The Cellular Architecture of the Larval Zebrafish Tectum, as Revealed by Gal4 Enhancer Trap Lines

We have carried out a Gal4 enhancer trap screen in zebrafish, and have generated 184 stable transgenic lines with interesting expression patterns throughout the nervous system. Of these, three display clear expression in the tectum, each with a distinguishable and stereotyped distribution of Gal4 expressing cells. Detailed morphological analysis of single cells, using a genetic “Golgi-like” labelling method, revealed four common cell types (superficial, periventricular, shallow periventricular, and radial glial), along with a range of other less common neurons. The shallow periventricular (PV) and a subset of the PV neurons are tectal efferent neurons that target various parts of the reticular formation. We find that it is specifically PV neurons with dendrites in the deep tectal neuropil that target the reticular formation. This indicates that these neurons receive the tectum's highly processed visual information (which is fed from the superficial retinorecipient layers), and relay it to premotor regions. Our results show that the larval tectum, both broadly and at the single cell level, strongly resembles a miniature version of its adult counterpart, and that it has all of the necessary anatomical characteristics to inform motor responses based on sensory input. We also demonstrate that mosaic expression of GFP in Gal4 enhancer trap lines can be used to describe the types and abundance of cells in an expression pattern, including the architectures of individual neurons. Such detailed anatomical descriptions will be an important part of future efforts to describe the functions of discrete tectal circuits in the generation of behavior

Anatomy and function of retinorecipient arborization fields in zebrafish

In 1994, Burrill and Easter described the retinal projections in embryonic and larval zebrafish, introducing the term “arborization fields” (AFs) for the retinorecipient areas. AFs were numbered from 1 to 10 according to their positions along the optic tract. With the exception of AF10 (neuropil of the optic tectum), annotations of AFs remained tentative. Here we offer an update on the likely identities and functions of zebrafish AFs after successfully matching classical neuroanatomy to the digital Max Planck Zebrafish Brain Atlas. In our system, individual AFs are neuropil areas associated with the following nuclei: AF1 with the suprachiasmatic nucleus; AF2 with the posterior parvocellular preoptic nucleus; AF3 and AF4 with the ventrolateral thalamic nucleus; AF4 with the anterior and intermediate thalamic nuclei; AF5 with the dorsal accessory optic nucleus; AF7 with the parvocellular superficial pretectal nucleus; AF8 with the central pretectal nucleus; and AF9d and AF9v with the dorsal and ventral periventricular pretectal nuclei. AF6 is probably part of the accessory optic system. Imaging, ablation, and activation experiments showed contributions of AF5 and potentially AF6 to optokinetic and optomotor reflexes, AF4 to phototaxis, and AF7 to prey detection. AF6, AF8 and AF9v respond to dimming, and AF4 and AF9d to brightening. While few annotations remain tentative, it is apparent that the larval zebrafish visual system is anatomically and functionally continuous with its adult successor and fits the general cyprinid pattern. This study illustrates the synergy created by merging classical neuroanatomy with a cellular-resolution digital brain atlas resource and functional imaging in larval zebrafish

Characterization of Genetically Targeted Neuron Types in the Zebrafish Optic Tectum

The optically transparent larval zebrafish is ideally suited for in vivo analyses of neural circuitry controlling visually guided behaviors. However, there is a lack of information regarding specific cell types in the major retinorecipient brain region of the fish, the optic tectum. Here we report the characterization of three previously unidentified tectal cell types that are specifically labeled by dlx5/6 enhancer elements. In vivo laser-scanning microscopy in conjunction with ex vivo array tomography revealed that these neurons differ in their morphologies, synaptic connectivity, and neurotransmitter phenotypes. The first type is an excitatory bistratified periventricular interneuron that forms a dendritic arbor in the retinorecipient stratum fibrosum et griseum superficiale (SFGS) and an axonal arbor in the stratum griseum centrale (SGC). The second type, a GABAergic non-stratified periventricular interneuron, extends a bushy arbor containing both dendrites and axons into the SGC and the deepest sublayers of the SFGS. The third type is a GABAergic periventricular projection neuron that extends a dendritic arbor into the SGC and a long axon to the torus semicircularis, medulla oblongata, and anterior hindbrain. Interestingly, the same axons form en passant synapses within the deepest neuropil layer of the tectum, the stratum album centrale. This approach revealed several novel aspects of tectal circuitry, including: (1) a glutamatergic mode of transmission from the superficial, retinorecipient neuropil layers to the deeper, output layers, (2) the presence of interneurons with mixed dendrite/axon arbors likely involved in local processing, and (3) a heretofore unknown GABAergic tectofugal projection to midbrain and hindbrain. These observations establish a framework for studying the morphological and functional differentiation of neural circuits in the zebrafish visual system

