Identification de nouveaux gènes cibles du facteur proneural Ngn1 et analyse de leurs fonctions dans la détermination et la spécification neuronale chez le poisson zèbre

Lors de ma thèse, j'ai étudié les activités spécifiques de facteurs de transcription à domaine bHLH, appelés gènes proneuraux, qui sont nécessaires et suffisants pour la formation des neurones. Les deux familles de facteurs proneuraux, achaete-scute et atonal, ont des activités différentes dans la formation des neurones. Les facteurs proneuraux sont des facteurs de transcription, il est donc possible que leurs activités spécifiques reflètent la régulation d'un ensemble de gènes cibles spécifique. Afin de mieux comprendre ces divergences d'activité, nous avons étudié la régulation du gène deltaA. L'analyse fonctionnelle du promoteur de deltaA a permis d'identifier un cluster de trois E-Box non-redondantes qui agissent en coopération pour permettre la fixation de Ngn1 et la régulation de deltaA. Cette étude met en évidence un nouveau mécanisme de régulation des gènes cibles par les facteurs proneuraux de la famille neurogenin. Dans le but de mieux comprendre l'activité spécifique de Ngn1, nous avons étudié la fonction de deux gènes cibles spécifiques, neurod4 et cxcr4b. Dans la placode olfactive, les expressions précoces de neurod4 et cxcr4b sont dépendantes de Ngn1. Nous avons montré qu’en l'absence de ngn1 et neurod4, les neurones olfactifs sont perdus indiquant que ces deux gènes sont redondants pour la neurogenése olfactive. Ensuite, nous avons montré que l'expression précoce de cxcr4b, qui est perdue dans le mutant ngn1, est nécessaire pour la projection des axones des neurones olfactifs sur le bulbe olfactif. Ainsi, cxcr4b est exprimé dans les progéniteurs neuraux mais sa fonction est requise dans les neurones différentiés pour contrôler le comportement cellulaire du neurone.During my thesis, I have studied specific activity of bHLH transcription factor called proneural genes which are necessary and sufficient for the formation of neurons. The two families of proneural genes: achaete-scute and atonal have divergent activities in the formation of neurons. Proneural genes are transcription factor, making it likely that divergent activity between atonal and achaete-scute family comes from transcriptional regulation of different targets genes. To understand these divergences, we have begun a study of deltaA gene regulation. The functional analysis of the deltaA promoter led us to identify a cluster of three non-redundant E-Box which act in a cooperative manner to allow DNA binding and regulation of deltaA by Ngn1. This study provides a novel mechanism for the regulation of target genes by proneural genes of the neurogenin family. In order to have a better understanding of specific Ngn1 activity in control of neuronal identity, I have studied the function of two specific Ngn1 target genes, neuroD4 and cxcr4b. In the developing olfactory placode, early neurod4 or cxcr4b expression is dependent of Ngn1. I have show that in absence of Ngn1 and Neurod4, olfactory neurons are totally lost indicating that these two genes act in a redundant manner to control olfactory neurogenesis. I have show that early cxcr4b expression which is lost in ngn1 mutant is necessary to allow axonal projection of olfactory neurons on the olfactory bulb. Interestingly, cxcr4b is expressed only in neural progenitor but his function is required in differentiated neurons. Ngn1 activity is necessary in neural progenitor to control neuron behaviour

Sox10 contributes to the balance of fate choice in dorsal root ganglion progenitors

