponding to rat placental GHRH. The placental GHRH mRNA codes for a pre-pro-GHRH identical to that found in the hypothalamus, suggesting that the mature placental GHRH is identical to its hypothalamic counterpart. Nevertheless, the placental and the hypothalamic GHRH mRNAs differ in the region corresponding to the untranslated exon 1 because of the use of an alternative promoter in the placenta located 10 kilobases upstream from the hypothalamic promoter. A combined mechanism involving the use of tissue-specific alternative promoters and the differential splicing of exon 1 generates the mature GHRH transcript in placenta and hypothalamus. Multiple transcription initiation sites have been found in the placental GHRH mRNA, which correlates to the lack of a consensus TATA box in the promoter region.