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. 2014 Feb;32(2):558-71.
doi: 10.1002/stem.1583.

Zebrafish Stem/Progenitor Factor msi2b Exhibits Two Phases of Activity Mediated by Different Splice Variants

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Free PMC article

Zebrafish Stem/Progenitor Factor msi2b Exhibits Two Phases of Activity Mediated by Different Splice Variants

Tatiana Hochgreb-Hägele et al. Stem Cells. .
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Abstract

The Musashi (Msi) family of RNA-binding proteins is important in stem and differentiating cells in many species. Here, we present a zebrafish gene/protein trap line gt(msi2b-citrine)(ct) (57) (a) that expresses a Citrine fusion protein with endogenous Msi2b. Our results reveal two phases of Msi2b expression: ubiquitous expression in progenitor cells in the early embryo and later, tissue-specific expression in differentiating cells in the olfactory organ, pineal gland, and subpopulations of neurons in the central nervous system (CNS). Interestingly, this division between early and late phases is paralleled by differential expression of msi2b alternative splicing products. Whereas the full-length and long variant v3 Msi2b predominate at early stages, the later expression of variants in differentiating tissues appears to be tissue specific. Using the gt(msi2b-citrine)(ct) (57) (a), we characterized tissue-specific expression of Msi2b with cellular resolution in subsets of differentiating cells in the olfactory organ, pineal gland, CNS, and ventral neural tube. By performing transcription activator-like effectors nuclease-mediated biallelic genome editing or morpholino knockdown of Msi2b in zebrafish, our results show that early inactivation of Msi2b results in severe embryonic defects including hypertrophy of the ventricles and shortening of the body, consistent with an important role in cell proliferation and survival. Moreover, specific inactivation of Msi2b full-length indicates that this species is essential for the early role of Msi2b. This line provides a valuable tool both for live imaging of the endogenous Msi2b at subcellular resolution and manipulation of Msi2b-expressing cells.

Keywords: Musashi; differentiating; neuronal progenitor; zebrafish.

