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A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs (1) and recruits general transcription factors to initiate transcription (2). The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle (3-6). Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition (7). This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression (8), and determined the positions of H3K4me3-marked promoter-associated nucleosomes (9). Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (11) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription-independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable their expression in the two regulatory environments. The dissection of overlapping core promoter determinants represents a framework for future studies of promoter structure and function across different regulatory contexts., Mapping of TSSs using cap analysis of gene expression (CAGE) (8) identified two major promoter classes with respect to TSS precision (10,11): 'sharp' promoters with one predominant TSS, often associated [...]

The chaperones Unc45b and Hsp90a are essential for folding of myosin in organisms ranging from worms to humans. We show here that zebrafish Unc45b, but not Hsp90a, binds to the putative cytidine deaminase Apobec2 (Apo2) in an interaction that requires the Unc45/Cro1 p/She4p-related (UCS) and central domains of Unc45b. Morpholino oligonucleotide-mediated knockdown of the two related proteins Apo2a and Apo2b causes a dystrophic phenotype in the zebrafish skeletal musculature and impairs heart function. These phenotypic traits are shared with mutants of unc45b, but not with hsp90a mutants. Apo2a and -2b act nonredundantly and bind to each other in vitro, which suggests a heteromeric functional complex. Our results demonstrate that Unc45b and Apo2 proteins act in a Hsp90a-independent pathway that is required for integrity of the myosepta and myofiber attachment. Because the only known function of Unc45b is that of a chaperone, Apo2 proteins may be clients of Unc45b but other yet unidentified processes cannot be excluded. doi/ 10.1083/jcb.200912125

The formation of thick filaments in striated muscle involves the chaperones Hsp90a and Unc45. We show that Unc45b and Hsp90a, two zebrafish orthologues, colocalize with myosin during myofibrillo-genesis and associate with the Z line when myofibril assembly is completed. In response to stress or damage to the myofiber, Unc45b and Hsp90a dissociate from the Z line and transiently associate with myosin. Although chaperone activity of Unc45b requires the full-length pro tein, only the central and Unc45-Cro1p-She4p domains are required to anchor it to the Z line, and multiple subdomains mediate association with nascent myosin. We propose that the Z line serves as a reservoir for chaperones, allowing a rapid mobilization in response to muscle damage. Our data are consistent with a differential affinity model as an explanation for the shuttling of the chaperones between the Z line and myosin.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.11.034 Byline: Saradavey Vinothkumar, Sepand Rastegar, Masanari Takamiya, Raymond Ertzer, Uwe Strahle Keywords: Retina; Zebrafish; Enhancer; Shh; Gene regulation Abstract: The signaling molecule Sonic hedgehog (Shh) is required for differentiation of the vertebrate retina. In the developing zebrafish retina, shh expression is initiated at the ventronasal region, from where it spreads as a wave through the retina. To investigate the molecular mechanism underlying this coordinated expression of shh, we mapped the cis-regulatory region and identified a novel regulatory sequence in the first intron of the shh locus. This sequence contains binding sites for the transcription factors Erm and Pea3 that are known transducers of Fgf signaling. Mutation of the binding sites or knockdown of Pea3 and Erm abolishes transgene expression, indicating that Fgf signaling regulates shh expression in the retina. We provide evidence that Fgf3 and -8 control initiation of expression, while Fgf19 is crucial for the propagation of transgene expression through the retina. Inhibitor experiments indicate a continued requirement of FGF and Hedgehog (Hh) signaling for transgene expression after initiation at the ventronasal aspect of the retina. We propose a model, in which Fgf3 and -8 initiate expression and Fgf19 and Shh signals cooperate subsequently to promote establishment of expression throughout the retina. Author Affiliation: Institute for Toxicology and Genetics, Forschungszentrum Karlsruhe, Postfach 3640, 76021 Karlsruhe, Germany Article History: Received 7 September 2007; Revised 19 November 2007; Accepted 27 November 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.05.014 Byline: Christelle Etard (a)(b), Martine Behra (a)(d), Nadine Fischer (a), David Hutcheson (b), Robert Geisler (c), Uwe Strahle (a)(b) Keywords: Zebrafish; Chaperone; Co-chaperone; Muscle damage; hsp90a; UCS factor Abstract: Contraction of muscles is mediated by highly organized arrays of myosin motor proteins. We report here the characterization of a mutation of a UCS gene named steif/unc-45b that is required for the formation of ordered myofibrils in both the skeletal and cardiac muscles of zebrafish. We show that Steif/Unc-45b interacts with the chaperone Hsp90a in vitro. The two genes are co-expressed in the skeletal musculature and knockdown of Hsp90a leads to impaired myofibril formation in the same manner as lack of Steif/Unc-45b activity. Transcripts of both genes are up-regulated in steif mutants suggesting co-regulation of the two genes. Our data indicate a requirement of Steif/unc-45b and Hsp90a for the assembly of the contractile apparatus in the vertebrate skeletal musculature. Author Affiliation: (a) Institut de Genetique et de Biologie Moleculaire et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch Cedex, CU de Strasbourg, France (b) Institute for Toxicology and Genetics, Forschungszentrum Karlsruhe, Postfach 3640, 76021 Karlsruhe, Germany (c) Max-Planck-Institut fur Entwicklungsbiologie, Spemannstr 35, D-72076 Tubingen, Germany (d) NIH/NHGRI Building 50, Bethesda, Maryland, USA Article History: Received 2 March 2007; Revised 9 May 2007; Accepted 15 May 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.11.004 Byline: Raymond Ertzer (a)(b), Ferenc Muller (a)(b), Yavor Hadzhiev (b), Saradavey Rathnam (b), Nadine Fischer (a), Sepand Rastegar (a)(b), Uwe Strahle (a)(b) Keywords: Embryonic shield; Notochord; Floor plate; Zona limitans intrathalamica; Hypothalamus; Zebrafish; Sonic hedgehog; Transgene; Enhancer Abstract: In zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution. Author Affiliation: (a) Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France (b) Institute of Toxicology and Genetics, Research Center Karlsruhe, PoB 3640, 76021 Karlsruhe, Germany Article History: Received 24 April 2006; Revised 30 October 2006; Accepted 4 November 2006