The development of functional peripheral ganglia requires a balance of specification of both neuronal and glial components. In the developing dorsal root ganglia (DRGs), these compo- nents form from partially-restricted bipotent neuroglial precursors derived from the neural crest. Work in mouse and chick has identified several factors, including Delta/Notch signal- ing, required for specification of a balance of these components. We have previously shown in zebrafish that the Sry-related HMG domain transcription factor, Sox10, plays an unex- pected, but crucial, role in sensory neuron fate specification in vivo. In the same study we described a novel Sox10 mutant allele, sox10baz1, in which sensory neuron numbers are elevated above those of wild-types. Here we investigate the origin of this neurogenic pheno- type. We demonstrate that the supernumerary neurons are sensory neurons, and that enteric and sympathetic neurons are almost absent just as in classical sox10 null alleles; peripheral glial development is also severely abrogated in a manner similar to other sox10 mutant alleles. Examination of proliferation and apoptosis in the developing DRG reveals very low levels of both processes in wild-type and sox10baz1, excluding changes in the bal- ance of these as an explanation for the overproduction of sensory neurons. Using chemical inhibition of Delta-Notch-Notch signaling we demonstrate that in embryonic zebrafish, as in mouse and chick, lateral inhibition during the phase of trunk DRG development is required to achieve a balance between glial and neuronal numbers. Importantly, however, we show that this mechanism is insufficient to explain quantitative aspects of the baz1 phenotype. The Sox10(baz1) protein shows a single amino acid substitution in the DNA binding HMG domain; structural analysis indicates that this change is likely to result in reduced flexibility in the HMG domain, consistent with sequence-specific modification of Sox10 binding to DNA. Unlike other Sox10 mutant proteins, Sox10(baz1) retains an ability to drive neurogenin1 transcription. We show that overexpression of neurogenin1 is sufficient to produce supernu- merary DRG sensory neurons in a wild-type background, and can rescue the sensory neu- ron phenotype of sox10 morphants in a manner closely resembling the baz1 phenotype. We conclude that an imbalance of neuronal and glial fate specification results from the Sox10 (baz1) protein\u2019s unique ability to drive sensory neuron specification whilst failing to drive glial development. The sox10baz1 phenotype reveals for the first time that a Notch-dependent lat- eral inhibition mechanism is not sufficient to fully explain the balance of neurons and glia in the developing DRGs, and that a second Sox10-dependent mechanism is necessary. Sox10 is thus a key transcription factor in achieving the balance of sensory neuronal and glial fates

Endogenous retinal neural stem cell reprogramming for neuronal regeneration

In humans, optic nerve injuries and associated neurodegenerative diseases are often followed by permanent vision loss. Consequently, an important challenge is to develop safe and effective methods to replace retinal neurons and thereby restore neuronal functions and vision. Identifying cellular and molecular mechanisms allowing to replace damaged neurons is a major goal for basic and translational research in regenerative medicine. Contrary to mammals, the zebrafish has the capacity to fully regenerate entire parts of the nervous system, including retina. This regenerative process depends on endogenous retinal neural stem cells, the Müller glial cells. Following injury, zebrafish Müller cells go back into cell cycle to proliferate and generate new neurons, while mammalian Müller cells undergo reactive gliosis. Recently, transcription factors and microRNAs have been identified to control the formation of new neurons derived from zebrafish and mammalian Müller cells, indicating that cellular reprogramming can be an efficient strategy to regenerate human retinal neurons. Here we discuss recent insights into the use of endogenous neural stem cell reprogramming for neuronal regeneration, differences between zebrafish and mammalian Müller cells, and the need to pursue the identification and characterization of new molecular factors with an instructive and potent function in order to develop theurapeutic strategies for eye diseases

Genetic deciphering of the antagonistic activities of the melanin-concentrating hormone and melanocortin pathways in skin pigmentation.

The genetic origin of human skin pigmentation remains an open question in biology. Several skin disorders and diseases originate from mutations in conserved pigmentation genes, including albinism, vitiligo, and melanoma. Teleosts possess the capacity to modify their pigmentation to adapt to their environmental background to avoid predators. This background adaptation occurs through melanosome aggregation (white background) or dispersion (black background) in melanocytes. These mechanisms are largely regulated by melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH), two hypothalamic neuropeptides also involved in mammalian skin pigmentation. Despite evidence that the exogenous application of MCH peptides induces melanosome aggregation, it is not known if the MCH system is physiologically responsible for background adaptation. In zebrafish, we identify that MCH neurons target the pituitary gland-blood vessel portal and that endogenous MCH peptide expression regulates melanin concentration for background adaptation. We demonstrate that this effect is mediated by MCH receptor 2 (Mchr2) but not Mchr1a/b. mchr2 knock-out fish cannot adapt to a white background, providing the first genetic demonstration that MCH signaling is physiologically required to control skin pigmentation. mchr2 phenotype can be rescued in adult fish by knocking-out pomc, the gene coding for the precursor of α-MSH, demonstrating the relevance of the antagonistic activity between MCH and α-MSH in the control of melanosome organization. Interestingly, MCH receptor is also expressed in human melanocytes, thus a similar antagonistic activity regulating skin pigmentation may be conserved during evolution, and the dysregulation of these pathways is significant to our understanding of human skin disorders and cancers