Hardwiring of fine synaptic layers in the zebrafish visual pathway

<p>Abstract</p> <p>Background</p> <p>Neuronal connections are often arranged in layers, which are divided into sublaminae harboring synapses with similar response properties. It is still debated how fine-grained synaptic layering is established during development. Here we investigated two stratified areas of the zebrafish visual pathway, the inner plexiform layer (IPL) of the retina and the neuropil of the optic tectum, and determined if activity is required for their organization.</p> <p>Results</p> <p>The IPL of 5-day-old zebrafish larvae is composed of at least nine sublaminae, comprising the connections between different types of amacrine, bipolar, and ganglion cells (ACs, BCs, GCs). These sublaminae were distinguished by their expression of cell type-specific transgenic fluorescent reporters and immunohistochemical markers, including protein kinase Cβ (PKC), parvalbumin (Parv), zrf3, and choline acetyltransferase (ChAT). In the tectum, four retinal input layers abut a laminated array of neurites of tectal cells, which differentially express PKC and Parv. We investigated whether these patterns were affected by experimental disruptions of retinal activity in developing fish. Neither elimination of light inputs by dark rearing, nor a D, L-amino-phosphono-butyrate-induced reduction in the retinal response to light onset (but not offset) altered IPL or tectal lamination. Moreover, thorough elimination of chemical synaptic transmission with <it>Botulinum </it>toxin B left laminar synaptic arrays intact.</p> <p>Conclusion</p> <p>Our results call into question a role for activity-dependent mechanisms – instructive light signals, balanced <it>on </it>and <it>off </it>BC activity, Hebbian plasticity, or a permissive role for synaptic transmission – in the synaptic stratification we examined. We propose that genetically encoded cues are sufficient to target groups of neurites to synaptic layers in this vertebrate visual system.</p

Focusing on optic tectum circuitry through the lens of genetics

The visual pathway is tasked with processing incoming signals from the retina and converting this information into adaptive behavior. Recent studies of the larval zebrafish tectum have begun to clarify how the 'micro-circuitry' of this highly organized midbrain structure filters visual input, which arrives in the superficial layers and directs motor output through efferent projections from its deep layers. The new emphasis has been on the specific function of neuronal cell types, which can now be reproducibly labeled, imaged and manipulated using genetic and optical techniques. Here, we discuss recent advances and emerging experimental approaches for studying tectal circuits as models for visual processing and sensorimotor transformation by the vertebrate brain

Neuroendocrine transcriptional programs adapt dynamically to the supply and demand for neuropeptides as revealed in NSF mutant zebrafish

<p>Abstract</p> <p>Background</p> <p>Regulated secretion of specialized neuropeptides in the vertebrate neuroendocrine system is critical for ensuring physiological homeostasis. Expression of these cell-specific peptide markers in the differentiating hypothalamus commences prior to birth, often predating the physiological demand for secreted neuropeptides. The conserved function and spatial expression of hypothalamic peptides in vertebrates prompted us to search for critical neuroendocrine genes in newly hatched zebrafish larvae.</p> <p>Results</p> <p>We screened mutant 5 days post-fertilization zebrafish larvae that fail to undergo visually mediated background adaptation for disruption in hypothalamic <it>pomc </it>expression. To our surprise, the ATPase <it>N-ethylmaleimide sensitive factor </it>(<it>nsf</it>) was identified as an essential gene for maintenance of neuroendocrine transcriptional programs during the embryo-to-larva transition. Despite normal hypothalamic development in <it>nsf</it>^{<it>st</it>53}mutants, neuropeptidergic cells exhibited a dramatic loss of cell-specific markers by 5 days post-fertilization that is accompanied by elevated intracellular neuropeptide protein. Consistent with the role of NSF in vesicle-membrane fusion events and intracellular trafficking, cytoplasmic endoplasmic reticulum-like membranes accumulate in <it>nsf</it>^-/-hypothalamic neurons similar to that observed for <it>SEC18 </it>(<it>nsf ortholog</it>) yeast mutants. Our data support a model in which unspent neuropeptide cargo feedbacks to extinguish transcription in neuropeptidergic cells just as they become functionally required. In support of this model we found that <it>gnrh3 </it>transcripts remained unchanged in pre-migratory, non-functional gonadotropin-releasing hormone (GnRH) neurons in <it>nsf</it>^-/-zebrafish. Furthermore, <it>oxytocin-like </it>(<it>oxtl</it>, <it>intp</it>) transcripts, which are found in osmoreceptive neurons and persist in mutant zebrafish, drop precipitously after mutant zebrafish are acutely challenged with high salt.</p> <p>Conclusion</p> <p>Our analyses of <it>nsf </it>mutant zebrafish reveal an unexpected role for NSF in hypothalamic development, with mutant 5 days post-fertilization larvae exhibiting a stage-dependent loss of neuroendocrine transcripts and a corresponding accumulation of neuropeptides in the soma. Based on our collective findings, we speculate that neuroendocrine transcriptional programs adapt dynamically to both the supply and demand for neuropeptides to ensure adequate homeostatic responses.</p

