The present study shows the expression profile and function of the homeobox gene, satb2 during zebrafish embryonic development. Satb2 was ubiquitously expressed from the 1 cell stage to the 10-somite stage in zebrafish embryos. Satb2 showed stage-specific expression profiles such as in the pronephric duct at 24 hpf, the branchial arches at 36 hpf, and the ganglion cell layer of the retina and fins at 48 hpf. Additionally, satb2 knockdown embryos were arrested at 50–60% epiboly, and transplantation experiments with satb2 knockdown cells showed migration defects. Interestingly, satb2 knockdown cells also exhibited down-regulation of dynamin II and VAMP4, which are involved in exocytosis and endocytosis, respectively. Furthermore, satb2 knockdown cells have a disorganized actin distribution and an underdeveloped external yolk syncytial layer, both of which are involved in epiboly. These results suggest that satb2 has a functional role in epiboly. This role may potentially be the regulation of endo-exocytic vesicle transport-dependent cell migration and/or the regulation of the development of the yolk syncytial layer.

Previous studies have shown that Notch signaling not only regulates the number of early differentiating neurons, but also maintains proliferating neural precursors in the neural tube. Although it is well known that Notch signaling is closely related to the differentiation of adult neural stem cells, none of transgenic zebrafish provides a tool to figure out the relationship between Notch signaling and the differentiation of neural precursors. The goal of this study was to characterize Her4-positive cells by comparing the expression of a fluorescent Her4 reporter in Tg[her4-dRFP] animals with a GFAP reporter in Tg[gfap-GFP] adult zebrafish. BrdU incorporation indicated that dRFP-positive cells were proliferating and a double labeling assay revealed that a significant fraction of the Her4-dRFP positive population was also GFAP-GFP positive. Our observations suggest that a reporter line with Notch-dependent gene expression can provide a tool to examine proliferating neural precursors and/or neuronal/glial precursors in the development of the adult nervous system to examine the model in which Notch signaling maintains proliferating neural precursors in the neural tube.

In the central nervous system (CNS), giving rise to the diversity and the complexity of neurons is the spatial and temporal differentiation of neural stem cells and/or neural precursors. Here, we investigated the role of Jagged-mediated Notch signaling in the maintenance and differentiation of progenitor cells during late neurogenesis by analyzing the expression patterns of zebrafish jagged homologues, and by injecting their morpholinos. Expression of both jagged2 and jagged1b mRNA in the CNS suggested that they might be involved in control of differentiating neural progenitors in which they are involved later in development. In Jagged2 and Jagged1b knock-down embryos, the overall rate of cell division dramatically decreased, and the ectopic VeMe neurons were generated. The results suggest that Jagged-Notch signaling plays a critical role in the maintenance of proliferating neural precursors, and that the generation of late-born neurons, especially VeMe neurons, is regulated by the interplay between Jagged2 and Jagged1b.

Mind bomb (Mib) is an E3 ubiquitin ligase that activates the Notch signaling pathway. A previous study demonstrated that the generation of late-born GABAergic neurons may be regulated by the interplay between Mib and retinoic acid (RA). However, the relationship between Mib function and the retinoid pathway during the generation of late-born motor neurons remains unclear. We investigated the differentiation of neural progenitors into motor neurons by inhibition of Notch signaling and administration of RA to Tg[hsp70-Mib:EGFP] embryos. The number of motor neurons in the ventral spinal cord increased or decreased depending on the temporal inhibition of Mib-mediated Notch signaling. Inhibition of the retinoid pathway by citral treatment had a synergistic effect with overexpression of Mib:EGFP on the generation of ectopic motor neurons. Additionally, the proteolytic fragment of Mib was detected in differentiated P19 cells following treatment with RA. Our observations imply that the function of Mib may be attenuated by the retinoid pathway, and that Mib-mediated Notch signaling and the retinoid pathway play critical roles in the spatiotemporal differentiation of motor neurons.

Lateral inhibition is known to be mediated by Delta-associated Notch signaling pathway (Kunisch et al., 1994). Within equivalent cells in proneural domain, signal-sending cells expressed Delta induce Notch receptor in neighboring cells, which become neuroblasts or sensory organ precursors (Bray, 1998). It is well known that Hairy Enhancer-of-split, a transcriptional repressor, is expressed by the transcriptional complex consist of notch intracellular domain (NICD), CBF1/Su(H)/Lag1 (CSL) and Mastermind (Mam) in neighboring cells (Bray, 1998). In neighboring cells of neural ectoderm, Ngn1 as a proneural gene is repressed by a Hairy Enhancer-of-split-related protein (Kim et al., 1997; Takke et al., 1999). In contrast to neighboring cells, Mind bomb (Mib) promotes ubiquitination and endocytosis of Delta with extracellular domain of Notch (NECD) in signal-sending cells (Itoh et al., 2003). Jagged have been identified as a ligand of Notch receptors (Lindsell et al., 1995). Zebrafish Jagged2 expressed in the domain located progenitor cells of motor neuron (pMN domain) activate Notch and maintain a population of proliferating progenitors in the p3 domain (Yeo & Chitnis, 2007). Jagged-mediated Notch signaling regulate the temporal generation of late-born neurons, such as secondary motor neurons (sMN) and GABAergic KA” neurons in the ventral spinal cord (Yeo & Chitnis, 2007). The gain-of-function study using Tg[hsp70-Mib:EGFP] showed that the majority of cells in the p3 domain differentiated into KA” neurons after heat-shock during late neurogenesis (Ryu et al., 2015). These observations imply that Jagged may also be a substrate for Mib. Our preliminary data showed that Jagged2 was ubiquitinated by Mib in P19 cells, but not cytosolic form of intracellular domain of zebrafish Jagged2 (J2ICD) (unpublished data). This observation implicate that an adaptor might be required for ubiquitinating membrane-bounded Jagged ICD by cytosolic Mib. By performing yeast-two hybrid screen using zebrafish Jagged2 ICD as a bait, zebrafish PEX1 was isolated and identified a potential adaptor between Mib and Jagged2 ICD.