Figures

Figure 1
Figure 1. A novel gene trap line depicts expression of Msi2b in zebrafish
(A) Schematic diagram of the FlipTrap strategy. The FlipTrap construct contains an artificial exon consisting of citrine ORF sequence lacking start and stop codons (green), flanked by rassf8 splice acceptor (SA) and donor (SD) sites. Genomic integration of the construct is mediated by Tol2 transposase. In the gt(msi2b-citrine)ct57a line, the FlipTrap construct has inserted in the chromosome 15, within the intron between exons 6 and 7 of msi2b. The endogenous splicing machinery recognizes the splice acceptor (SA) and splice donor (SD) sites flanking the citrine sequence (dashed lines), resulting in Citrine-fusion with endogenous protein. (B) Schematic diagram of zebrafish msi2b splice variants. Members of the Msi family of RNA-binding proteins are characterized by the presence of two RNA-binding domains (gray boxes) that include two short highly conserved motifs called RNP-1 and RNP-2 (asterisks). Alternative splicing of msi2b produces 3 splice variants (v1, v2, v3). (C) Detection of Msi2b protein fused with Citrine-tag reveals Msi2b the presence and relative abundance of variants at the protein level. Expression of Msi2b variants by western blot in gt(msi2b-citrine)ct57a embryos at 56 hpf. Alpha-Tubulin is used as a load control.
Figure 2
Figure 2. Comparison among members of the Msi family of RNA-binding proteins
(A) Phylogenetic tree of Musashi proteins in vertebrates was generated by ClustalX analysis. Human, hMSI1 and hMSI2; mouse, mMSI1 and mMSI2; Xenopus, xMsi1 and xMsi2 and Zebrafish, zMsi1, zMsi2a, and zMsi2b. Zebrafish and human RNA-binding protein, zElav3 and hELAV3, were included in the analysis as outgroups. In zebrafish, Msi2 has diverged further into zMsi2a and zMsi2b. The numbers on the nodes of the tree represent the bootstrap values from Neighbor joining analysis. (B) Alignment of zMsi2b protein sequence with mouse and human MSI2. The RNA-binding domains (green boxes) are highly conserved between zMsi2b and mammalian MSI2, with only two amino acids substitutions in the first domain. Sequence divergences consist of an additional sequence of amino acids between positions 263 and 343 of zMsi2b sequence. Full conservation of residues are indicated by asterisk (*); “:” and “.” indicate full conservation of ‘strong’ and ‘weaker’ residues, respectively.
Figure 3
Figure 3. msi2b splice variants are differentially expressed in zebrafish
(A) Schematic diagram of msi2b splice variants. Target regions for anti-sense riboprobes (orange brackets) and qPCR (blue) were designed to specifically detect the distinct msi2b splice variants. (B) Quantitative analysis of msi2b variants by qPCR. cDNA template from zebrafish embryos at 24 hpf (n=10, light blue) and 56 hpf (n=10, dark blue). Values are expressed in 2-dCt±SD, calculated as a ratio to beta-actin expression. (C-E”) Expression of msi2b splice variants by whole mount in situ hybridization. Lateral views. Arrows point to areas of msi2b expression in the ventricles (v) and olfactory region (o). Low level, ubiquitous expression of msi2b full-length (v1) and the splice variant v3 at the 1-2 somite-stage (ss) (C,C”). At this stage, expression of v2 is not detected (C’). The full-length msi2b (v1) is expressed at low levels in the head, with tissue-specific expression that corresponds to the expression of Msi2b-Citrine in the epiphysis (ep, *), olfactory placode and CNS at 24 hpf (D), and appearing in the ventral neural tube at 30 hpf (E). msi2b v2 (D’) and v3 (D”) are expressed in the otic vesicle (ov) and lining of the brain ventricles at 24 hpf. At 30 hpf, v2 expression is downregulated in the otic vesicle and localizes to the brain ventricle and CNS (E’), while v3 expression is enhanced in the otic vesicles, ventricles and forebrain (E”). Scale bars: 1 μm. (F-F”) Subcellular localization of msi2b transcripts by fluorescent in situ hybridization. Transcripts of msi2b variants (red) are localized in the cytoplasm of expressing cells in the neural tube (full arrowheads, E,F) and ventricles (empty arrowheads, E’,F’, and E”,F”), rather than the nuclei counterstained by DAPI (blue). Scale bars: 10 μm.
Figure 4
Figure 4. Msi2b is expressed in distinct neuronal populations in zebrafish
Confocal imaging of live gt(msi2b-citrine)ct57a embryos depicts cytoplasmic expression of Msi2b in zebrafish embryos (green) at 30 hpf (A-E’), 52 hpf (F-I’) and 5 dpf (J-L). Counterstaining by BODIPYTR (red). Olfactory region, o; pallium, p; epiphysis, ep; otic vesicle, ov; neural tube, nt; notochord, n; somite, s; telencephalon, t; forebrain, fb; dorsal telencephalon, dt; midbrain-hindbrain boundary, mhb; otoliths, ot; anterior, median and posterior cristae are respectively ac, mc and pc. Scale bars: 10 μm. (A, B) Frontal view. Single plane imaging (A) and respective confocal projection (B) of Msi2b-Citrine expression in the olfactory placode and pallium, with lower levels of expression in the forebrain. (C-E’) Lateral views. (C) Msi2b is highly expressed in the olfactory placode. (D) Expression of Msi2b in three distinct subpopulations of neurons in the ventral neural tube highly expressing cells within (white arrowhead) and dorsally (asterisks) to the floor plate, and low expression in the periphery of the neural tube (dashed white arrow). (E, E’) Low levels of Mis2b are detected in the somites. (F, G-I’) Lateral views. (F’,G”) Coronal sections. (F) Msi2b is expressed in the epiphysis, olfactory organs, otic vesicles and CNS (white arrow). (F’, F”) Frontal views, optical sections. Expression in the dorsal thalamus and epiphysis as well as in the forebrain and olfactory organs. (G) Msi2b is expressed in the ventral cells of the hindbrain. (H) Expression in the otic vesicle is localized to cells of the otoliths and in the central cells of the cristae of the semicircular canals. (I, I’) Expression of Msi2b in the ventral cells of the neural tube (white arrowhead). Cells dorsal to the floor plate (asterisks) and low expression in the periphery of the neural tube (dashed white arrow). (J) Dorsal view of zebrafish head at 5dpf. Msi2b is expressed in the habenula (K) and is restricted to a subset of neurons in the epiphysis (L).
