A Hox gene mutation that triggers Nonsense-mediated RNA decay and affects alternative splicing during Drosophila development

Nonsense mutations are usually assumed to affect protein function by generating truncated protein products. Nonetheless, it is now clear that these mutations affect not just protein synthesis but also messenger RNA stability. The surveillance mechanism responsible for the detection and degradation of 'nonsense' RNA messages is termed nonsense-mediated RNA decay (NMD). Essential biochemical components of the NMD machinery have been defined in several species. Here we identify the Drosophila orthologue of one of these factors, Upf1, and document its expression during embryogenesis. To test whether NMD acts during Drosophila development, we make use of a mutation that introduces a stop codon into a variably spliced exon of the Hox gene Ultrabithorax (Ubx). Using real-time quantitative RT-PCR we demonstrate that Ubx transcripts containing the premature stop codon are expressed at lower levels than their wild type counterpart. Unexpectedly, we also find that the same mutation significantly increases the levels of a Ubx splicing isoform that lacks the exon containing the premature termination codon. These findings indicate that NMD is operational during Drosophila development and suggest that nonsense mutations may affect development by altering the spectrum of splicing products formed, as well as by reducing or eliminating protein synthesis

The role of homeotic genes in the specification of the Drosophila gonad

AbstractBackground: In Drosophila, the gonads are composed of two cell populations: the germ line, derived from the pole cells, and a somatic component, derived from the mesoderm of abdominal segments 5–8. Formation of the gonad requires the function of a specific homeotic gene, abdominal-A (abd-A). Other genes of the bithorax complex, Ultrabithorax (Ubx) or Abdominal-B (Abd-B), cannot substitute for this requirement when abd-A is removed.Results We show here that, in embryos lacking the entire bithorax complex, experimentally induced expression of either ABD-A or UBX protein in the mesoderm will rescue the expression of a gonad-specific marker, 412 RNA. Ubiquitous expression of these homeotic proteins within the mesoderm results in the formation of ectopic gonad tissue anterior to the normal location of the gonads. In the absence of any endogenous bithorax-complex gene expression, however, mesoderm expressing gonad markers still condenses preferentially in the posterior segments of the abdomen, even in the absence of pole cells.Conclusion The specific requirement for abd-A and not Ubx in gonad development does not reflect differences in the properties of the proteins that these genes encode, but presumably reflects differences in their regulation. In normal development, the restriction of gonad formation to the posterior abdomen does not depend on the overlap of abd-A and Abd-B expression, but must depend on the regulation of abd-A and Ubx in the sub-population of the mesoderm that forms the gonad. Factors other than homeotic gene expression provide some cues that direct gonadal mesoderm to condense in the correct location

An anterior medial cell population with an apical-organ-like transcriptional profile that pioneers the central nervous system in the centipede Strigamia maritima.

The apical plate of primary marine larvae is characterized by a common set of transcription factors comprising six3, rx, hbn, nk2.1 and FoxQ2. It harbours the apical organ, a neural and ciliary structure with neurosecretory properties. Recent studies in lophotrochozoans have found that apical organ cells form the anterior tip of the developing central nervous system. We identify an anterior medial tissue in the embryonic centipede head that shares the transcriptional profile of the apical plate of marine larvae, including nested domains of FoxQ2 and six3 expression. This domain gives rise to an anterior medial population of neural precursors distinct from those arising within the segmental neuroectoderm. These medial cells do not express achaete scute homologue in proneural clusters, but express collier, a marker for post mitotic cells committed to a neural fate, while they are still situated in the surface ectodermal layer. They then sink under the surface to form a compact cell cluster. Once internalized these cells extend axons that pioneer the primary axonal scaffold of the central nervous system. The same cells express phc2, a neural specific prohormone convertase, which suggests that they form an early active neurosecretory centre. Some also express markers of hypothalamic neurons, including otp, vtn and vax1. These medial neurosecretory cells of the centipede are distinct from those of the pars intercerebralis, the anterior neurosecretory part of the insect brain. The pars intercerebralis derives from vsx positive placodal-like invagination sites. In the centipede, vsx expressing invaginating ectoderm is situated bilaterally adjacent to the medial pioneer cell population. Hence the pars intercerebralis is present in both insect and centipede brains, whereas no prominent anterior medial cluster of pioneer neurons is present in insects. These observations suggest that the arthropod brain retained ancestrally an anterior medial population of neurosecretory cells homologous to those of the apical plate in other invertebrate phyla, but that this cell population has been lost or greatly reduced in insects.OtherThis is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.ydbio.2014.09.02

Hox go omics: insights from Drosophila into Hox gene targets

Microarray analysis reveals hundreds of hitherto unsuspected Hox gene targets

Formation and subdivision of the head field in the centipede Strigamia maritima, as revealed by the expression of head gap gene orthologues and hedgehog dynamics.

