Alain Vincent

Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre

Drosophila haemocytes (blood cells) originate from a specialized haematopoietic organ—the lymph gland. Larval haematopoietic progenitors (prohaemocytes) give rise to three types of circulating haemocytes: plasmatocytes, crystal cells and lamellocytes. Lamellocytes, which are devoted to encapsulation of large foreign bodies, only differentiate in response to specific immune threats, such as parasitization by wasps. Here we show that a small cluster of signalling cells, termed the PSC (posterior signalling centre), controls the balance between multipotent prohaemocytes and differentiating haemocytes, and is necessary for the massive differentiation of lamellocytes that follows parasitization. Communication between the PSC and haematopoietic progenitors strictly depends on the PSC-restricted expression of Collier, the Drosophila orthologue of mammalian early B-cell factor. PSC cells act, in a non-cell-autonomous manner, to maintain JAK/STAT signalling activity in prohaemocytes, preventing their premature differentiation. Serrate-mediated Notch signalling from the PSC is required to maintain normal levels of col transcription. The key role of the PSC in controlling blood cell homeostasis is reminiscent of interactions between haematopoietic progenitors and their micro-environment in vertebrates, thus further highlighting the interest of Drosophila as a model system for studying the evolution of haematopoiesis and cellular innate immunity.

The COE--Collier/Olf1/EBF--transcription factors: structural conservation and diversity of developmental functions.

One major conclusion of studies in Developmental Biology during the last two decades is that, despite profound anatomical differences, the building of vertebrate and arthropod bodies relies on the same fundamental molecular networks, including conserved cell signalling and transcription-regulatory cascades. Rodent Early B-Cell Factor/Olfactory-1 and Drosophila Collier belong to a recently defined, novel family of transcription factors, the Collier/Olf1/EBF (COE) proteins which have a unique DNA-binding domain. Early investigations revealed that, despite their high degree of sequence identity, the different vertebrate and invertebrate COE proteins play a variety of developmental roles. We review here the current evidence for this diversity of COE functions, including in the specification and differentiation of various neuronal populations. We also discuss the existence of an evolutionarily conserved pathway linking Notch signalling and COE regulatory functions in various developmental decisions.

XCoe2, a transcription factor of the Col/Olf-1/EBF family involved in the specification of primary neurons in Xenopus

BACKGROUNDnPrimary neurogenesis in Xenopus is a model for studying the control of neural cell fate decisions. The specification of primary neurons appears to be driven by transcription factors containing a basic region and a helix-loop-helix (HLH) motif: expression of Xenopus neurogenin-related-1 (X-ngnr-1) defines the three prospective domains of primary neurogenesis, and expression of XNeuroD coincides with neuronal differentiation. The transition between neuronal competence and stable commitment to a neuronal fate remains poorly characterised, however.nnnRESULTSnDrosophila Collier and rodent early B-cell factor/olfactory-1 define a family of HLH transcription factors containing a previously unknown type of DNA-binding domain. We isolated an orthologous gene from Xenopus, Xcoe2, which is expressed in precursors of primary neurons. Xcoe2 is transcribed after X-ngnr-1 and before XNeuroD. Overexpression of a dominant-negative mutant of XCoe2 prevented neuronal differentiation. Conversely, overexpressed wild-type Xcoe2 could promote ectopic differentiation of neurons, in both the neural plate and the epidermis. In contrast to studies with X-ngnr-1 or XNeuroD, the supernumerary neurons induced by Xcoe2 appeared in a salt-and-pepper pattern, resulting from the activation of X-Delta1 expression and feedback regulation by lateral inhibition.nnnCONCLUSIONSnXCoe2 may play a pivotal role in the transcriptional cascade that specifies primary neurons in Xenopus embryos: by maintaining Delta-Notch signalling, XCoe2 stabilises the higher neural potential of selected progenitor cells that express X-ngnr-1, ensuring the transition between neural competence and irreversible commitment to a neural fate; and it promotes neuronal differentiation by activating XNeuroD expression, directly or indirectly.

MOLECULAR CLONING OF ZCOE2, THE ZEBRAFISH HOMOLOG OF XENOPUS XCOE2 AND MOUSE EBF-2, AND ITS EXPRESSION DURING PRIMARY NEUROGENESIS

Xcoe2 is a recently identified HLH transcription factor of the Xenopus primary neurogenesis pathway, which is necessary downstream of Neurogenin to stabilize neuroblast determination (Dubois, L. et al., 1998. Curr. Biol. 8, 199-209). We report here the embryonic expression pattern of Zcoe2, its zebrafish homolog. As observed for Xcoe2, Zcoe2 is strongly expressed in a subset of the neurogenin1- (ngn1-) positive primary neuroblasts of the spinal cord. In the anterior neural plate, in contrast, Zcoe2 is expressed earlier and more widely than ngn1. This pattern is strongly maintained in the presumptive mesencephalon and rhombomeres 1-4 until the 2-3-somite stage. This expression of Zcoe2 in the brain anlage calls for a re-analysis in zebrafish of the functional relationship demonstrated in Xenopus between Coe2 and Neurogenin factors. At later stages, Zcoe2 is expressed in early forming neurons of the anterior brain and is a marker of the olfactory placodes.

