Expression of dorsal-ventral genes during early development of Rhynchosciara americana embryos.

The establishment of dorsal-ventral polarity in Drosophila is a complex process which involves the action of maternal and zygotically expressed genes. Interspecific differences in the expression pattern of some of these genes have been described in other species. Here we present the expression of dorsal-ventral genes during early embryogenesis in the lower dipteran Rhynchosciara americana. The expression of four genes, the ventralizing genes snail (sna) and twist (twi) and the dorsalizing genes decapentaplegic (dpp) and zerknullt (zen), was investigated by whole-mount in situ hybridization. Sense and antisense mRNA were transcribed in vitro using UTP-digoxigenin and hybridized at 55 degrees C with dechorionated fixed embryos. Staining was obtained with anti-digoxigenin alkaline phosphatase-conjugated antibody revealed with NBT-BCIP solution. The results showed that, in general, the spatial-temporal expression of R. americana dorsal-ventral genes is similar to that observed in Drosophila, where twi and sna are restricted to the ventral region, while dpp and zen are expressed in the dorsal side. The differences encountered were subtle and probably represent a particular aspect of dorsal-ventral axis determination in R. americana. In this lower dipteran sna is expressed slightly later than twi and dpp expression is expanded over the lateral ectoderm during cellular blastoderm stage. These data suggest that the establishment of dorsal-ventral polarity in R. americana embryos follows a program similar to that observed in Drosophila melanogaster.

Expression of dorsal-ventral genes during early development of Rhynchosciara americana embryos

The establishment of dorsal-ventral polarity in Drosophila is a complex process which involves the action of maternal and zygotically expressed genes. Interspecific differences in the expression pattern of some of these genes have been described in other species. Here we present the expression of dorsal-ventral genes during early embryogenesis in the lower dipteran Rhynchosciara americana. The expression of four genes, the ventralizing genes snail (sna) and twist (twi) and the dorsalizing genes decapentaplegic (dpp) and zerknüllt (zen), was investigated by whole-mount in situ hybridization. Sense and antisense mRNA were transcribed in vitro using UTP-digoxigenin and hybridized at 55°C with dechorionated fixed embryos. Staining was obtained with anti-digoxigenin alkaline phosphatase-conjugated antibody revealed with NBT-BCIP solution. The results showed that, in general, the spatial-temporal expression of R. americana dorsal-ventral genes is similar to that observed in Drosophila, where twi and sna are restricted to the ventral region, while dpp and zen are expressed in the dorsal side. The differences encountered were subtle and probably represent a particular aspect of dorsal-ventral axis determination in R. americana. In this lower dipteran sna is expressed slightly later than twi and dpp expression is expanded over the lateral ectoderm during cellular blastoderm stage. These data suggest that the establishment of dorsal-ventral polarity in R. americana embryos follows a program similar to that observed in Drosophila melanogaster.

Stripe forming architecture of the gap gene system.

In this report, we show that gap genes encode exactly one set of pair-rule stripes, which occur in the native even-skipped position. The core of this work is a detailed analysis that shows how this conclusion follows from the arrangement of gap domains in the embryo. This analysis shows that: (1) pattern forming information is transmitted from gap to pair-rule genes by means of a nonredundant set of morphogenetic gradients, and (2) the stripe forming capability of the gap genes is constrained by the arrangement of these gradients and by the fact that each gap domain consists of a pair of correlated gradients. We also show that in the blastoderm, the regulatory sign of a transcriptional regulator is unlikely to change in a concentration dependent manner. The principal analytic tool used to establish these results is the gene circuit method. Here, this method is applied to examine hybrid data sets consisting of real gene expression data for four gap genes and hypothetical pair-rule expression data generated by translating native even-skipped data along the anterior-posterior axis. In this way, we are able to investigate the stripe forming capabilities of the gap gene system in the complete absence of pair-rule cross regulation. We close with an inference about evolutionary development. It is argued that the constraints on gap gene architecture identified here are a consequence of selective pressures that minimize the number of gap genes required to determine segments in long-germ band insects.

Embryonic expression of the engrailed homologue of Rhynchosciara americana.

The segment polarity gene engrailed is involved in the determination of segment posterior identity in Drosophila. engrailed has been largely used for comparative developmental studies due to its evolutionary conservation from nematodes to humans. By in situ hybridization of an engrailed cDNA probe from Drosophila to polytene chromosomes of fourth instar larvae of Rhynchosciara americana we have shown that engrailed-like sequences must be localized in band 6 of chromosome A in this species. The pattern of engrailed protein expression during R. americana embryo development is diffuse at first evolving into a nuclear striped pattern after quite a length of time. In addition, our results suggest a possible developmentally regulated molecular modification of engrailed protein in R. americana embryos.

Targeted ribozymes reveal a conserved function of the Drosophila paired gene in sensory organ development.

The Drosophila paired (prd) gene, the founding member of the PAX gene family, is required for normal embryonic segmentation and is re-expressed later in development in the head and developing CNS. As for most embryonically active genes, global defects resulting from loss of early prd function obscure an analysis of the role of later expression phases. We used inducible targeted ribozymes to functionally 'knock-out' prd at late stages. When prd protein levels in the head are reduced in this fashion, the maxillary chemosensory ventral organs fail to develop and dorsal-lateral cirri rows are disrupted. These studies reveal a role for prd in sensory organ development that appears to be conserved in PAX genes throughout the animal kingdom.

Multiple regulatory elements direct the complex expression pattern of the Drosophila segmentation gene paired.

The paired (prd) gene of Drosophila belongs to the pair-rule class of segmentation genes involved in establishing the metameric organization of the Drosophila body plan. The complex expression pattern of prd has previously been shown to depend upon a number of segmentation genes, including gap and pair-rule genes. In an attempt to characterize and analyze the regulatory regions necessary and sufficient for prd expression, we have identified an 18-kb genomic fragment, consisting of the transcribed portion of prd and 10 kb of 5'- and 5 kb of 3'-flanking region, that is able to rescue prd mutant embryos to full viability. Analysis of a series of prd-lacZ fusion constructs containing progressively reduced lengths of prd 5'-flanking sequences delimits different cis-regulatory regions. The entire 5'-flanking region directs fusion gene expression in a pattern similar, but not identical, to the endogenous prd protein pattern. This 10-kb fragment contains both activator and repressor regions that mediate the establishment of the seven-stripe prd pattern, as well as the splitting into anterior and posterior stripes for the 14-stripe expression phase. The prd intron in combination with a minimal upstream region (0.15 kb) is able to direct low levels of prd-lacZ fusion gene expression in stripes. Information for expression of the anterior dorsal spot and of the early seven-stripe pattern is located downstream of the prd coding region. We propose that regulation of prd by pair-rule and gap gene products is mediated by upstream and downstream cis-regulatory elements. Regulation during separate but overlapping phases of expression by separable regulatory regions might be a general characteristic of segmentation genes.