Background There have been few studies of head patterning in non-insect arthropods, and even in the insects, much is not yet understood. In the fly Drosophila three head gap genes, orthodenticle (otd), buttonhead (btd) and empty spiracles (ems) are essential for patterning the head. However, they do not act through the same pair-rule genes that pattern the trunk from the mandibular segment backwards. Instead they act through the downstream factors collier (col) and cap‘n’collar (cnc), and presumably other unknown factors. In the beetle Tribolium, these same gap and downstream genes are also expressed during early head development, but in more restricted domains, and some of them have been shown to be of minor functional importance. In the spider Parasteatoda tepidariorum, hedgehog (hh) and otd have been shown to play an important role in head segmentation. Results We have investigated the expression dynamics of otx (otd), SP5/btd, ems, and the downstream factors col, cnc and hh during early head development of the centipede Strigamia maritima. Our results reveal the process of head condensation and show that the anteroposterior sequence of specific gene expression is conserved with that in insects. SP5/btd and otx genes are expressed prior to and during head field formation, whereas ems is not expressed until after the initial formation of the head field, in an emerging gap between SP5/btd and otx expression. Furthermore, we observe an early domain of Strigamia hh expression in the head field that splits to produce segmental stripes in the ocular, antennal and intercalary segments. Conclusions The dynamics of early gene expression in the centipede show considerable similarity with that in the beetle, both showing more localised expression of head gap genes than occurs in the fly. This suggests that the broad overlapping domains of head gap genes observed in Drosophila are derived in this lineage. We also suggest that the splitting of the early hh segmental stripes may reflect an ancestral and conserved process in arthropod head patterning. A remarkably similar stripe splitting process has been described in a spider, and in the Drosophila head hh expression starts from a broad domain that transforms into three stripes

Germ cells of the centipede Strigamia maritima are specified early in embryonic development.

We provide the first systematic description of germ cell development with molecular markers in a myriapod, the centipede Strigamia maritima. By examining the expression of Strigamia vasa and nanos orthologues, we find that the primordial germ cells are specified from at least the blastoderm stage. This is a much earlier embryonic stage than previously described for centipedes, or any other member of the Myriapoda. Using these genes as markers, and taking advantage of the developmental synchrony of Strigamia embryos within single clutches, we are able to track the development of the germ cells throughout embryogenesis. We find that the germ cells accumulate at the blastopore; that the cells do not internalize through the hindgut, but rather through the closing blastopore; and that the cells undergo a long-range migration to the embryonic gonad. This is the first evidence for primordial germ cells displaying these behaviours in any myriapod. The myriapods are a phylogenetically important group in the arthropod radiation for which relatively little developmental data is currently available. Our study provides valuable comparative data that complements the growing number of studies in insects, crustaceans and chelicerates, and is important for the correct reconstruction of ancestral states and a fuller understanding of how germ cell development has evolved in different arthropod lineages.This is the final published version. It was originally published by Elsevier in Development Biology here: http://www.sciencedirect.com/science/article/pii/S0012160614002991. DOI: 10.1016/j.ydbio.2014.06.003

A Double Segment Periodicity Underlies Segment Generation in Centipede Development

AbstractThe number of leg-bearing segments in centipedes varies extensively, between 15 and 191, and yet it is always odd [1, 2]. This suggests that segment generation in centipedes involves a stage with double segment periodicity and that evolutionary variation in segment number reflects the generation of these double segmental units. However, previous studies have revealed no trace of this [3–5]. Here we report the expression of two genes, an odd-skipped related gene (odr1) and a caudal homolog, that serve as markers for early steps of segment formation in the geophilomorph centipede, Strigamia maritima. Dynamic expression of odr1 around the proctodaeum resolves into a series of concentric rings, revealing a pattern of double segment periodicity in overtly unsegmented tissue. Initially, the expression of the caudal homolog mirrors this double segment periodicity, but shortly before engrailed expression and overt segmentation, the intercalation of additional stripes generates a repeat with single segment periodicity. Our results provide the first clues about the causality of the unique and fascinating “all-odd” pattern of variation in centipede segment numbers and have implications for the evolution of the mechanisms of arthropod segmentation

Arthropod segmentation.