Non-SMC condensin I complex proteins control chromosome segregation and survival of proliferating cells in the zebrafish neural retina.

BACKGROUND: The condensation of chromosomes and correct sister chromatid segregation during cell division is an essential feature of all proliferative cells. Structural maintenance of chromosomes (SMC) and non-SMC proteins form the condensin I complex and regulate chromosome condensation and segregation during mitosis. However, due to the lack of appropriate mutants, the function of the condensin I complex during vertebrate development has not been described. RESULTS: Here, we report the positional cloning and detailed characterization of retinal phenotypes of a zebrafish mutation at the cap-g locus. High resolution live imaging reveals that the progression of mitosis between prometa- to telophase is delayed and that sister chromatid segregation is impaired upon loss of CAP-G. CAP-G associates with chromosomes between prometa- and telophase of the cell cycle. Loss of the interaction partners CAP-H and CAP-D2 causes cytoplasmic mislocalization of CAP-G throughout mitosis. DNA content analysis reveals increased genomic imbalances upon loss of non-SMC condensin I subunits. Within the retina, loss of condensin I function causes increased rates of apoptosis among cells within the proliferative ciliary marginal zone (CMZ) whereas postmitotic retinal cells are viable. Inhibition of p53-mediated apoptosis partially rescues cell numbers in cap-g mutant retinae and allows normal layering of retinal cell types without alleviating their aberrant nuclear sizes. CONCLUSION: Our findings indicate that the condensin I complex is particularly important within rapidly amplifying progenitor cell populations to ensure faithful chromosome segregation. In contrast, differentiation of postmitotic retinal cells is not impaired upon polyploidization.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Non-SMC condensin I complex proteins control chromosome segregation and survival of proliferating cells in the zebrafish neural retina

<p>Abstract</p> <p>Background</p> <p>The condensation of chromosomes and correct sister chromatid segregation during cell division is an essential feature of all proliferative cells. Structural maintenance of chromosomes (SMC) and non-SMC proteins form the condensin I complex and regulate chromosome condensation and segregation during mitosis. However, due to the lack of appropriate mutants, the function of the condensin I complex during vertebrate development has not been described.</p> <p>Results</p> <p>Here, we report the positional cloning and detailed characterization of retinal phenotypes of a zebrafish mutation at the <it>cap-g </it>locus. High resolution live imaging reveals that the progression of mitosis between prometa- to telophase is delayed and that sister chromatid segregation is impaired upon loss of CAP-G. CAP-G associates with chromosomes between prometa- and telophase of the cell cycle. Loss of the interaction partners CAP-H and CAP-D2 causes cytoplasmic mislocalization of CAP-G throughout mitosis. DNA content analysis reveals increased genomic imbalances upon loss of non-SMC condensin I subunits. Within the retina, loss of condensin I function causes increased rates of apoptosis among cells within the proliferative ciliary marginal zone (CMZ) whereas postmitotic retinal cells are viable. Inhibition of p53-mediated apoptosis partially rescues cell numbers in <it>cap-g </it>mutant retinae and allows normal layering of retinal cell types without alleviating their aberrant nuclear sizes.</p> <p>Conclusion</p> <p>Our findings indicate that the condensin I complex is particularly important within rapidly amplifying progenitor cell populations to ensure faithful chromosome segregation. In contrast, differentiation of postmitotic retinal cells is not impaired upon polyploidization.</p