Centre de Biologie du Développement

Gene regulatory networks in Drosophila haematopoiesis and myogenesis

Overview

Two main research projects are currently developed in our group using Drosophila as a model system : i) The control of definitive hematopoiesis and the cellular immune responses to parasitism ; ii) Specification and realisation of skeletal muscle identity in the embryo. The historical link between these two projects is the characterisation, in 1996 of a new head-segmentation gene, collier (col) (Crozatier et al., 1996, ). This in turn led to the identification of a novel family of metazoan transcription factors, the COE proteins (COE for Drosophila Collier and vertebrate EBF (Early B-Cell Factor) (Dubois and Vincent, 2001), which are characterised by the presence of a DNA binding domain specific to this family associated with an atypical HLH dimerisation motif. Multiple genome sequence analysis shows that there exists a single coe gene in all metazoans, except for vertebrates where 4 genes have been identified (Daburon et al., 2008). Phenotypic analyses of ebf/coe mutants in Drosophila C. elegans and mouse indicate that COE proteins play a variety of different developmental roles, correlating with a variety of tissue and/or cell lineage-specific expression sites (see for example Crozatier et al., 2002; Crozatier and Vincent, 2008).

Hematopoieis and the cellular immune response to parasitism.

Drosophila has become a major model system to study innate immunity. The Drosophila immune defence relies upon the synthesis of anti-microbial peptides and the activity of specialised cells, the hemocytes : plasmatocytes involved in phagocytosis ; cristal cells involved in immune-triggered melanisation; lamellocytes which neutralise parasites too large to be phagocytosed. Unlike plasmatocytes and crystal cells, lamellocytes are not present in healthy larvae but differentiate in response to specific immune challenges such as parasitization by wasps. Drosophila larval hemocytes originate from a hematopoietic organ called the lymph gland (LG). In collaboration with Marie Meister, Strasbourg, we found several years ago that Collier activity is required for formation of a signalling center in the LG, termed the PSC, and the differentiation of lamellocytes following parasitization (Crozatier et al., PLoS Biology 2004). A turning point was our discovery that the PSC controls, in a non cell autonomous manner, the balance between multipotent hemocyte precursors and differentiating hemocytes (Krzemien et al., Nature, 2007). The Drosophila PSC thus acts as a niche in controlling blood cell homeostasis, similar to the vertebrate hematopoietic for survival and self-renewing of Hematopoietic Stem Cells in the bone marrow (Crozatier et al., 2007, Krzemien et al., 2010a, Crozatier and Vincent, Disease Model and Mechanisms, 2011). The maintenance of a pool of pro-hemocytes is dependent upon JAK-STAT signallin and is dependent upon the activity of Latran, a truncated, dominant-negative cytokine type 1 receptor. The role of Latran in the LG thus uncovered a novel mechanism for modulating JAK/STAT signalling in innate immune responses (Makki et al., PloS Biology, 2010). Lineage analyses showed that a pool of proliferative, intermediate progenitors accounts for increased hemocyte differentiation at metamorphosis (Krzemien et al., 2010b). Transcriptome analyses of LGs in different mutant conditions (in preparation) revealed that Bone Morphogenetic Protein (BMP) and Wg (Wnt) signalling in PSC cells controls the niche size via regulation of dmyc. An increased number of PSC cells prevents hemocyte differentiation, demonstrating that niche size is a key parameter of hemocyte homeostasis (Pennetier et al., 2012). Hedgehog and Wg signaling was independently shown to be required to maintain pro-hemocytes (Mandal et al., 2007 ; Sinenko et al., 2009). Starting from these observations, our current project is identifying parameters of developmental plasticity of prohemocytes and the niche function in physiological and stress conditions. The niche cells are specified by the transcription factor Collier (Col)/Early B-cell factor (EBF) (Crozatier et al., 2004). In mouse, EBF2 activity and BMP and Wnt signaling in osteoblasts is required for the proper numbers of niche cells and HSCs (Kieslinger et al., Cell 2010). Our recent data showing that Col coordinately regulates the PSC cell number and signaling to prohemocytes (Pennetier et al., 2012) revealed new parallels between Drosophila and mammalian hematopoiesis. Genetic manipulation of PSC function is thus expected to provide novel clues as to how the mammalian bone marrow niche controls the balance between HSC self-renewal and differentiation.

Specification of muscle identity in the Drosophila embryo

Textbook drawings of human anatomy illustrate the diversity of body muscles that are essential for coordinated movements. The genetic and molecular bases of this muscle diversity remain, however, largely unknown. The rather simple Drosophila larval musculature - every (hemi)-segment of the Drosophila larva contains about 30 different somatic muscles, each composed of a single multinucleate syncitial fibre- makes it an ideal model to study this process. Each muscle displays its own identity which can be described as its specific position and orientation with respect to the dorso-ventral (D/V) and antero-posterior (A/P) axes, size, attachment sites to the epidermis and innervations. Each Drosophila muscle is seeded by a founder cell (FC). FCs display the unique property of being able to undergo multiple rounds of fusion with fusion competent myoblasts (FCMs). FCs are born from the asymmetric division of progenitor cells which are themselves selected by Notch (N)-mediated lateral inhibition from promuscular clusters specified at fixed positions within the somatic mesoderm (see Fig). The current view is that muscle identity reflects the expression by each PC/FC of a specific combination of “identity” transcription factors (iTFs). Using as paradigm a specific muscle lineage, the dorsal DA3 (Dorsal Acute 3) muscle and expression of two iTFs, Collier (Col) and Nautilus/d-MyoD (Nau), we have recently shown that the “transcriptional identity” is propagated from the FC to nuclei of FCM recruited by the growing myofibre during the fusion process (Dubois et al., Development, 2007). Col and Nau combinatorially control the shape of dorso-lateral Drosophila muscles (Enriquez et al., Dev. Biol. 2012). Finally, homeotic (Hox) proteins play a decisive role in establishing the pattern of Drosophila muscles by implementing the expression of iTFs at the progenitor stage (Enriquez et al., Development 2010). Ongoing studies aim at identifying muscle "identity realisator" genes and further deciphering the molecular and cellular mechanisms of progenitor specification, in the frame of comparative studies with vertebrate myogenesis.