A study on the behavior of neural stem cells in the brain of zebrafish is discussed. Immunodetection of markers is done in the study.

Many studies led to the hypothesis that acetylcholinesterase (AChE) and the closely related enzyme butyrylcholinesterase may play other non-hydrolytic roles during development. Comparison of data from in vivo studies performed on invertebrate and vertebrate genetic models is discussed.

The floor plate is an organising centre that controls neural differentiation and axonogenesis in the neural tube. The axon guidance molecule Netrin1 is expressed in the floor plate of zebrafish embryos. To elucidate the regulatory mechanisms underlying expression in the floor plate, we scanned the netrin1 locus for regulatory regions and identified an enhancer that drives expression in the floor plate and hypochord of transgenic embryos. The expression of the transgene is ectopically activated by Cyclops (Nodal) signals but does not respond to Hedgehog signals. The winged-helix transcription factor foxA2 (also HNF3[beta], axial) is expressed in the notochord and floor plate. We show that knock-down of FoxA2 leads to loss of floor plate, while notochord and hypochord development is unaffected, suggesting a specific requirement of FoxA2 in the floor plate. The transgene is ectopically activated by FoxA2, and expression of FoxA2 leads to rescue of floor plate differentiation in mutant embryos that are deficient in Cyclops signalling. Zebrafish and mouse use different signalling systems to specify floor plate. The zebrafish netrin1 regulatory region also drives expression in the floor plate of mouse and chicken embryos. This suggests that components of the regulatory circuits controlling expression in the floor plate are conserved and that FoxA2--given its importance for midline development also in the mouse--may be one such component. Key Words: cyclops; Nodal; floor plate; hypochord; netrin1; foxA2 (HNF3[beta], axial).

