Christian Stigloher

MicroRNA-9 directs late organizer activity of the midbrain-hindbrain boundary

The midbrain-hindbrain boundary (MHB) is a long-lasting organizing center in the vertebrate neural tube that is both necessary and sufficient for the ordered development of midbrain and anterior hindbrain (midbrain-hindbrain domain, MH). The MHB also coincides with a pool of progenitor cells that contributes neurons to the entire MH. Here we show that the organizing activity and progenitor state of the MHB are co-regulated by a single microRNA, miR-9, during late embryonic development in zebrafish. Endogenous miR-9 expression, initiated at late stages, selectively spares the MHB. Gain- and loss-of-function studies, in silico predictions and sensor assays in vivo demonstrate that miR-9 targets several components of the Fgf signaling pathway, thereby delimiting the organizing activity of the MHB. In addition, miR-9 promotes progression of neurogenesis in the MH, defining the MHB progenitor pool. Together, these findings highlight a previously unknown mechanism by which a single microRNA fine-tunes late MHB coherence via its co-regulation of patterning activities and neurogenesis.

Axonal projections originating from raphe serotonergic neurons in the developing and adult zebrafish, Danio rerio, using transgenics to visualize raphe-specific pet1 expression

Serotonin is a major central nervous modulator of physiology and behavior and plays fundamental roles during development and plasticity of the vertebrate central nervous system (CNS). Understanding the developmental control and functions of serotonergic neurons is therefore an important task. In all vertebrates, prominent serotonergic neurons are found in the superior and inferior raphe nuclei in the hindbrain innervating most CNS regions. In addition, all vertebrates except for mammals harbor other serotonergic centers, including several populations in the diencephalon. This, in combination with the intricate and wide distribution of serotonergic fibers, makes it difficult to sort out serotonergic innervation originating from the raphe from that of other serotonergic cell populations. To resolve this issue, we isolated the regulatory elements of the zebrafish raphe‐specific gene pet1 and used them to drive expression of an eGFP transgene in the raphe population of serotonergic neurons. With this approach together with retrograde tracing we 1) describe in detail the development, anatomical organization, and projection pattern of zebrafish pet1‐positive neurons compared with their mammalian counterparts, 2) identify a new serotonergic population in the ventrolateral zebrafish hindbrain, and 3) reveal some extent of functional subdivisions within the zebrafish superior raphe complex. Together, our results reveal for the first time the specific innervation pattern of the zebrafish raphe and, thus, provide a new model and various tools to investigate further the role of raphe serotonergic neurons in vertebrates. J. Comp. Neurol. 512:158–182, 2009.

Segregation of telencephalic and eye-field identities inside the zebrafish forebrain territory is controlled by Rx3

Anteroposterior patterning of the vertebrate forebrain during gastrulation involves graded Wnt signaling, which segregates anterior fields (telencephalon and eye) from the diencephalon. How the telencephalic and retinal primordia are subsequently subdivided remains largely unknown. We demonstrate that at late gastrulation the Paired-like homeodomain transcription factor Rx3 biases cell specification choices towards the retinal fate within a population of bipotential precursors of the anterior forebrain: direct cell tracing demonstrates that retinal precursors acquire a telencephalic fate in embryos homozygous for the rx3-null allele ckhne2611, characterized by an enlarged telencephalon and a lack of eyes. Chimera analyses further indicate that this function of Rx3 is cell autonomous. Transfating of the eye field in the absence of Rx3 function correlates with a substantial posterior expansion of expression of the Wnt antagonist Tlc and the winged-helix transcription factor Foxg1. These results suggest that the process segregating the telencephalic and eye fields is isolated from diencephalic patterning, and is mediated by Rx3.

The serotonergic phenotype is acquired by converging genetic mechanisms within the zebrafish central nervous system

To gain knowledge about the developmental origin of serotonergic precursors and the regulatory cascades of serotonergic differentiation in vertebrates, we determined the spatiotemporal expression profile of the Ets‐domain transcription factor‐encoding gene pet1 in developing and adult zebrafish. We show that it is an early, specific marker of raphe serotonergic neurons, but not of other serotonergic populations. We then use pet1 expression together with tracing techniques to demonstrate that serotonergic neurons of rhombomeres (r) 1–2 largely originate from a progenitor pool at the midbrain–hindbrain boundary. Furthermore, by combining expression analyses of pet1 and the raphe tryptophan hydroxylase (Tph2) with rhombomere identity markers, we show that anterior and posterior hindbrain clusters of serotonergic precursors are separated by r3, rather than r4 as in other vertebrates. Our findings establish the origin of r1–2 serotonergic precursors, and strengthen the evidence for molecular, ontogenic and phylogenic heterogeneities among the vertebrate brain serotonergic cell populations. Developmental Dynamics 236:1072–1084, 2007.

