Abnormal Nuclear Pore Formation Triggers Apoptosis in the Intestinal Epithelium of elys-Deficient Zebrafish

Abstract

Background & Aims Zebrafish mutants generated by ethylnitrosourea (ENU)-mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, “flotte lotte” (flo), displays striking defects in intestinal, liver, pancreas and eye formation at 78hpf. In this study we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype. Methods Positional cloning was employed to map the flo mutation. Sub-cellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, confocal and electron microscopy. Results The molecular lesion in flo is a nonsense mutation in the elys (embryonic large molecule derived from yolk sac) gene which encodes a severely truncated protein lacking the Elys C-terminal AT-hook DNA binding domain. Recently, ELYS has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Though elys mRNA is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally-expressed elys mRNA until 24hpf. From 72hpf, elys mRNA expression is restricted to proliferating tissues, including the intestinal epithelium, pancreas, liver and eye. Cells in these tissues display disrupted nuclear pore formation; ultimately intestinal epithelial cells undergo apoptosis. Conclusion Our results demonstrate that Elys regulates digestive organ formation.

The zebrafish flotte lotte mutant reveals that the local retinal environment promotes the differentiation of proliferating precursors emerging from their stem cell niche

“…1G,H). Consistent with the identity of Elys as a component of the Nup107-160 nuclear pore complex, which is also required for kinetochore function, nuclear pores are disrupted and their components aggregated in retinal cells (Davuluri et al, 2008;de Jong-Curtain et al, 2009) (see Fig. S1 in the supplementary material).…”

“…1A,B) (Wallace et al, 2005). Other than small eyes, mutants show no overt phenotypes until later stages, when defects in intestinal development lead to larval death (Davuluri et al, 2008;de Jong-Curtain et al, 2009;Wallace et al, 2005). In histological sections, the phenotype of flo mutants could first be identified at 2 dpf by the presence of occasional acellular holes in the retina (Fig.…”

“…Embryos were genotyped as described (de Jong-Curtain et al, 2009), except that the digestion products were resolved on a 2.5% Metasieve agarose (Flowgen) gel in Tris-borate-EDTA buffer (Sigma).…”

“…The flo locus encodes Elys (also known as Ahctf1) (Davuluri et al, 2008;de Jong-Curtain et al, 2009), a component of the Nup107-160 complex that localises to kinetochores during mitosis and participates in the reformation of functional nuclear pores immediately after mitosis (Rasala et al, 2006). Elys is required for cell cycle progression, playing key roles in kinetochore function (Mishra et al, 2010) and in the regulation of mitotic entrance and exit (Davuluri et al, 2008;Fernandez and Piano, 2006;Franz et al, 2007;Galy et al, 2006;Gillespie et al, 2007).…”

A chemical enterocolitis model in zebrafish larvae that is dependent on microbiota and responsive to pharmacological agents

“…Development of the eye, containing rapidly proliferative tissue, was used as an indicator of extra-intestinal effects of TNBS exposure on growth rate (de Jong-Curtain et al, 2009;Parichy et al, 2009). Measurement of larval eye diameter revealed no significant difference between TNBS-treated and control samples (97.4% 6 1.0% and 100% 6 1.2%, respectively, P ¼ 0.10).…”