A vital part of the endoderm formation pathway, CASANOVA, has been found through study of zebrafish to be encoded by a novel sox-related gene, cas. An HMG box-containing gene, 10J3, which is expressed in the endoderm, was isolated in a subtractive screen for Nodal-responsive genes for Tar-responsive genes.

Glucocorticoid receptors (GRs) are found in a variety of cell types and its levels vary considerably depending on the tissue. To explain the differential expression of GRs, the mouse GR gene was isolated and characterized. The results showed that the mouse GR contains 11 exons of which the first three can be alternatively spliced to a common second exon. The organization of the translated genes was similar to the chicken progesterone, human estrogen and human androgen receptor genes. The gene could be transcribed from three different promoters whose presence and activities were not as conserved as the intron-exon structure of the gene.

Previous reports have linked Neurogenin 1 with the general promotion of neurogenesis and gliogenesis. A new study in zebrafish, supported by recent findings in Drosophila, has now challenged this idea by demonstrating that the activity of Neurogenin 1 is limited to activation of development of two classes of sensory neurons.

Polydactyly is one of the most frequent inherited defects of the limbs characterized by supernumerary digits and high‐genetic heterogeneity. Among the many genes involved, either in isolated or syndromic forms, eight have been implicated in postaxial polydactyly (PAP). Among those, IQCE has been recently identified in a single consanguineous family. Using whole‐exome sequencing in patients with uncharacterized ciliopathies, including PAP, we identified three families with biallelic pathogenic variations in IQCE. Interestingly, the c.895_904del (p.Val301Serfs*8) was found in all families without sharing a common haplotype, suggesting a recurrent mechanism. Moreover, in two families, the systemic phenotype could be explained by additional pathogenic variants in known genes (TULP1, ATP6V1B1). RNA expression analysis on patients' fibroblasts confirms that the dysfunction of IQCE leads to the dysregulation of genes associated with the hedgehog‐signaling pathway, and zebrafish experiments demonstrate a full spectrum of phenotypes linked to defective cilia: Body curvature, kidney cysts, left–right asymmetry, misdirected cilia in the pronephric duct, and retinal defects. In conclusion, we identified three additional families confirming IQCE as a nonsyndromic PAP gene. Our data emphasize the importance of taking into account the complete set of variations of each individual, as each clinical presentation could finally be explained by multiple genes. [ABSTRACT FROM AUTHOR]

Cyclopia is a class of human birth-defect syndromes known as holoprosencephaly. The biochemical defects underlying the malformation are not well known. Feldman and colleagues, Sampath and colleages and Rebagliati and colleagues have analyzed cyclopic zebra fish mutants, which gives an insight into the processes that establish the ventral regions of the brain and spinal cord.

The zebrafish (Danio rerio) is a well-established vertebrate model organism. Its embryos are used extensively in biology and medicine to perform chemical screens to identify drug candidates or to evaluate teratogenicity and embryotoxicity of substances. Behavioral readouts are increasingly used to assess the effects of compounds on the nervous system. Early stage zebrafish show characteristic behavioral features at stages between 30 and 42 hours post fertilization (hpf) when exposed to a short and bright light flash. This so-called Photomotor Response (PMR) is a reaction of the nervous system of the fish and can be used as a marker in screenings for neuroactive chemicals. To probe a broad and diverse chemical space, many different substances have to be tested and repeated observations are necessary to warrant statistical significance of the results. Although PMR-based chemical screens must use a large number of specimens, there is no sophisticated, automated high-throughput platform available which ensures minimal human intervention. Here we report a PMR platform that was developed by combining an improved automatic sample handling with a remotely controllable microscope setup and an image analysis pipeline. Using infrared illumination during automatic sample preparation, we were able to eliminate excess amounts of visible light that could potentially alter the response results. A remotely controlled microscope setup allows us to screen entire 96-well microtiter plates without human presence that could disturb the embryos. The development of custom video analysis software, including single egg detection, enables us to detect variance among treated specimens and extract easy to interpret numerical values representing the PMR motion. By testing several neuroactive compounds we validated the workflow that can be used to analyze more than one thousand zebrafish eggs on a single 96-well plate.