The Presynaptic Dense Projection of the Caenorhabiditis elegans Cholinergic Neuromuscular Junction Localizes Synaptic Vesicles at the Active Zone through SYD-2/Liprin and UNC-10/RIM-Dependent Interactions

The active zone (AZ) of chemical synapses is a specialized area of the presynaptic bouton in which vesicles fuse with the plasma membrane and release neurotransmitters. Efficient signaling requires synaptic vesicles (SVs) to be recruited, primed, and retained at the AZ, in close proximity to voltage-dependent calcium channels that are activated during presynaptic depolarization. The electron-dense specializations at the AZ might provide a molecular platform for the spatial coordination of these different processes. To investigate this hypothesis, we examined high-resolution three-dimensional models of Caenorhabditis elegans cholinergic neuromuscular junctions generated by electron tomography. First, we found that SVs are interconnected within the bouton by filaments similar to those described in vertebrates. Second, we resolved the three-dimensional structure of the dense projection centered in the AZ. The dense projection is a more complex structure than previously anticipated, with filaments radiating from a core structure that directly contact SVs in the interior of the bouton as well as SVs docked at the plasma membrane. Third, we investigated the functional correlate of these contacts by analyzing mutants disrupting two key AZ proteins: UNC-10/RIM and SYD-2/liprin. In both mutants, the number of contacts between SVs and the dense projection was significantly reduced. Similar to unc-10 mutants, the dependence of SV fusion on extracellular calcium concentration was exacerbated in syd-2 mutants when compared with the wild type. Hence, we propose that the dense projection ensures proper coupling of primed vesicles with calcium signaling by retaining them at the AZ via UNC-10/RIM and SYD-2/liprin-dependent mechanisms.

Expression of hairy/enhancer of split genes in neural progenitors and neurogenesis domains of the adult zebrafish brain.

All subdivisions of the adult zebrafish brain maintain niches of constitutive neurogenesis, sustained by quiescent and multipotent progenitor populations. In the telencephalon, the latter potential neural stem cells take the shape of radial glia aligned along the ventricle and are controlled by Notch signalling. With the aim of identifying new markers of this cell type and of comparing the effectors of embryonic and adult neurogenesis, we focused on the family of hairy/enhancer of split [E(spl)] genes. We report the expression of seven hairy/E(spl) (her) genes and the new helt gene in three neurogenic areas of the adult zebrafish brain (telencephalon, hypothalamus, and midbrain) in relation to radial glia, proliferation, and neurogenesis. We show that the expression of most her genes in the adult brain characterizes quiescent radial glia, whereas only few are expressed in progenitor domains engaged in active proliferation or neurogenesis. The low proliferation status of most her‐positive progenitors contrasts with the embryonic nervous system, in which her genes are expressed in actively dividing progenitors. Likewise, we demonstrate largely overlapping expression domains of a set of her genes in the adult brain, which is in striking contrast to their distinct embryonic expression profiles. Overall, our data provide a consolidated map of her expression, quiescent glia, proliferation, and neurogenesis in these various subdivisions of the adult brain and suggest distinct regulation and function of Her factors in the embryonic and adult contexts. J. Comp. Neurol. 519:1748–1769, 2011.

Fgf signaling in the zebrafish adult brain: association of Fgf activity with ventricular zones but not cell proliferation.

The zebrafish adult brain contains numerous neural progenitors and is a good model to approach the general mechanisms of adult neural stem cell maintenance and neurogenesis. Here we use this model to test for a correlation between Fgf signaling and cell proliferation in adult progenitor zones. We report expression of Fgf signals (fgf3,4,8a,8b,17b), receptors (fgfr1–4), and targets (erm, pea3, dusp6, spry1,2,4, and P‐ERK) and document that genes of the embryonic fgf8 synexpression group acquire strikingly divergent patterns in the adult brain. We further document the specific expression of fgf3, fgfr1–3, dusp6, and P‐ERK in ventricular zones, which contain neural progenitors. In these locations, however, a comparison at the single‐cell level of fgfr/P‐ERK expression with bromo‐deoxy‐uridine (BrdU) incorporation and the proliferation marker MCM5 indicates that Fgf signaling is not specifically associated with proliferating progenitors. Rather, it correlates with the ventricular radial glia state, some of which only are progenitors. Together these results stress the importance of Fgf signaling in the adult brain and establish the basis to study its function in zebrafish, in particular in relation to adult neurogenesis. J. Comp. Neurol. 510:422–439, 2008.