Invasive meningococcal disease-induced myocarditis in critically ill adult patients: initial presentation and long-term outcome

International audienc

Cell-type heterogeneity in the early zebrafish olfactory epithelium is generated from progenitors within preplacodal ectoderm

International audienc

Habenular Neurogenesis in Zebrafish Is Regulated by a Hedgehog, Pax6 Proneural Gene Cascade

<div><p>The habenulae are highly conserved nuclei in the dorsal diencephalon that connect the forebrain to the midbrain and hindbrain. These nuclei have been implicated in a broad variety of behaviours in humans, primates, rodents and zebrafish. Despite this, the molecular mechanisms that control the genesis and differentiation of neural progenitors in the habenulae remain relatively unknown. We have previously shown that, in zebrafish, the timing of habenular neurogenesis is left-right asymmetric and that in the absence of Nodal signalling this asymmetry is lost. Here, we show that habenular neurogenesis requires the homeobox transcription factor Pax6a and the redundant action of two proneural bHLH factors, Neurog1 and Neurod4. We present evidence that Hedgehog signalling is required for the expression of <i>pax6a</i>, which is in turn necessary for the expression of <i>neurog1</i> and <i>neurod4</i>. Finally, we demonstrate by pharmacological inhibition that Hedgehog signalling is required continuously during habenular neurogenesis and by cell transplantation experiments that pathway activation is required cell autonomously. Our data sheds light on the mechanism underlying habenular development that may provide insights into how Nodal signalling imposes asymmetry on the timing of habenular neurogenesis.</p></div

Hh signalling is required for the expression of <i>pax6a</i>, <i>neurog1</i> and <i>neurod4</i> in the Habenular nuclei.

<p>Confocal sections (A-F) or 15μm maximum projections (A’-F’) showing the heads of control treated embryos (A-A’, n = 9; C-C’, n = 6; E-E’, n = 6) or those treated from 16 hpf with cyclopamine (B-B’, n = 10, D-D’, n = 7, F-F’, n = 6) after a whole-mount <i>in situ</i> hybridization against <i>pax6a</i> (A,A’,B,B’), <i>neurog1</i> (C,C’,D,D’) and <i>neurod4</i> (E,E’,F,F’) (red) and immunostaining against HuC/D protein (green); cell nuclei staining (grey) makes visible brain structures in the confocal sections (A-F). The overall morphology of the head appears normal in cyclopamine treated embryos and the expression of HuC/D does not appear to be affected in the telencephalon (Tel), in the epiphysis (*) nor in the tectum (Tc). In contrast, HuC/D expression is absent or strongly reduced in the habenular domain (Hb, white brackets) of cyclopamine treated embryos. The expression of <i>pax6a</i> is strongly reduced (B-B’, n = 10/10) in the habenular domain (Hb, white brackets) of cyclopamine treated embryos. The expression of <i>neurog1</i> and <i>neurod4</i> is also abrogated specifically in the habenular domain of cyclopamine treated embryos (D-D’, n = 7/7, F-F’, n = 6/6). All embryos are at 36 hpf. Embryos are viewed dorsally with anterior up.</p

Pax6a but not Pax6b are required for habenular neurogenesis.

<p>Whole-mount <i>in situ</i> hybridization against <i>cxcr4b</i> (A-D) or <i>brn3a</i> (E-H) showing the epithalamus of wild type (A,E), <i>pax6b</i>^<i>sa86</i> mutant embryos (B,F), <i>pax6a</i> morpholino injected embryos (C,G) or <i>pax6b</i>^<i>sa8</i>;<i>pax6a</i> morphant embryos (D,H) at 36 (A-D) or 48 (E-H) hpf. Whereas the expression of <i>cxcr4b</i> and <i>brn3a</i> appears unaffected in <i>pax6b</i>^<i>sa86</i> mutant embryos (B, n = 6/6 and F, n = 5/5) compared to the expression in wild type controls (A and E, n = 5/5), expression of both genes is abrogated after injection of morpholinos against <i>pax6a</i> in either wild type (C, n = 4/6 and G, n = 6/8) or <i>pax6b</i>^<i>sa8</i> embryos (D and H, n = 4/4). Embryos are viewed dorsally with anterior up.</p