The Zebrafish Anillin-eGFP Reporter Marks Late Dividing Retinal Precursors and Stem Cells Entering Neuronal Lineages.

Monitoring cycling behaviours of stem and somatic cells in the living animal is a powerful tool to better understand tissue development and homeostasis. The tg(anillin:anillin-eGFP) transgenic line carries the full-length zebrafish F-actin binding protein Anillin fused to eGFP from a bacterial artificial chromosome (BAC) containing Anillin cis-regulatory sequences. Here we report the suitability of the Anillin-eGFP reporter as a direct indicator of cycling cells in the late embryonic and post-embryonic retina. We show that combining the anillin:anillin-eGFP with other transgenes such as ptf1a:dsRed and atoh7:gap-RFP allows obtaining spatial and temporal resolution of the mitotic potentials of specific retinal cell populations. This is exemplified by the analysis of the origin of the previously reported apically and non-apically dividing late committed precursors of the photoreceptor and horizontal cell layers.

Temporal and spatial expression pattern of Nnat during mouse eye development.

BACKGROUND: Neuronatin (Nnat) was initially identified as a highly expressed gene in neonatal mammalian brain. In this study, we analyze the spatial and temporal expression pattern of Nnat during mouse eye development as well as in the adult. METHODS: The expression of Nnat was analyzed on mRNA as well as protein level. The presence of Nnat transcripts in the adult retina was examined using reverse transcription-polymerase chain reaction (RT-PCR). Nnat protein expression was evaluated by Western blot and immunohistochemistry during eye development at embryonic day (E) 12, 15, 16 and postnatal day (P) 7, 14, 30 and 175 (adult). RESULTS: Immunohistochemical studies of the developing mouse eye revealed Nnat expression in embryonic and adult neuroretina as well as in corneal epithelial, stromal, endothelial cells and in lens epithelium. Expression of Nnat was detected from E12 onwards and was also present in adult eyes. CONCLUSIONS: The expression pattern suggests that Nnat may play an important role during eye development and in the maintenance of mature eye.

Asymmetric inheritance of the apical domain and self-renewal of retinal ganglion cell progenitors depend on Anillin function.

Divisions that generate one neuronal lineage-committed and one self-renewing cell maintain the balance of proliferation and differentiation for the generation of neuronal diversity. The asymmetric inheritance of apical domains and components of the cell division machinery has been implicated in this process, and might involve interactions with cell fate determinants in regulatory feedback loops of an as yet unknown nature. Here, we report the dynamics of Anillin - an essential F-actin regulator and furrow component - and its contribution to progenitor cell divisions in the developing zebrafish retina. We find that asymmetrically dividing retinal ganglion cell progenitors position the Anillin-rich midbody at the apical domain of the differentiating daughter. anillin hypomorphic conditions disrupt asymmetric apical domain inheritance and affect daughter cell fate. Consequently, the retinal cell type composition is profoundly affected, such that the ganglion cell layer is dramatically expanded. This study provides the first in vivo evidence for the requirement of Anillin during asymmetric neurogenic divisions. It also provides insights into a reciprocal regulation between Anillin and the ganglion cell fate determinant Ath5, suggesting a mechanism whereby the balance of proliferation and differentiation is accomplished during progenitor cell divisions in vivo.

The myosin-interacting protein SMYD1 is essential for sarcomere organization.

Assembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity - mainly responsible for the nuclear function of SMYD1 - is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosin-binding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1-myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly.

Protein kinase D2 controls cardiac valve formation in zebrafish by regulating histone deacetylase 5 activity.

BACKGROUND: The molecular mechanisms that guide heart valve formation are not well understood. However, elucidation of the genetic basis of congenital heart disease is one of the prerequisites for the development of tissue-engineered heart valves. METHODS AND RESULTS: We isolated here a mutation in zebrafish, bungee (bng(jh177)), which selectively perturbs valve formation in the embryonic heart by abrogating endocardial Notch signaling in cardiac cushions. We found by positional cloning that the bng phenotype is caused by a missense mutation (Y849N) in zebrafish protein kinase D2 (pkd2). The bng mutation selectively impairs PKD2 kinase activity and hence Histone deacetylase 5 phosphorylation, nuclear export, and inactivation. As a result, the expression of Histone deacetylase 5 target genes Krüppel-like factor 2a and 4a, transcription factors known to be pivotal for heart valve formation and to act upstream of Notch signaling, is severely downregulated in bungee (bng) mutant embryos. Accordingly, the expression of Notch target genes, such as Hey1, Hey2, and HeyL, is severely decreased in bng mutant embryos. Remarkably, downregulation of Histone deacetylase 5 activity in homozygous bng mutant embryos can rescue the mutant phenotype and reconstitutes notch1b expression in atrioventricular endocardial cells. CONCLUSIONS: We demonstrate for the first time that proper heart valve formation critically depends on Protein kinase D2-Histone deacetylase 5-Krüppel-like factor signaling.

Reconstitution of defective protein trafficking rescues Long-QT syndrome in zebrafish.

Inherited cardiac arrhythmias are caused by genetic defects in ion channels and associated proteins. Mutations in these channels often do not affect their biophysical properties, but rather interfere with their trafficking to the cell membrane. Accordingly, strategies that could reroute the mutated channels to the membrane should be sufficient to restore the electrical properties of the affected cells, thereby suppressing the underlying arrhythmia. We identified here both, embryonic and adult zebrafish breakdance (bre) as a valuable model for human Long-QT syndrome. Electrocardiograms of adult homozygous bre mutants exhibit significant QT prolongation caused by delayed repolarization of the ventricle. We further show that the bre mutation (zERG(I59S)) disrupts ERG protein trafficking, thereby reducing the amount of active potassium channels on the cell membrane. Interestingly, improvement of channel trafficking by cisapride or dimethylsulfoxid is sufficient to reconstitute ERG channels on the cell membrane in a manner that suffices to suppress the Long-QT induced arrhythmia in breakdance mutant zebrafish. In summary, we show for the first time that therapeutic intervention can cure protein trafficking defects and the associated cardiac arrhythmia in vivo.