Figure 5
Figure 5. Second phase of Msi2b expression is localized in the olfactory organ and pineal gland
Confocal images of co-expression of neuronal markers (red) with Msi2b-citrine (green) in gt(msi2bcitrine)ct57a line. Scale bars: 10 μm. (A-C) Msi2b is expressed in the HuC/D-positive neurons (red) in the olfactory bulb at 36 hpf. (D-F) GABA-positive neurons (red) in the olfactory bulb at 36 hpf. Lateral view. (G-I) Low levels of Pax6 expression in (red) in the olfactory bulb at 54 hpf. (J-L) Expression of HuC/D (red) in a subset of Msi2b positive neuron in the pineal gland at 36 hpf. (M-O) GABA (red) is co-expressed with high levels of Msi2b in the pineal gland at 36 hpf. (P-R) Strong expression of Pax6 (red) in neurons of the pineal gland at 54 hpf.
Figure 6
Figure 6. Msi2b expression in the ventral neural tube encompasses distinct cell types in zebrafish
(A-N) Cross sections of gt(msi2b-citrine)ct57a embryos depict distribution of Msi2b-positive cells in the neural tube (nt, dotted line). Arrowheads point to areas of co-expression of Msi2b-Citrine (green) with the respective neuronal marker (red). Somites, s; olfactory bulb, o. Scale bars: 10 μm. (A-C) Citrine is highly expressed by a subset of ventral cells closely associated with the midline, while differentiated neurons reside in the dorsal-lateral region of the neural tube, as determined by expression of HuC/D. (D-F) Citrine-positive cells correspond to a subset of olig2-positive cells, as determined by co-expression with dsRed in the Tg(olig2:dsred) transgenic line. (G-I) Crosses with Tg(ngn1:RFP) indicates that Msi2b-positive cells in the olfactory organs correspond to a population of neuronal progenitors. (J-L) Immunodetection reveals that ventral-most Msi2-positive cells express GABA (red). (M,N) Cross-section of neural tube of gt(msi2b-citrine)ct57a embryos treated with EdU (red) at 24 hpf and observed at 32 hpf. Incorporation of EdU (red) is observed in the cells adjacent to Msi2b-positive (green) cells of the dorsal neural tube in the trunk level and in the cells adjacent to the midline in the tail. (O,O’) Lateral views of the neural tube of gt(msi2b-citrine)ct57a embryos injected with Cherry-H2B mRNA at the 1-cell stage. Scale bars: 10 μm. (O) Expression of Cherry is ubiquitously expressed in the neural tube of embryos at 52 hpf. (O’) At 72 hpf, Cherry-H2B labeling is retained by few cells in the neural tube, including some Msi2b-positive cells (white arrows).
Figure 7
Figure 7. Msi2b loss-of-function by morpholino knockdown and biallelic TALEN-induced somatic mutation
(A) Schematic diagram of the binding sites for Msi2b translation blocking (ATG) and splice-blocking (i7/E8) morpholinos (MO) as well as the binding sites for E2/i2 TALEN. E2/i2 TALEN was designed to target the splice donor site of Msi2b exon 2. Binding of msi2b i7/E8-MO results in alternative splicing events (i, ii) by disrupting splicing between exons 7 and 8 of Msi2b. Numbered boxes represent exons. Asterisks depict RNP octomer recognition sequences (RNPs). (B) Schematic diagram of genome editing by E2/i2 TALEN in zebrafish. TALEN E2 (blue arrow) and TALEN i2 (green arrow) respectively bind to regions of exon 2 (E2) and intron 2 (i2) flanking the splice donor (SD) of msi2 E2. Boxed sequences within E2 (blue) and i2 (green) represent the sites of DNA cleavage by FokI nuclease in the TALEN E2/i2 mutant embryos (n=16 clones). The gradient color represents the frequency of DNA cleavage within the region for the mutant embryos analyzed. Nucleotide deletions (squares) and replacements (letters) induced by E2/i2 TALENs are represented under the target sequence. PCR amplification from genomic DNA, cloning and sequencing of multiple clones was obtained for each embryo. (B’) Schematic diagram of alternative splice events produced by i7/E8 splice-blocking morpholino. Sequences of msi2b exon 7 (E7, green), exon 8 (E8, yellow), exon 9 (E9, orange) and intron 7 (i7, gray) and corresponding translation sequence. Alternative splicing between E7 and E9 (i) or persistence of intron 7 (ii) results in a premature stop signal and truncated of Msi2b protein similar to v2. (C-E”) Inactivation of all Msi2b variants by translation-blocking morphants or E2/i2 TALEN as well as specific truncation of Msi2b full-length (v1) by i7/E8 morpholino in zebrafish is characterized by ventricular edema (red dashed lines), shortening of the body length and atrophic or absent eyes. Scale bars: 500 μm. (F-G’) Knockdown of msi2b in zebrafish indicates that Msi2b plays an early role in embryonic development. Similar early embryonic phenotype, with truncated body length and tissue degeneration, was observed after specific knockdown of full-length by co-injection of i7/E8 MO and p53 MO (F) and mutagenesis of msi2b by E2/i2 TALEN (G), as compared to embryos co-injected with control MO and p53 MO (F’) or a single TALEN E2 L1 as a control (G’). Scale bars: 100 μm. (H-J) Confocal 3D projection of the olfactory organ in Tg(omp-RFP)/Tg(trpc2-Venus) zebrafish embryos at 52 hpf. Knockdown of msi2b expression by ATG+p53 MO (H) or i7E8+p53 MO (I), as compared to control embryos (J). Scale bars: 10 μm. (K-M) Quantitative analysis of the distribution of resulting phenotypes after inactivation of msi2b in zebrafish. Asterisks indicate statistical significance, *P<0.001.

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