Identification of neural progenitor pools by E(Spl) factors in the embryonic and adult brain.

The maintenance of progenitor cells is a crucial aspect of central nervous system development and maturation, and bHLH transcription factors of the E(Spl) subfamily are involved in this process in all vertebrates studied to date. In the zebrafish embryonic neural plate, a large number of E(Spl) genes (her genes) are at play. We review recent data on this point, and propose a model where distinct subsets of these genes define different progenitor subtypes. Analysis of her genes expression in the adult zebrafish brain suggests that part of the embryonic her cascade might also be reused to define progenitors during adulthood. Further, available evidence on orthologous genes in the mouse (Hes genes) point to different modes of Hes regulation depending on cell location within the embryonic neural tube, perhaps associated with distinct progenitor types in this species as well. Out of these comparisons emerges a simple model of neural stem cell maintenance applicable from embryonic development until adulthood as well as across species. This working model suggests the directions for future experiments.

Pleiotropic effects in Eya3 knockout mice

BackgroundIn Drosophila, mutations in the gene eyes absent (eya) lead to severe defects in eye development. The functions of its mammalian orthologs Eya1-4 are only partially understood and no mouse model exists for Eya3. Therefore, we characterized the phenotype of a new Eya3 knockout mouse mutant.ResultsExpression analysis of Eya3 by in-situ hybridizations and β-Gal-staining of Eya3 mutant mice revealed abundant expression of the gene throughout development, e.g. in brain, eyes, heart, somites and limbs suggesting pleiotropic effects of the mutated gene. A similar complex expression pattern was observed also in zebrafish embryos.The phenotype of young adult Eya3 mouse mutants was systematically analyzed within the German Mouse Clinic. There was no obvious defect in the eyes, ears and kidneys of Eya3 mutant mice. Homozygous mutants displayed decreased bone mineral content and shorter body length. In the lung, the tidal volume at rest was decreased, and electrocardiography showed increased JT- and PQ intervals as well as decreased QRS amplitude. Behavioral analysis of the mutants demonstrated a mild increase in exploratory behavior, but decreased locomotor activity and reduced muscle strength. Analysis of differential gene expression revealed 110 regulated genes in heart and brain. Using real-time PCR, we confirmed Nup155 being down regulated in both organs.ConclusionThe loss of Eya3 in the mouse has no apparent effect on eye development. The wide-spread expression of Eya3 in mouse and zebrafish embryos is in contrast to the restricted expression pattern in Xenopus embryos. The loss of Eya3 in mice leads to a broad spectrum of minor physiological changes. Among them, the mutant mice move less than the wild-type mice and, together with the effects on respiratory, muscle and heart function, the mutation might lead to more severe effects when the mice become older. Therefore, future investigations of Eya3 function should focus on aging mice.

Positive modulation of a Cys-loop acetylcholine receptor by an auxiliary transmembrane subunit

Auxiliary subunits regulate the trafficking, localization or gating kinetics of voltage- and ligand-gated ion channels by associating tightly and specifically with pore-forming subunits. However, no auxiliary subunits have been identified for members of the Cys-loop receptor superfamily. Here we identify MOLO-1, a positive regulator of levamisole-sensitive acetylcholine receptors (L-AChRs) at the Caenorhabditis elegans neuromuscular junction. MOLO-1 is a one-pass transmembrane protein that contains a single extracellular globular domain—the TPM domain, found in bacteria, plants and invertebrates, including nonvertebrate chordates. Loss of MOLO-1 impairs locomotion and renders worms resistant to the anthelmintic drug levamisole. In molo-1 mutants, L-AChR-dependent synaptic transmission is reduced by half, while the number and localization of receptors at synapses remain unchanged. In a heterologous expression system, MOLO-1 physically interacts with L-AChRs and directly enhances channel gating without affecting unitary conductance. The identification of MOLO-1 expands the mechanisms for generating functional and pharmacological diversity in the Cys-loop superfamily.