Egfr-signaling in Oryzias latipes - Characterization of inducible oncogene gain-of-function and receptor loss-of-function Medaka lines

One of the major growth-promoting factors in animal development and growth is the epidermal growth factor receptor (Egfr). The Egfr pathway is involved in proliferation, cell survival, migration and differentiation and is one of the most commonly deregulated pathways in cancer. Medaka fish have two copies of the egfr gene, egfr a and b, which have so far not been analyzed for their particular function and redundancy. To understand the role of Egfr signaling in the neural stem cell zones of the retina and the brain, we performed a whole mount in situ hybridization screen for egfrs, selected ligands as well as selected downstream signal transducers. We found that both egfrs are broadly and overlapping expressed during development and more specifically in proliferative zones of the brain and the ciliary marginal zone in hatchlings. To determine the effect of enhanced growth signaling on the retinal stem and progenitor cells we created a versatile toolbox for targeted induction of Egfr-related oncogenes (Xmrk, myrAKT, and K-Ras12V) in cell types of interest. To this end, we tagged the Egfr variant Xmrk with a fluorescent protein and demonstrated its oncogenicity in a somitic overproliferation assay. A Cre-loxP based induction of oncogenes in single cells in somites and also in the retina was successfully achieved in transient and stable lines, and multiple lines were generated. The analysis of oncogenic effects on RSCs was however hampered by the loss of visible expression in later generations in the retina and brain. To analyze the specific role of Egfra in development and growth I generated a CRISPR/Cas9 based knockout (KO) disrupting the open reading frame by GFP-plasmid insertion or deletion and analyzed the resulting phenotype. Egfra-KO fish develop and reproduce normally but have a defect in oogenesis. Oocytes derived from homozygous Egfra-KO females contain multiple sperm entry sites (micropyles), which causes polyspermy and therefore lethality. The characterization of the polyspermy phenotype, as well as the oogenesis defect in adult ovaries, points towards a role of Egfra in the communication between the granulosa cell layer and the oocyte to specify the micropyle. By generation of genetic loss and gain of function tools for Egfra and related oncogenes, this work will contribute to understanding the general and the more specific role of Egfr signaling in growth and development of the Medaka fish

Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina.

The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic origin in the medaka retina. To analyze whether NSC potency is affected by developmental progression, as reported for the mammalian brain, we developed an inducible toolkit for clonal labeling and non-invasive fate tracking. We used this toolkit to address post-embryonic stem cells in different tissues and to functionally differentiate transient progenitor cells from permanent, bona fide stem cells in the retina. Using temporally controlled clonal induction, we showed that post-embryonic retinal NSCs are exclusively multipotent and give rise to the complete spectrum of cell types in the neural retina. Intriguingly, and in contrast to any other vertebrate stem cell system described so far, long-term analysis of clones indicates a preferential mode of asymmetric cell division. Moreover, following the behavior of clones before and after external stimuli, such as injuries, shows that NSCs in the retina maintained the preference for asymmetric cell division during regenerative responses. We present a comprehensive analysis of individual post-embryonic NSCs in their physiological environment and establish the teleost retina as an ideal model for studying adult stem cell biology at single cell resolution.This is the final version of the article. It has been published by The Company of Biologists Ltd in Development here: http://dev.biologists.org/content/early/2014/08/19/dev.109892.long

Revisiting the origin of interleukin 1 in anamniotes and sub-functionalization of interleukin 1 in amniotes

The cytokine interleukin 1 (IL-1) is an evolutionary innovation of vertebrates. Fish and amphibian have one IL1 gene, while mammals have two copies of IL1, IL1A and IL1B, with distinct expression patterns and differences in their proteolytic activation. Our current understanding of the evolutionary history of IL-1 is mainly based on phylogenetic analysis, but this approach provides no information on potentially different functions of IL-1 homologues, and it remains unclear which biological activities identified for IL-1α and IL-1β in mammals are present in lower vertebrates. Here, we use in vitro and in vivo experimental models to examine the expression patterns and cleavage of IL-1 proteins from various species. We found that IL-1 in the teleost medaka shares the transcriptional patterns of mammalian IL-1α, and its processing also resembles that of mammalian IL-1α, which is sensitive to cysteine protease inhibitors specific for the calpain and cathepsin families. By contrast, IL-1 proteins in reptiles also include biological properties of IL-1β. Therefore, we propose that the duplication of the ancestral IL1 gene led to the segregation of expression patterns and protein processing that characterizes the two extant forms of IL-1 in mammals