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Identification of a novel intronic enhancer responsible for the transcriptional regulation of musashi1 in neural stem/progenitor cells.

Kawase S, Imai T, Miyauchi-Hara C, Yaguchi K, Nishimoto Y, Fukami S, Matsuzaki Y, Miyawaki A, Itohara S, Okano H - Mol Brain (2011)

Bottom Line: When neuronal differentiation was induced in embryoid body (EB)-derived neurosphere cells, reporter signals were detected in Msi1-positive NSCs and GFAP-positive astrocytes, but not in MAP2-positive neurons.A regulatory element for Msi1 transcription in NS/PCs is located in the sixth intron of the Msi1 gene.The 595-bp D5E2 intronic enhancer can transactivate Msi1 gene expression with cell-type specificity markedly similar to the endogenous Msi1 expression patterns.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, Japan.

ABSTRACT

Background: The specific genetic regulation of neural primordial cell determination is of great interest in stem cell biology. The Musashi1 (Msi1) protein, which belongs to an evolutionarily conserved family of RNA-binding proteins, is a marker for neural stem/progenitor cells (NS/PCs) in the embryonic and post-natal central nervous system (CNS). Msi1 regulates the translation of its downstream targets, including m-Numb and p21 mRNAs. In vitro experiments using knockout mice have shown that Msi1 and its isoform Musashi2 (Msi2) keep NS/PCs in an undifferentiated and proliferative state. Msi1 is expressed not only in NS/PCs, but also in other somatic stem cells and in tumours. Based on previous findings, Msi1 is likely to be a key regulator for maintaining the characteristics of self-renewing stem cells. However, the mechanisms regulating Msi1 expression are not yet clear.

Results: To identify the DNA region affecting Msi1 transcription, we inserted the fusion gene ffLuc, comprised of the fluorescent Venus protein and firefly Luciferase, at the translation initiation site of the mouse Msi1 gene locus contained in a 184-kb bacterial artificial chromosome (BAC). Fluorescence and Luciferase activity, reflecting the Msi1 transcriptional activity, were observed in a stable BAC-carrying embryonic stem cell line when it was induced toward neural lineage differentiation by retinoic acid treatment. When neuronal differentiation was induced in embryoid body (EB)-derived neurosphere cells, reporter signals were detected in Msi1-positive NSCs and GFAP-positive astrocytes, but not in MAP2-positive neurons. By introducing deletions into the BAC reporter gene and conducting further reporter experiments using a minimized enhancer region, we identified a region, "D5E2," that is responsible for Msi1 transcription in NS/PCs.

Conclusions: A regulatory element for Msi1 transcription in NS/PCs is located in the sixth intron of the Msi1 gene. The 595-bp D5E2 intronic enhancer can transactivate Msi1 gene expression with cell-type specificity markedly similar to the endogenous Msi1 expression patterns.

