A zebrafish nanos-related gene is essential for the development of primordial germ cells

Abstract

Asymmetrically distributed cytoplasmic determinants collectively termed germ plasm have been shown to play an essential role in the development of primordial germ cells (PGCs). Here, we report the identification of a nanos-like (nanos1) gene, which is expressed in the germ plasm and in the PGCs of the zebrafish. We find that several mechanisms act in concert to restrict the activity of Nanos1 to the germ cells including RNA localization and control over the stability and translatability of the RNA. Reducing the level of Nanos1 in zebrafish embryos revealed an essential role for the protein in ensuring proper migration and survival of PGCs in this vertebrate model organism.

Figure 1

The zebrafish nanos1 (nos1) gene encodes a nanos-like zinc finger protein. (a) The predicted amino acid sequence of Nanos1 (Nos1) protein. The characteristic C-terminal CCHC CCHC zinc finger domain with its conserved residues (red) is underlined. This is the only region that shows high homology to nanos genes of other organisms. (b) Schematic representation of the cDNA of nos1. The cDNA contains 29 base pairs (bp) of 5′UTR (orange). The 480 bp-long open reading frame is followed by 637 bp of 3′UTR (orange). The red box within the coding region depicts the position of the zinc finger domain. (c) Comparison of the amino acid sequence of the zinc finger domain among Nanos-like proteins from Danio rerio (Nos1), Drosophila melanogaster (Nanos), and Xenopus laevis (Xcat-2). The amino acid residues comprising the conserved CCHC zinc finger are labeled in red, and residues that are identical in the three proteins are labeled in yellow. The total length of the Nanos1 protein is more similar to that of the Xcat-2 protein than to the Nanos proteins of Drosophila and C. elegans.

Figure 2

Spatial and temporal distribution of nos1 RNA and Nos1 protein. (a–f) Expression of nos1 mRNA during the first 5 d of zebrafish development. nos1 mRNA is undetectable by the fifth day of development. (g) Expression of vasa mRNA in a 5-day-old embryo. Inserts in f and g show a magnification of the region where the PGCs are normally found. (h) Developmental Northern blot of nos1. nos1 RNA is present in oocytes and in embryos before MBT (the 4- and 256-cell stages). The level of the transcript declines and it is barely detectable by the fifth day of development.

Figure 3

nos1 RNA distribution and protein expression are regulated by its 3′UTR. (a–h) Embryos injected with 40 pg of GFP-nos1 3′UTR RNA, or with 40 pg of GFP–globin RNA (i–p). (a–d,i–l) Fluorescent pictures of live embryos injected with the constructs described above. The earliest time point when the germ cells can be distinguished from somatic cells is during early gastrulation stages (insert in b). (e–h,m–p) Whole-mount in situ hybridization of embryos injected with the constructs described above using GFP as a probe. (e–h) Specific degradation in the soma and stabilization in the PGCs is observed for RNAs containing the nos1 3′UTR. (q) A 24-hour-old embryo injected with 100 pg of DsRed-nos1 3′UTR RNA at the one-cell stage showing red fluorescent in the PGC cluster with trailing posterior cells found along the yolk extension. Cell membranes were labeled by coinjecting 10 pg of EGFP-F-globin RNA. (r) Specific expression of DsRed in the PGCs of a 3-day-old embryo (dorsal view) injected with DsRed-nos1 3′UTR RNA at the one-cell stage.

Figure 4

The function of Nos1 in PGC development in zebrafish. (a) The Nos1 protein is localized to perinuclear granules. Embryos injected with mRNA of GFP fused in frame to nos1 (200 pg of GFP-FLnos1) show localization of Nos1 to Vasa-containing granules (Knaut et al. 2000), while some of the protein appears to be distributed in the cytoplasm. (b) Defects in PGC migration and reduction in PGC number in nos1 morpholino-injected embryos. Embryos were injected with 60 pg of GFP-nos1 3′UTR and 800 pg of nos1 morpholinos (right panel) or with 800 pg of control morpholinos (left panel) and were photographed at the end of the first day of development. The insert in the right panel shows a magnification of a single dying ectopic PGC. The red arrow indicates the position of the cell within the embryo. (c) Loss of PGCs as a result of increased amounts of injected morpholinos. PGCs were visualized by whole-mount in situ hybridization using vasa as a probe at the 24-hpf stage. The proportion of PGC number in the nos1 morpholino-injected embryos relative to that of control morpholino-injected embryos is given for each experimental point (9–20 embryos were analyzed per point). (d) Loss of PGCs in nos1 morpholino-injected embryos during development. The number of PGCs was determined using vasa as an in situ hybridization probe at the indicated developmental stages. Even at the highest concentration used, the initial number of PGCs was not affected by the injected nos1 morpholinos (for each point 10–20 embryos were analyzed). (e) nos1 morpholino-injected embryos can be rescued by coinjection of nos1 mRNA that cannot be bound by the nos1 morpholino (FLnos1mut). (f) PGCs of nos1 morpholino-injected embryos are found in ectopic positions and die. The total number of PGCs decreases from the 3-somite stage to the 24-hpf stage, and most of the cell death can be attributed to cells found in ectopic locations (9–16 embryos were analyzed for each experimental point). Despite the severe PGC phenotype, whole-mount in situ hybridization of 3-somite stage embryos injected with morpholinos and stained with myoD, papC, pax2.1, pax 8 (all in brown) and vasa (blue) show that somatic development is largely unaffected (right panel, ectopic cells are labeled with arrows). (g) A dying PGC in nos1 morpholino-injected embryos show the characteristic morphology of apoptotic cells. Embryos were injected with nos1 morpholinos as in b (right panel) and their PGCs were followed in live embryos at the end of gastrulation (1-somite stage). One of the two PGCs remains alive and exhibits normal morphology, while the other cell (arrow) undergoes cell death. Pseudocolors represent GFP intensity from blue (low) to green, yellow, red, and white (high).