Control of multidendritic neuron differentiation in Drosophila: The role of Collier

Proper sampling of sensory inputs critically depends upon neuron morphogenesis and expression of sensory channels. The highly stereotyped organisation of the Drosophila peripheral nervous system (PNS) provides a model to study neuronal determination and morphogenesis. Here, we report that Collier/Knot (Col/Kn), the Drosophila member of the COE family of transcription factors, is transiently expressed in the subset of multidendritic arborisation (da) sensory neurons that display an highly branched dendritic arborisation, class IV neurons. When lacking Col activity, class IV da neurons are formed but display a reduced dendrite arborisation. Col control on dendrite branching is distinct from that exerted by Cut, another transcription factor expressed in class IV neurons and necessary for proper dendrite morphogenesis. Col is also required for the class IV da-specific expression of pickpocket (ppk), which encodes a degenerin/epithelial sodium channel subunit required for larval locomotion. Characterisation of the col upstream region identified a 9-kb cis-regulatory region driving col expression in all class IV md neurons, even though these originate from two types of sensory precursor cells. Altogether, these findings indicate that col is required in at least two distinct programs that control the morphological and sensory specificity of Drosophila md neurons.

Drosophila melanogaster poly(A)-binding protein: cDNA cloning reveals an usually long 3′-untranslated region of the mRNA, also present in other eukaryotic species

Abstract Two classes of cDNAs encoding the Drosophila melanogaster poly(A)-binding protein (PABP), which differ in length due to different positions of their respective 3′ ends, were isolated by screening an embryonic cDNA library. These cDNAs hybridize to a single chromosomal site at position 55B on the right arm of the second chromosome. A unique 3.8-kb PABP mRNA species was detected, indicating that ‘long’ cDNAs correspond to full-length cDNAs and that the 3′-untranslated region of the D. melanogaster mRNA is close to 1.5 kb long. The PABP transcript accumulates in oocytes, is maternally inherited by the embryo and present at every developmental stage tested. The D. melanogaster PABP cDNAs contain a 1722-nt ORF encoding a 64-kDa protein. This protein contains four RNA-binding domains which show limited primary sequence divergence during evolution, in contrast to the C-terminal third of the protein. The strikingly long 3′-untranslated region of the D. melanogaster PABP mRNA is shown to exist also in other eukaryotes including vertebrate species. It suggests that important regulatory sequences intrinsic to the PABP mRNA are present within this 3′-untranslated region.

The dual function of ovo/shavenbaby in germline and epidermis differentiation is conserved between Drosophila melanogaster and the olive fruit fly Bactrocera oleae

The olive fruit fly Bactrocera oleae (B. oleae) is a major olive damaging pest in the Mediterranean area. As a first molecular analysis of a developmental gene in this insect, we characterised the ovo/shavenbaby (ovo/svb) gene. In Drosophila, ovo/svb encodes a family of transcription regulators with two distinct functions: ovo is required for female germline differentiation and svb controls morphogenesis of epidermal cells. Here, we report the cloning and characterisation of ovo/svb in B. oleae, showing that the ovo genomic organisation and complex pattern of germline transcription have been conserved between distantly related Dipterae. We further show that B. oleae svb embryonic expression precisely prefigures the pattern of larval trichomes, supporting the conclusion that regulatory changes in svb transcription underlie evolutionary diversification of trichome patterns seen among Dipterae.

Separate cis-regulatory sequences control expression of serendipity beta and janus A, two immediately adjacent Drosophila genes.

The genes janus (Jan) A and B, and serendipity (sry) β and λ are two pairs of duplicated genes that are adjacent to each other on the third chromosome of Drosophila melanogaster. The jan A and sry β genes are expressed throughout development in both males and females. They are transcribed in opposite orientations from start sites separated by only 173 by of DNA. We report here the complete sequence of the jan A and B genes in Drosophila pseudoobscura, a species distantly related to D. melanogaster in which the overall organization of the sry β, Jan A and jan B genes is identical to that in D. melanogaster. Sequence comparison of the jan A-sry β intergenic region and 5-transcribed domain of each gene between D. melanogaster and D. pseudoobscura reveals short stretches of conserved sequences that may correspond to cis-acting regulator elements. In order to test the possibility that some cis-acting regulatory sequences are shared by the two genes, we carried out a deletion analysis of the jan A/sry β intergenic region in D. melanogaster using transgenic lacZ fusion genes. Our results show that sry β cis-acting sequences are located in the (-117; + 137) 5′-region of the gene and that jan A cis-regulatory sequences are included in the (-56; +151) 5′-domain of this gene. Together these data indicate that in spite of the physical proximity of the jan A and sry β genes, their transcription is regulated by separate cis-acting sequences.