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Msi1-ffLuc expression corresponds with Msi1-positive NS/PCs and astrocytes in Msi1-ffLuc ESC-derived neural cells. (A) The experimental protocol for embryonic stem cell (ESC) differentiation with a retinoic-acid (RA) conditioned medium. ESCs formed embryoid bodies (EB) after 6 days in floating culture. (B) The expression of Msi1-ffLuc on day 0 of ESCs and day 6 of EBs is shown in the left panels. GFP was not expressed in ESCs and RA-untreated EBs(-RA) but was expressed in RA-treated EB(+RA). Nine Msi1-ffLuc ES cell lines were analysed for GFP fluorescence in each condition indicated (right panel). Scale bar: 100 μm. FLU: firefly luciferase light unit. (C) Dissociated day-6 EBs (+RA) derived from Msi1-ffLuc ES cell line (Red line in left panel) were assessed by flow cytometry and divided into H, M, and L fractions according to their GFP intensity. Each fraction was then analysed by qRT-PCR. The GFP, Msi1, and Nestin mRNA transcripts were enriched in the H fraction (L = 1 in each fraction). Black line in left panel shows day-6 EBs (+RA) derived from wild type ES cell line. (D), (E) secondary neurospheres were dissociated, cultured in differentiation medium for 4 days, and immunostained. Anti-GFP reactivity for GFP(+) cells correlated with Nestin(+) and endogenous Msi1(+) cells, but not with MAP2(+) mature neurons with long processes (D, Scale bar: 50 um). Vertical arrowheads show Marker(+)/GFP(+) cells. The immunofluorescent intensity of each marker was represented by scatter blots (E). The vertical axis shows the intensity of Msi1 and of cell-type-specific markers for neurons (β-III Tubulin), astrocytes (GFAP) and NS/PCs (Nestin). The horizontal axis shows the endogenous Msi1 (upper panel) and GFP (lower panel) intensities. The proportion (%) of marker(+) cells among GFP(+) cells is indicated in the bottom of each box. Note that the GFP/Marker double-positive cells were highly observed for Msi1, GFAP, and Nestin, but the β-III Tubulin-positive neurons had weak GFP expression.
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Figure 2: Msi1-ffLuc expression corresponds with Msi1-positive NS/PCs and astrocytes in Msi1-ffLuc ESC-derived neural cells. (A) The experimental protocol for embryonic stem cell (ESC) differentiation with a retinoic-acid (RA) conditioned medium. ESCs formed embryoid bodies (EB) after 6 days in floating culture. (B) The expression of Msi1-ffLuc on day 0 of ESCs and day 6 of EBs is shown in the left panels. GFP was not expressed in ESCs and RA-untreated EBs(-RA) but was expressed in RA-treated EB(+RA). Nine Msi1-ffLuc ES cell lines were analysed for GFP fluorescence in each condition indicated (right panel). Scale bar: 100 μm. FLU: firefly luciferase light unit. (C) Dissociated day-6 EBs (+RA) derived from Msi1-ffLuc ES cell line (Red line in left panel) were assessed by flow cytometry and divided into H, M, and L fractions according to their GFP intensity. Each fraction was then analysed by qRT-PCR. The GFP, Msi1, and Nestin mRNA transcripts were enriched in the H fraction (L = 1 in each fraction). Black line in left panel shows day-6 EBs (+RA) derived from wild type ES cell line. (D), (E) secondary neurospheres were dissociated, cultured in differentiation medium for 4 days, and immunostained. Anti-GFP reactivity for GFP(+) cells correlated with Nestin(+) and endogenous Msi1(+) cells, but not with MAP2(+) mature neurons with long processes (D, Scale bar: 50 um). Vertical arrowheads show Marker(+)/GFP(+) cells. The immunofluorescent intensity of each marker was represented by scatter blots (E). The vertical axis shows the intensity of Msi1 and of cell-type-specific markers for neurons (β-III Tubulin), astrocytes (GFAP) and NS/PCs (Nestin). The horizontal axis shows the endogenous Msi1 (upper panel) and GFP (lower panel) intensities. The proportion (%) of marker(+) cells among GFP(+) cells is indicated in the bottom of each box. Note that the GFP/Marker double-positive cells were highly observed for Msi1, GFAP, and Nestin, but the β-III Tubulin-positive neurons had weak GFP expression.

Mentions: We next examined how well the BAC reporter signal corresponds to endogenous Msi1 expression during the neural differentiation of ESCs. For this purpose, we generated ESC lines with stably-integrated Msi1 BAC DNA. We introduced Msi1 BAC DNA into parent (line EB3 tg14) ESCs. Forty-seven stable lines were established and characterized by their neural differentiation, using the scheme shown in Figure 2A. ESCs can be induced into neural lineages, including NSCs, by treatment with retinoic acid (RA) at a low concentration (10-8M). In embryoid body (EB)-forming cells, NS/PCs expressing Msi1 and/or Nestin can be efficiently induced by treatment with 10-8M RA [49]. We cultured recombinant ESCs in floating conditions to form EBs, added 10-8M RA to the culture medium after 2 days to induce neural differentiation, and cultured the cells for 4 more days (Figure 2A). Recombinant ES clones bearing Msi1 BAC DNA frequently showed GFP fluorescence on EB day 6 with RA (+RA), while GFP was not expressed in ESCs and RA-untreated EBs(-RA) (Figure 2B left panel). Firefly Luciferase activity, reflecting the Msi1 transcription activity, was measured in EBs treated with RA and without RA. The average luminescent activity per total protein increased about 10-fold in the treated EBs (+RA) over that found in undifferentiated recombinant ESCs (Figure 2B right panel). Luciferase activity in treated EBs (+RA) was about 5-fold greater than in untreated EBs (-RA) (Figure 2B right panel).