There is now compelling evidence that many arthropods pattern their segments using a clock-and-wavefront mechanism, analogous to that operating during vertebrate somitogenesis. In this Review, we discuss how the arthropod segmentation clock generates a repeating sequence of pair-rule gene expression, and how this is converted into a segment-polarity pattern by 'timing factor' wavefronts associated with axial extension. We argue that the gene regulatory network that patterns segments may be relatively conserved, although the timing of segmentation varies widely, and double-segment periodicity appears to have evolved at least twice. Finally, we describe how the repeated evolution of a simultaneous (Drosophila-like) mode of segmentation within holometabolan insects can be explained by heterochronic shifts in timing factor expression plus extensive pre-patterning of the pair-rule genes.BBSRC grant BB/P009336/1 in Cambridge (MA and EC) BBSRC grant BB/L020092/1 in Leeds (ADP) Trinity College JRF to Erik Clark in Cambridge EMBO Long term fellowship to Erik Clark in Harvar

Oncopeltus fasciatus zen is essential for serosal tissue function in katatrepsis

AbstractUnlike most Hox cluster genes, with their canonical role in anterior–posterior patterning of the embryo, the Hox3 orthologue of insects has diverged. Here, we investigate the zen orthologue in Oncopeltus fasciatus (Hemiptera:Heteroptera). As in other insects, the Of-zen gene is expressed extraembryonically, and RNA interference (RNAi) experiments demonstrate that it is functionally required in this domain for the proper occurrence of katatrepsis, the phase of embryonic movements by which the embryo emerges from the yolk and adjusts its orientation within the egg. After RNAi knockdown of Of-zen, katatrepsis does not occur, causing embryos to complete development inside out. However, not all aspects of expression and function are conserved compared to grasshopper, beetle, and fly orthologues. Of-zen is not expressed in the extraembryonic tissue until relatively late, suggesting it is not involved in tissue specification. Within the extraembryonic domain, Of-zen is expressed in the outer serosal membrane, but unlike orthologues, it is not detectable in the inner extraembryonic membrane, the amnion. Thus, the role of zen in the interaction of serosa, amnion, and embryo may differ between species. Of-zen is also expressed in the blastoderm, although this early expression shows no apparent correlation with defects seen by RNAi knockdown

XX/XY System of Sex Determination in the Geophilomorph Centipede Strigamia maritima.

We show that the geophilomorph centipede Strigamia maritima possesses an XX/XY system of sex chromosomes, with males being the heterogametic sex. This is, to our knowledge, the first report of sex chromosomes in any geophilomorph centipede. Using the recently assembled Strigamia genome sequence, we identified a set of scaffolds differentially represented in male and female DNA sequence. Using quantitative real-time PCR, we confirmed that three candidate X chromosome-derived scaffolds are present at approximately twice the copy number in females as in males. Furthermore, we confirmed that six candidate Y chromosome-derived scaffolds contain male-specific sequences. Finally, using this molecular information, we designed an X chromosome-specific DNA probe and performed fluorescent in situ hybridization against mitotic and meiotic chromosome spreads to identify the Strigamia XY sex-chromosome pair cytologically. We found that the X and Y chromosomes are recognizably different in size during the early pachytene stage of meiosis, and exhibit incomplete and delayed pairing.This work was in part funded by Wellcome Trust (wellcome.ac.uk) Ph.D. studentship WT089615MA to JEG. Cytological experiments by MD and FM were funded by Grant IAA600960925 of the Grant Agency of The Czech Academy of Sciences (until 2013; gaav.cz) and by Grant 14- 22765S of the Czech Science Foundation (since 2014; gacr.cz). KS was supported by JSPS Excellent Young Researchers Overseas Visit Program (21– 7147; jsps.go.jp).This is the final version of the article. It first appeared from the Public Library of Science (PLOS) via https://doi.org/10.1371/journal.pone.015029