DEAD-Box Protein Ddx46 Is Required for the Development of the Digestive Organs and Brain in Zebrafish

Abstract

Spatially and temporally controlled gene expression, including transcription, several mRNA processing steps, and the export of mature mRNA to the cytoplasm, is essential for developmental processes. It is well known that RNA helicases of the DExD/H-box protein family are involved in these gene expression processes, including transcription, pre-mRNA splicing, and rRNA biogenesis. Although one DExD/H-box protein, Prp5, a homologue of vertebrate Ddx46, has been shown to play important roles in pre-mRNA splicing in yeast, the in vivo function of Ddx46 remains to be fully elucidated in metazoans. In this study, we isolated zebrafish morendo (mor), a mutant that shows developmental defects in the digestive organs and brain, and found that it encodes Ddx46. The Ddx46 transcript is maternally supplied, and as development proceeds in zebrafish larvae, its ubiquitous expression gradually becomes restricted to those organs. The results of whole-mount in situ hybridization showed that the expression of various molecular markers in these organs is considerably reduced in the Ddx46 mutant. Furthermore, splicing status analysis with RT-PCR revealed unspliced forms of mRNAs in the digestive organ and brain tissues of the Ddx46 mutant, suggesting that Ddx46 may be required for pre-mRNA splicing during zebrafish development. Therefore, our results suggest a model in which zebrafish Ddx46 is required for the development of the digestive organs and brain, possibly through the control of pre-mRNA splicing.

Ddx46 Is Required for Multi-Lineage Differentiation of Hematopoietic Stem Cells in Zebrafish

“…Our previous study has shown that Ddx46, a member of the DEAD-box RNA helicase family, is required for the development of digestive organs and the brain, possibly by regulating pre-mRNA splicing [20]. Here, we show zebrafish Ddx46 expression in HSCs during development.…”

“…We have previously shown that Ddx46 hi2137/hi2137 mutants have defects in the formation of digestive organs and the brain [20]. In the course of the phenotypic analyses of Ddx46 hi2137/ hi2137 mutants, we also found that the expression of hematopoietic markers was downregulated in the mutants.…”

“…The stained embryos/larvae were embedded in 0.5% low melting temperature agarose in 1/3 Ringer's solution and imaged on an Olympus FV1000-D confocal microscope. Following the wholemount in situ hybridization, TUNEL staining, or immunohistochemical staining, Ddx46 hi2137/hi2137 mutants were confirmed by genotyping as described previously [20].…”

“…Possible explanations for the reduction of tal1-, runx1-, or cmyb-expressing HSCs in Ddx46 hi2137/hi2137 larvae are the upregulation of cell death or the downregulation of cell proliferation. We first examined cell death of HSCs in the AGM and CHT from 36 hpf to 4 dpf by TUNEL analysis and acridine orange staining because massive apoptosis was observed in digestive organs and the brain of Ddx46 hi2137/ hi2137 larvae at 3 dpf, and these larvae cannot survive beyond 5 dpf [20]. From 24 hpf to 4 dpf, increased cell death was not detected in the AGM or CHT of the Ddx46 hi2137/hi2137 larvae ( Fig.…”

“…We have previously reported that pre-mRNA splicing of specific genes is defective in Ddx46 hi2137/hi2137 mutants [20]; hence, we examined the pre-mRNA splicing of gata1a and and Ddx46 hi2137/hi2137 larvae at 48 hpf, 3 dpf, and 4 dpf. Ddx46 hi2137/ + larvae: n = 7/7 (48 hpf), n = 5/5 (3 dpf), n = 5/5 (4 dpf); Ddx46 hi2137/ hi2137 larvae: n = 5/5 (48 hpf), n = 6/6 (3 dpf), n = 8/8 (4 dpf).…”