Identification of a novel intronic enhancer responsible for the transcriptional regulation of musashi1 in neural stem/progenitor cells.

Kawase S, Imai T, Miyauchi-Hara C, Yaguchi K, Nishimoto Y, Fukami S, Matsuzaki Y, Miyawaki A, Itohara S, Okano H - Mol Brain (2011)

Msi1-ffLuc expression corresponds with Msi1-positive NS/PCs and astrocytes in Msi1-ffLuc ESC-derived neural cells. (A) The experimental protocol for embryonic stem cell (ESC) differentiation with a retinoic-acid (RA) conditioned medium. ESCs formed embryoid bodies (EB) after 6 days in floating culture. (B) The expression of Msi1-ffLuc on day 0 of ESCs and day 6 of EBs is shown in the left panels. GFP was not expressed in ESCs and RA-untreated EBs(-RA) but was expressed in RA-treated EB(+RA). Nine Msi1-ffLuc ES cell lines were analysed for GFP fluorescence in each condition indicated (right panel). Scale bar: 100 μm. FLU: firefly luciferase light unit. (C) Dissociated day-6 EBs (+RA) derived from Msi1-ffLuc ES cell line (Red line in left panel) were assessed by flow cytometry and divided into H, M, and L fractions according to their GFP intensity. Each fraction was then analysed by qRT-PCR. The GFP, Msi1, and Nestin mRNA transcripts were enriched in the H fraction (L = 1 in each fraction). Black line in left panel shows day-6 EBs (+RA) derived from wild type ES cell line. (D), (E) secondary neurospheres were dissociated, cultured in differentiation medium for 4 days, and immunostained. Anti-GFP reactivity for GFP(+) cells correlated with Nestin(+) and endogenous Msi1(+) cells, but not with MAP2(+) mature neurons with long processes (D, Scale bar: 50 um). Vertical arrowheads show Marker(+)/GFP(+) cells. The immunofluorescent intensity of each marker was represented by scatter blots (E). The vertical axis shows the intensity of Msi1 and of cell-type-specific markers for neurons (β-III Tubulin), astrocytes (GFAP) and NS/PCs (Nestin). The horizontal axis shows the endogenous Msi1 (upper panel) and GFP (lower panel) intensities. The proportion (%) of marker(+) cells among GFP(+) cells is indicated in the bottom of each box. Note that the GFP/Marker double-positive cells were highly observed for Msi1, GFAP, and Nestin, but the β-III Tubulin-positive neurons had weak GFP expression.
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Figure 2: Msi1-ffLuc expression corresponds with Msi1-positive NS/PCs and astrocytes in Msi1-ffLuc ESC-derived neural cells. (A) The experimental protocol for embryonic stem cell (ESC) differentiation with a retinoic-acid (RA) conditioned medium. ESCs formed embryoid bodies (EB) after 6 days in floating culture. (B) The expression of Msi1-ffLuc on day 0 of ESCs and day 6 of EBs is shown in the left panels. GFP was not expressed in ESCs and RA-untreated EBs(-RA) but was expressed in RA-treated EB(+RA). Nine Msi1-ffLuc ES cell lines were analysed for GFP fluorescence in each condition indicated (right panel). Scale bar: 100 μm. FLU: firefly luciferase light unit. (C) Dissociated day-6 EBs (+RA) derived from Msi1-ffLuc ES cell line (Red line in left panel) were assessed by flow cytometry and divided into H, M, and L fractions according to their GFP intensity. Each fraction was then analysed by qRT-PCR. The GFP, Msi1, and Nestin mRNA transcripts were enriched in the H fraction (L = 1 in each fraction). Black line in left panel shows day-6 EBs (+RA) derived from wild type ES cell line. (D), (E) secondary neurospheres were dissociated, cultured in differentiation medium for 4 days, and immunostained. Anti-GFP reactivity for GFP(+) cells correlated with Nestin(+) and endogenous Msi1(+) cells, but not with MAP2(+) mature neurons with long processes (D, Scale bar: 50 um). Vertical arrowheads show Marker(+)/GFP(+) cells. The immunofluorescent intensity of each marker was represented by scatter blots (E). The vertical axis shows the intensity of Msi1 and of cell-type-specific markers for neurons (β-III Tubulin), astrocytes (GFAP) and NS/PCs (Nestin). The horizontal axis shows the endogenous Msi1 (upper panel) and GFP (lower panel) intensities. The proportion (%) of marker(+) cells among GFP(+) cells is indicated in the bottom of each box. Note that the GFP/Marker double-positive cells were highly observed for Msi1, GFAP, and Nestin, but the β-III Tubulin-positive neurons had weak GFP expression.
Mentions: We next examined how well the BAC reporter signal corresponds to endogenous Msi1 expression during the neural differentiation of ESCs. For this purpose, we generated ESC lines with stably-integrated Msi1 BAC DNA. We introduced Msi1 BAC DNA into parent (line EB3 tg14) ESCs. Forty-seven stable lines were established and characterized by their neural differentiation, using the scheme shown in Figure 2A. ESCs can be induced into neural lineages, including NSCs, by treatment with retinoic acid (RA) at a low concentration (10-8M). In embryoid body (EB)-forming cells, NS/PCs expressing Msi1 and/or Nestin can be efficiently induced by treatment with 10-8M RA [49]. We cultured recombinant ESCs in floating conditions to form EBs, added 10-8M RA to the culture medium after 2 days to induce neural differentiation, and cultured the cells for 4 more days (Figure 2A). Recombinant ES clones bearing Msi1 BAC DNA frequently showed GFP fluorescence on EB day 6 with RA (+RA), while GFP was not expressed in ESCs and RA-untreated EBs(-RA) (Figure 2B left panel). Firefly Luciferase activity, reflecting the Msi1 transcription activity, was measured in EBs treated with RA and without RA. The average luminescent activity per total protein increased about 10-fold in the treated EBs (+RA) over that found in undifferentiated recombinant ESCs (Figure 2B right panel). Luciferase activity in treated EBs (+RA) was about 5-fold greater than in untreated EBs (-RA) (Figure 2B right panel).

