DNGR-1-tracing marks an ependymal cell subset with damage-responsive neural stem cell potential

Cells with latent stem ability can contribute to mammalian tissue regeneration after damage. Whether the central nervous system (CNS) harbors such cells remains controversial. Here, we report that DNGR-1 lineage tracing in mice identifies an ependymal cell subset, wherein resides latent regenerative potential. We demonstrate that DNGR-1-lineage-traced ependymal cells arise early in embryogenesis (E11.5) and subsequently spread across the lining of cerebrospinal fluid (CSF)-filled compartments to form a contiguous sheet from the brain to the end of the spinal cord. In the steady state, these DNGR-1-traced cells are quiescent, committed to their ependymal cell fate, and do not contribute to neuronal or glial lineages. However, trans-differentiation can be induced in adult mice by CNS injury or in vitro by culture with suitable factors. Our findings highlight previously unappreciated ependymal cell heterogeneity and identify across the entire CNS an ependymal cell subset wherein resides damage-responsive neural stem cell potential

Identification of Dmrt2a downstream genes during zebrafish early development using a timely controlled approach

This research was supported by FCT (Portugal) grant (PTDC/SAU-BID/119627/2010) given to L.S. L.S. was supported by an IF contract from FCT (Portugal). R.A.P. was supported by a PhD fellowship (SFRH/BD/87607/2012) from FCT (Portugal). Publication was sponsored by LISBOA-01-0145-FEDER-007391, project co-funded by FEDER through POR Lisboa 2020 - Programa Operacional Regional de Lisboa, PORTUGAL 2020 and by Fundacao para a Ciencia e a Tecnologia.BACKGROUND: Dmrt2a is a zinc finger like transcription factor with several roles during zebrafish early development: left-right asymmetry, synchronisation of the somite clock genes and fast muscle differentiation. Despite the described functions, Dmrt2a mechanism of action is unknown. Therefore, with this work, we propose to identify Dmrt2a downstream genes during zebrafish early development. RESULTS: We generated and validated a heat-shock inducible transgenic line, to timely control dmrt2a overexpression, and dmrt2a mutant lines. We characterised dmrt2a overexpression phenotype and verified that it was very similar to the one described after knockdown of this gene, with left-right asymmetry defects and desynchronisation of somite clock genes. Additionally, we identified a new phenotype of somite border malformation. We generated several dmrt2a mutant lines, but we only detected a weak to negligible phenotype. As dmrt2a has a paralog gene, dmrt2b, with similar functions and expression pattern, we evaluated the possibility of redundancy. We found that dmrt2b does not seem to compensate the lack of dmrt2a. Furthermore, we took advantage of one of our mutant lines to confirm dmrt2a morpholino specificity, which was previously shown to be a robust knockdown tool in two independent studies. Using the described genetic tools to perform and validate a microarray, we were able to identify six genes downstream of Dmrt2a: foxj1b, pxdc1b, cxcl12b, etv2, foxc1b and cyp1a. CONCLUSIONS: In this work, we generated and validated several genetic tools for dmrt2a and identified six genes downstream of this transcription factor. The identified genes will be crucial to the future understanding of Dmrt2a mechanism of action in zebrafish.publishersversionpublishe

Functional analysis of the zebrafish dorsal organizer

The Spemann's organizer is a region of the amphibian gastrula that will induce a second body axis when transplanted to ventral or lateral regions of a host embryo. The organizer can dorsalise mesoderm, induce convergent extension movements and specify neuroectoderm. Functional equivalents of the Spemann's organizer have been identified in other vertebrates by transplantation experiments. A region of the fish gastrula called the embryonic shield is thought to function as the dorsal organizer. Using a novel surgical method, I showed that the morphological shield can induce complete secondary axes when transplanted into the ventral germ ring of a host embryo. In induced secondary axes, the donor shield contributed to hatching gland, prechordal plate, notochord, floor plate and hypochord. When explanted shields were divided into deep and superficial fragments and separately transplanted, I found that deep tissue can induce ectopic axes with heads but lacking posterior tissues. I found that when only the morphological shield was removed, embryos recovered and were completely normal by 24 hours-post-fertilisation. Ablation of the morphological shield does not remove all goosecoid- and floating head-expressing cells, suggesting that the morphological shield does not comprise the entire organizer region. Removal of the morphological shield plus adjacent marginal tissue, however, led to loss of all shield derivatives, a cyclopean head, loss of floor plate and primary motomeurons and disrupted somite patterning. Embryos from which only the morphological shield was removed still had some goosecoid- and floating head-expressing cells. I have tested whether these residual shield cells were sufficient to form all shield derivatives or, alternatively, if adjacent non-shield tissues could be recruited to shield fate. After morphological shield removal, I found no increase in cell proliferation. Transplantation studies indicated, however, that non-shield tissue may be recruited to a shield fate. Finally, I have employed the shield removal and transplantation method to study two mutations: sneezy and silberblick/wnt11. Transplantation results indicate that sneezy acts autonomously within the shield derivatives. By contrast, silberblick/wnt11 acts non-autonomously in paraxial tissues to drive the convergent extension movement of axial mesoderm

Notch signalling is required for the formation of structurally stable muscle fibres in zebrafish

Accurate regulation of Notch signalling is central for developmental processes in a variety of tissues, but its function in pectoral fin development in zebrafish is still unknown.Here we show that core elements necessary for a functional Notch pathway are expressed in developing pectoral fins in or near prospective muscle territories. Blocking Notch signalling at different levels of the pathway consistently leads to the formation of thin, wavy, fragmented and mechanically weak muscles fibres and loss of stress fibres in endoskeletal disc cells in pectoral fins. Although the structural muscle genes encoding Desmin and Vinculin are normally transcribed in Notch-disrupted pectoral fins, their proteins levels are severely reduced, suggesting that weak mechanical forces produced by the muscle fibres are unable to stabilize/localize these proteins. Moreover, in Notch signalling disrupted pectoral fins there is a decrease in the number of Pax7-positive cells indicative of a defect in myogenesis.We propose that by controlling the differentiation of myogenic progenitor cells, Notch signalling might secure the formation of structurally stable muscle fibres in the zebrafish pectoral fin