A highly conserved cis-regulatory motif directs differential gonadal synexpression of Dmrt1 transcripts during gonad development

Abstract

Differential gene expression largely accounts for the coordinated manifestation of the genetic programme underlying embryonic development and cell differentiation. The 3' untranslated region (3'-UTR) of eukaryotic genes can contain motifs involved in regulation of gene expression at the post-transcriptional level. In the 3'-UTR of dmrt1, a key gene that functions in gonad development and differentiation, an 11-bp protein-binding motif was identified that mediates gonad-specific mRNA localization during embryonic and larval development of fish. Mutations that disrupt the 11-bp motif leading to in vitro protein-binding loss and selective transcript stabilization failure indicate a role for this motif in RNA stabilization through protein binding. The sequence motif was found to be conserved in most of the dmrt1 homologous genes from flies to humans suggesting a widespread conservation of this specific mechanism.

Figure 1.

A short highly conserved cis-regulatory motif located in dmrt1bY/dmrt1a 3′-UTRs regulates spatial and temporal expression during early development. (A–D) GFP expression of a reporter construct that contains Medaka dmrt1bY 3′-UTR during somitogenesis (A and B) and at hatching stage (C and D). (E–H) GFP expression of a control reporter construct that contains Xenopus β-globin 3′-UTR during somitogenesis (E and F) and at hatching stage (G and H). (I–Q) GFP expression of reporter constructs that contain either human dmrt1 3′-UTR (I–K) or takifugu dmrt1 3′-UTR (L–N) in Medaka embryos during somitogenesis (I, J and L, M) and at hatching stage (K and N). (O–Q) GFP expression of a reporter construct that contains Medaka dmrt1bY 3′-UTR in zebrafish embryos during somitogenesis (O and P) and at hatching stage (Q). (R–U) GFP expression in Medaka embryos of a reporter construct that contains Xenopus ß-globin 3′-UTR in which the Box was inserted. Specific GFP expression in PGCs is indicated (arrow heads).

Figure 2.

The dmrt1 box drives specific stability in the somatic mesoderm of the gonadal primordium as well as in a sub-population of PGCs. (A–F) RFP expression from dmrt1bY 3′-UTR containing capped RNA compared to germ cell-specific GFP expression due to the nanos 3′-UTR at stages 24–26 (A–C). (D–F) RFP expression compared to germ cell specific GFP expression achieved with vasa promoter around hatching stage. (G–N) Gonadal dmrt1bY 3′-UTR-driven GFP expression investigated in Sertoli cell specific DsRed expressing sox9prom:DsRed transgenic fish either at stage 34 (G, I and H, J) or just after hatching (K, M and L, N) in males (XY) and females (XX). Arrow heads indicate either putative somatic gonadal precursor cells (C) or different sub-populations of germ cells (A, B and D–N). Blue: DAPI or Olvas; Red: Sertoli cell specific expression (sox9prom:DsRed transgenic fish) and Green: gonadal dmrt1bY 3′-UTR-driven GFP expression.

Figure 3.

Tissue-specific and temporal-restricted expression by a combination of dmrt1 3′-UTR induced differential mRNA stability and translational regulation. (A) GFP:dmrt1bY 3′-UTR was injected. As controls, mRNA constructs such as GFP:nos 3′-UTR, olvas:GFP, GFP:zfvasa 3′-UTR and GFP:xlß-globin 3′-UTR were also injected to be able to compare the expression with the pattern of known post-transcriptional mechanisms such as micro-RNA mRNA induced decay, specific PGC translational regulation, and ubiquitous stability, respectively. (B) Subsequently in situ hybridization using an antisense GFP probe was performed at different stages of development to reveal the spatial distribution of the injected GFP:dmrt1bY 3′-UTR RNAs. GFP fluorescence and the distribution of RNA were followed at different stages of development. Arrowheads and circles indicate where the PGCs are or should be located respectively. (C) Luciferase expression in different cell lines transfected with a dmrt1bY 3′-UTR containing construct reveals that translation of the reporter gene was significantly enhanced by the presence of dmrt1bY 3′-UTR in Medaka spermatogonial cells in contrast to either Medaka embryonic stem or fibroblast cells.

Figure 4.

A RNA-binding factor that recognizes the Box could be responsible for primordial gonad restricted stability. (A) Electrophoretic Mobility Shift Assay (EMSA) using cell extracts from three different cell lines (i) a medaka spermatogonial cell line (Sg3), (ii) a medaka embryonic stem cell line (MES-1) and (iii) a mouse sertoli cell line (TM4) and the 11-mer box as a probe shows shift for Sg3 and TM4 cells. (B) A mutated version of the box [Mut(3)-Box] used as competitor (1–1, 1–5 and 1–10 ratios) did not interfere significantly with the binding, indicating the specificity of the binding. (C) As control, using the spermatogonial cell line, competition of radioactively and non-radioactively labeled box probes resulted in progressive loss of the apparent shift (1–1, 1–5 and 1–10 ratios). (D) Different mutated versions of the box [Mut(2–5)-Box] were then tested for binding, and resulted in apparent different binding affinities. (E–H) Relative robustness of the shift was then tested by mean of competition assays among the different mutated versions of the box (E: 1–1, 1–5 and 1–10; F: 1–1, 1–2 and 1–5; G and H: 1–1 and 1–5 ratios).