Lin41/Trim71 is essential for mouse development and specifically expressed in postnatal ependymal cells of the brain

Abstract

Lin41/Trim71 is a heterochronic gene encoding a member of the Trim-NHL protein family, and is the original, genetically defined target of the microRNA let-7 in C. elegans. Both the LIN41 protein and multiple regulatory microRNA binding sites in the 3' UTR of the mRNA are highly conserved from nematodes to humans. Functional studies have described essential roles for mouse LIN41 in embryonic stem cells, cellular reprogramming and the timing of embryonic neurogenesis. We have used a new gene trap mouse line deficient in Lin41 to characterize Lin41 expression during embryonic development and in the postnatal central nervous system (CNS). In the embryo, Lin41 is required for embryonic viability and neural tube closure. Nevertheless, neurosphere assays suggest that Lin41 is not required for adult neurogenesis. Instead, we show that Lin41 promoter activity and protein expression in the postnatal CNS is restricted to ependymal cells lining the walls of the four ventricles. We use ependymal cell culture to confirm reestablishment of Lin41 expression during differentiation of ependymal progenitors to post-mitotic cells possessing motile cilia. Our results reveal that terminally differentiated ependymal cells express Lin41, a gene to date associated with self-renewing stem cells.

Keywords: Lin41; Trim71; ependyma; gene trap; neural tube closure; neurogenesis.

Figure 1

Characterization of a Lin41 gene trap mouse line. (A) Scheme of the wild type Lin41 gene and the gene-trap cassette insertion in the second intron. (B) Western blot from whole E9.5 embryo lysates with genotypes indicated above each lane. The filter was developed with anti-Lin41 and anti-Vinculin antibodies as loading control (left). (C) X-Gal staining of wild type, heterozygote, and homozygote knockout embryos, from E9.5 to E12.5. Knockout animals display a failure in neural tube closure and die after E9.5 (panel g: Lin41^gt/gt E9.5 embryo not stained, dotted line and arrowhead indicate the open neural fold), encapsulating and reabsorbing by E12.5. Embryos not to scale. (D) Sagittal E10.5 Lin41^+/gt mouse embryo section, X-Gal stained; neuroepithelium and limb buds are strongly positive (arrowheads). (E) Sagittal E12.5 Lin41^+/gt mouse embryo section; vestiges of X-Gal signal remain in the neuroepithelium (arrowhead).

Figure 2

Lin41 is present in the adult SVZ but not SVZ-derived neurospheres (NSPs). (A) X-Gal staining of anterior and posterior coronal sections of an adult heterozygote brain, boxes indicate fields containing the lateral ventricle shown at higher magnification (right). Diffuse staining of the hippocampus most likely reflects background and not specific X-Gal staining (see Supplementary Figure 1D). A view of the third ventricle is also provided for comparison (scale bar 100 μm). (B) X-Gal staining of NSPs cultured from wild type and heterozygote SVZs. Scale bar 100 μm. (C) RT-PCR from cDNA of NSP and positive control P19; ß-Actin was used as loading control. (D) Western blot of wild type and heterozygote NSPs and positive control P19 lysates as indicated; antibodies and their corresponding signals are indicated on the left (Vinculin serves as loading control).

Figure 3

LIN41 protein is expressed in ependymal cells. (A–F) Immunostaining of adult wild type brain coronal sections with antibodies for Lin41 (red) with overview of lateral ventricles (A,B) and higher magnification co-staining with ependymal markers as follows (green) CD133 (C), CD24 (D), acetylated α-tubulin (ac. tubulin, E), and GFAP (F). Scale bars 10 μm except (C), 5 μm. Arrowheads indicate double positive cells (C–E) or GFAP negative cell (F). L, lateral; LV, lateral ventricle; M, medial.

Figure 4

Whole mount lateral ventricle preparation. (A) Whole mount of adult brain from heterozygote and wild type mice stained for X-Gal (blue). Arrows indicate dorsal and anterior coordinates. Scale bar 1 mm. Bottom left panel shows higher magnification view of the X-Gal staining in the lateral wall of the ventricle (scale bar 100 μm). (B) Immunostaining of Lin41 (red) in a wild type sample prepared in parallel. Scale bar 100 μm.

Figure 5

Lin41 promoter activity in primary ependymal cultures. X-Gal staining of cultures derived from wild type (left column, panels A,C,E,G) and heterozygote (right column, panels B,D,F,H) newborn brain tissue are compared. Time in culture is expressed in days in vitro (DIV, left). Scale bar 100 μm.

Figure 6

Comparison of ependymal LIN41 expression in whole mount immunostaining and primary cell culture. Immunostaining of lateral wall whole mounts from adult wild type mouse brain (left column) and primary ependymal culture derived from newborn brain tissue at DIV15 (right column). LIN41 immunostaining is in red, marker antibodies in green. S100ß is an ependymal marker (A,B), acetylated α-tubulin (ac.tubulin) stains the cilia (C,D), ß-Catenin is used to label the cell-cell contacts and γ-tubulin the basal bodies of the cilia (E,F). FoxJ1 is a nuclear transcription factor responsible for the multicilia differentiation program (G,H). Scale bar 10 μm except for (B), with dashed line 40 μm.