Bottom Line: When neuronal differentiation was induced in embryoid body (EB)-derived neurosphere cells, reporter signals were detected in Msi1-positive NSCs and GFAP-positive astrocytes, but not in MAP2-positive neurons.A regulatory element for Msi1 transcription in NS/PCs is located in the sixth intron of the Msi1 gene.The 595-bp D5E2 intronic enhancer can transactivate Msi1 gene expression with cell-type specificity markedly similar to the endogenous Msi1 expression patterns.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, Japan.

ABSTRACT

Background: The specific genetic regulation of neural primordial cell determination is of great interest in stem cell biology. The Musashi1 (Msi1) protein, which belongs to an evolutionarily conserved family of RNA-binding proteins, is a marker for neural stem/progenitor cells (NS/PCs) in the embryonic and post-natal central nervous system (CNS). Msi1 regulates the translation of its downstream targets, including m-Numb and p21 mRNAs. In vitro experiments using knockout mice have shown that Msi1 and its isoform Musashi2 (Msi2) keep NS/PCs in an undifferentiated and proliferative state. Msi1 is expressed not only in NS/PCs, but also in other somatic stem cells and in tumours. Based on previous findings, Msi1 is likely to be a key regulator for maintaining the characteristics of self-renewing stem cells. However, the mechanisms regulating Msi1 expression are not yet clear.

Results: To identify the DNA region affecting Msi1 transcription, we inserted the fusion gene ffLuc, comprised of the fluorescent Venus protein and firefly Luciferase, at the translation initiation site of the mouse Msi1 gene locus contained in a 184-kb bacterial artificial chromosome (BAC). Fluorescence and Luciferase activity, reflecting the Msi1 transcriptional activity, were observed in a stable BAC-carrying embryonic stem cell line when it was induced toward neural lineage differentiation by retinoic acid treatment. When neuronal differentiation was induced in embryoid body (EB)-derived neurosphere cells, reporter signals were detected in Msi1-positive NSCs and GFAP-positive astrocytes, but not in MAP2-positive neurons. By introducing deletions into the BAC reporter gene and conducting further reporter experiments using a minimized enhancer region, we identified a region, "D5E2," that is responsible for Msi1 transcription in NS/PCs.

Conclusions: A regulatory element for Msi1 transcription in NS/PCs is located in the sixth intron of the Msi1 gene. The 595-bp D5E2 intronic enhancer can transactivate Msi1 gene expression with cell-type specificity markedly similar to the endogenous Msi1 expression patterns.

Show MeSH
Related in: MedlinePlus