The RNA-binding protein Musashi is required intrinsically to maintain stem cell identity

Abstract

A key goal of regenerative medicine is an understanding of the genetic factors that define the properties of stem cells. However, stem cell research in mammalian tissue has been hampered by a paucity of stem cell-specific markers. Although increasing evidence suggests that members of the Musashi (Msi) family of RNA-binding proteins play important functions in progenitor cells, it remains unclear whether there is a stem cell-autonomous requirement for Msi because of an inability to distinguish stem cells from early-lineage cells in mammalian tissues. Here, using the Drosophila testis as a model system for the study of stem cell regulation, we show specific evidence for a cell-autonomous requirement for Msi family proteins in regulating stem cell differentiation, leading to the identification of an RNA-binding protein required for spermatogonial stem cell maintenance. We found that loss of Msi function disrupts the balance between germ-line stem cell renewal and differentiation, resulting in the premature differentiation of germ-line stem cells. Moreover, we found that, although Msi is expressed in both somatic and germ cells, Msi function is required intrinsically in stem cells for maintenance of stem cell identity. We also discovered a requirement for Msi function in male meiosis, revealing that Msi has distinct roles at different stages of germ cell differentiation. We describe the complementary expression patterns of the murine Msi paralogues Msi1 and Msi2 during spermatogenesis, which support the idea of distinct, evolutionarily conserved roles of Msi.

Fig. 1.

Msi function is required for maintenance of early germ cells. (A) Schematic of spermatogenesis in the testis apex. GSCs (red) and CPCs (yellow) are anchored around somatic hub cells (light blue). Gonialblasts (pink) and mitotically active spermatogonia (dark blue) outside of the stem cell niche are encapsulated by differentiated cyst cells (green). These cells constitute the apical proliferation center and stain intensely with DAPI in a wild-type testis (B; arrowhead shows hub). In msi¹-null mutants (C), hypomorphic msi² mutants (E), and transheterozygotes (msi^½) (D), fewer DAPI-labeled cells can be detected. (Scale bars: 50 μm.)

Fig. 2.

GSCs are not maintained in Msi mutants. (A) Vasa-expressing GSCs (red, dotted line) are anchored around hub (N-cadherin, blue). Dlg (green) outlines all cells. msi¹ (B), msi^½ (C), and msi² (D) have fewer GSCs but have Vasa-negative cells (thick arrows) abutting the hub. (E) Wild-type Broad expression (green) is observed in CPCs near the hub (N-cadherin, blue) and in differentiated cyst cells. (Inset) ptc-lacZ (β-galactosidase, red) marks hub and CPCs. In most cases differentiated cyst cells show increased Broad expression (green, arrow) compared with that observed in CPCs. msi¹ (F), msi^½ (G), and msi² (H) show Broad-expressing cyst cells abutting the hub (thick arrows). (I) Wild-type Eya expression (green) is observed in differentiated cyst cells but is absent from CPCs. (J) In msi¹ mutants, differentiated cyst cells expressing Eya abut the hub. (K) Wild-type third-instar gonad GSCs express high levels of Vasa (red, hub; blue, Fasciclin III). (L) msi¹ has fewer germ cells adjacent to the hub. (M) Scatter plot of GSC distribution in msi-null mutant and control pharate adults. Line represents the mean. (N) bam-GFP is excluded from wild-type GSCs (arrow), Vasa (red), bam-GFP (green), and spectrin/N-cadherin (blue), but msi¹ testes exhibit bam-GFP-positive germ cells abutting the hub (O, arrow). Arrowheads, somatic hub. (Scale bars: 10 μm.)

Fig. 3.

Msi is expressed in somatic and germ cells but is required intrinsically in GSCs for stem cell maintenance. (A) Low-magnification image of a wild-type third-instar testis stained with Msi (Left) and Fasciclin III (Right). Msi is expressed at low levels in the apical region, where gonial amplification normally occurs, but at higher levels in spermatocytes (horizontal line). A region of Msi down-regulation (arrow) before spermatocyte differentiation is observed. (Scale bar: 50 μm.) Arrowhead, hub. (B) Wild-type third-instar testis stained with Msi (green) and Vasa (red). (Scale bar: 10 μm.) Msi labels hub (arrowhead), GSCs (thick arrow), cyst cells (thin arrow), early germ cells (green line), and spermatocytes (white line). (B′) Grayscale image of B (Msi expression only) shows the differential expression pattern of Msi, with low levels observed in the apical region and high levels observed in spermatocytes. (B″) No Msi expression can be detected in msi¹-null third-instar gonads. (C) Msi expression persists in hub, cyst, and germ cells in adult testes. (C Inset) High-magnification image of apex of adult testis. (C′) Grayscale image of C (Msi expression only) shows differential expression pattern of Msi in adults. (C″) No Msi expression can be detected in msi¹ mutant adult testes. (D) Comparison of wild-type and msi¹ GSC clone maintenance over time. (E–G) msi¹ gonial clone observed 8 days after heat shock in the absence of a stem cell clone. (Scale bars: 10 μm.) (E and F) msi¹ germ cell clones are GFP-negative (E, dotted line) and Vasa-positive (F, dotted line). (G) Merged image; dotted line shows msi¹ gonial cell clones. No msi¹ GSC clones are observed because all GSCs abutting the hub (arrowhead, Fasciclin III) express GFP.

Fig. 4.

Msi is required for meiotic chromosome segregation and cytokinesis. (A–C) Phase-contrast micrographs of postmeiotic spermatids in wild type (A) and msi¹ (B–C). Arrowheads, mitochondrial derivatives; arrows, nuclei. (Scale bar: 10 μm.) (A) Onion-stage spermatids show one haploid nucleus and one mitochondrial derivative, each approximately the same diameter. (B) In msi¹ mutants, two or four nuclei per mitochondrial derivative are often detected. (C) Nuclei in msi¹ mutants are often different sizes (arrow). (D) Msi1 expressed in both adult murine spermatogonia (arrowhead) and Sertoli cells (arrow). (E) Msi2 expressed in both adult murine pachytene spermatocytes (arrowhead) and round spermatids (arrow). (Scale bars: 50 μm.)