The fragilis interferon-inducible gene family of transmembrane proteins is associated with germ cell specification in mice

Abstract

Background: Specification of primordial germ cells in mice depends on instructive signalling events, which act first to confer germ cell competence on epiblast cells, and second, to impose a germ cell fate upon competent precursors. fragilis, an interferon-inducible gene coding for a transmembrane protein, is the first gene to be implicated in the acquisition of germ cell competence.

Results: Here, we describe four additional fragilis-related genes, fragilis2-5, which are clustered within a 68 kb region in the vicinity of the fragilis locus on Chr 7. These genes exist in a number of mammalian species, which in the human are also clustered on the syntenic region on Chr 11. In the mouse, fragilis2 and fragilis3, which are proximate to fragilis, exhibit expression that overlaps with the latter in the region of specification of primordial germ cells. Using single cell analysis, we confirm that all these three fragilis-related genes are predominant in nascent primordial germ cells, as well as in gonadal germ cells.

Conclusion: The Fragilis family of interferon-inducible genes is tightly associated with germ cell specification in mice. Furthermore, its evolutionary conservation suggests that it probably plays a critical role in all mammals. Detailed analysis of these genes may also elucidate the role of interferons as signalling molecules during development.

Figure 1

The Fragilis family cluster on mouse Chr7, and the human homologues in the syntenic region on Chr11. In the mouse, the five Fragilis genes are clustered within a 68 kb region. All genes are encoded by two exons, and apart from fragilis2, they are located on the minus strand. In human, the four homologous genes, ENSG142056, Ifitm1 (9–27), Ifitm2 (1-8D) and Ifitm3 (1-8U), are clustered within a 26.5 kb stretch. No sequence information is currently available for the genomic region downstream the Ifitm2 gene (blank bar), although a mapped genomic DNA clone containing the three Ifitm genes has been reported [13]. The four human homologues are each encoded by two exons, but the length of the intronic sequence for Ifitm1 and Iftm3 is not known. Apart from Ifitm2, all human genes are encoded on the minus strand. The green circles represent ISRE consensus sequences.

Figure 2

Protein alignment of the Fragilis family and their homologues in human, cow and rat. Green bars indicate the location of the two predicted transmembrane domains, of which the first as well as the inter-domain stretch appear to be highly conserved throughout the four mammalian species. Identical amino acids are highlighted in dark grey, similar amino acids in light grey. The alignment was done using ClustlW.

Figure 3

Expression analysis of fragilis (a-f), fragilis2 (g-l) and fragilis3 (m-r) by whole mount in situ hybridisation. Pictures are taken as lateral view unless otherwise stated, with anterior to the left and posterior to the right. fragilis is expressed throughout the epiblast in E5.5 embryos (a) and in the region of germ cell specification at the base of the incipient and early allantoic bud at E7.5 (b, b' posterior view, c). At E8.5, signal is detected at the base and in the proximal third of the allantois as well as in the latero-anterior aspects of the brain (d rostral view, e anterior view). At E9.5, fragilis appears expressed in the mesoderm as well as in the pharyngeal arches (f). fragilis2 is detected throughout the epiblast at E5.5 (g). Expression seems thereafter downregulated but becomes again detectable in the posterior mesoderm and at the base of the incipient allantoic bud in E7.0 and E7.5 embryos (h, i, i' posterior view). At E8.5, expression is seen in caudal mesoderm (j, k posterior view), while at E9.5 expression is seen in the tailbud, the mesoderm caudal to the 15^th somite and the developing lung tissue (arrow, l). fragilis3 is expressed throughout the epiblast at E6.5 (m) and around E7.5 additionally in the region of PGC specification (n, n' posterior view, o). At E8.5, fragilis3 expression is seen throughout the embryo and appears intense in cells (arrow in q posterior view) at the base and within the proximal region of the allantois (p posterior view, q, r). asterix: allantois; black arrowhead: allantoic bud; h: developing heart; scale bars: 100 μm (a, b, g-i, m, n); 200 μm (c-e, o-q); 400 μm (f, j-l, r).

Figure 4

Expression analysis of fragilis2 by in situ hybridisation on sections. (a-d) transverse sections through the caudal region of an embryo at E9.5 (approx. 25 somites) at progressively rostral levels. At most caudal levels, fragilis2 expression is seen in cells of the neural tube, in the presomitic mesoderm, in single cells within the hindgut (arrowhead) and in the body wall. (b) staining at approx. 23^rd somite level is present within the forming somite, the body wall mesoderm and cells within the hindgut as well as the floorplate. (c) at approx. 21^st somite level, expression in the differentiating somites is reduced, while cells in the floor plate and within the hindgut remain fragilis2 mRNA positive. (d) at approx. the 13^th somite level, fragilis2 expression is absent from the somatic mesoderm as well as the neural tube. (e) sagittal section through an E10.5 embryo shows fragilis2 expression in developing lung tissue (asterix; higher magnification in f) and migrating cells along the hindgut anterior to the dorsal aorta (arrow). (g) shows a magnified view of fragilis2 mRNA expressing, migrating cells. da: dorsal aorta; fp: floor plate; g: gut; h: developing heart; nt: neural tube; s: somite; bw: body wall; scale bars: 150 μm (a-d); 1 mm (e); 400 μ m (f, g).

Figure 5

Expression analysis of the Fragilis family genes in single cells from the region of germ cell specification of E7.5 embryos. (a) shows PCR analysis of cDNAs from three, stella-positive founder PGCs and three surrounding, stella-negative somatic cells. Note that fragilis, fragilis2 and fragilis3 are expressed in PGCs and somatic cells, while fragilis4 and fragilis5 are not detected in any of the cells. (b) shows expression of fragilis, fragilis2 and fragilis3 in single cell cDNAs using Southernblot analysis. GAPDH was used as blotting control. (c) Semi-quantitative expression analysis of the Southernblot data shows that all three Fragilis genes are predominantly expressed in nascent PGCs compared to the somatic cells within the region.

Figure 6

Expression analysis of fragilis, fragilis2 and fragilis3 at E11.5/E12.5 in single cells from the genital ridge and by in situ hybridisation. (a) shows PCR analysis of cDNAs from three gonadal stella-positive germ cells and three surrounding, stella-negative somatic cells. While fragilis is detected only in the three germ cell clones, fragilis2 and fragilis3 are expressed in the germ cells as well as the somatic cells. (b) shows in situ hybridisation of urogenital ridges of E11.5/E12.5 embryos. While fragilis3 is expressed in the mesonephros as well as the genital ridge, fragilis and fragilis2 are restricted to the genital ridge. The staining pattern for fragilis appears punctate and restricted to single cells mimicking the pattern seen for the germ cell-specific stella gene. asterix: genital ridge; black arrowhead: mesonephros; scale bars: 400 μm.