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. 2016 Jun 21;113(25):E3509-18.
doi: 10.1073/pnas.1521341113. Epub 2016 Jun 2.

A premeiotic function for boule in the planarian Schmidtea mediterranea

Harini Iyer  1 , Melanie Issigonis  1 , Prashant P Sharma  2 , Cassandra G Extavour  2 , Phillip A Newmark  3
Affiliations

A premeiotic function for boule in the planarian Schmidtea mediterranea

Harini Iyer et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in Deleted in Azoospermia (DAZ), a Y chromosome gene, are an important cause of human male infertility. DAZ is found exclusively in primates, limiting functional studies of this gene to its homologs: boule, required for meiotic progression of germ cells in invertebrate model systems, and Daz-like (Dazl), required for early germ cell maintenance in vertebrates. Dazl is believed to have acquired its premeiotic role in a vertebrate ancestor following the duplication and functional divergence of the single-copy gene boule. However, multiple homologs of boule have been identified in some invertebrates, raising the possibility that some of these genes may play other roles, including a premeiotic function. Here we identify two boule paralogs in the freshwater planarian Schmidtea mediterranea Smed-boule1 is necessary for meiotic progression of male germ cells, similar to the known function of boule in invertebrates. By contrast, Smed-boule2 is required for the maintenance of early male germ cells, similar to vertebrate Dazl To examine if Boule2 may be functionally similar to vertebrate Dazl, we identify and functionally characterize planarian homologs of human DAZL/DAZ-interacting partners and DAZ family mRNA targets. Finally, our phylogenetic analyses indicate that premeiotic functions of planarian boule2 and vertebrate Dazl evolved independently. Our study uncovers a premeiotic role for an invertebrate boule homolog and offers a tractable invertebrate model system for studying the premeiotic functions of the DAZ protein family.

Keywords: DAZ; DAZL; Deleted in Azoospermia; germ cells; spermatogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Planarian boule1 and boule2 perform different functions in spermatogenesis. (A) Illustration of sexual planarian depicting the positions of reproductive structures. Ovaries are in red, testes are in blue, and germ-line stem cells are in green. (B and C) Colorimetric ISH showing boule1 and boule2 mRNA expression in the testes. (Scale bars, 1 mm.) FISH detects boule1 and boule2 expression in spermatogonial stem cells (SSCs), spermatogonia, and spermatocytes. (Scale bars, 50 μm.) Coexpression of boule transcripts with nanos+ SSCs is shown. (D) Animals fixed following two feedings of dsRNA spaced 4–5 d apart. Control (RNAi), boule1(RNAi), and boule2(RNAi) animals labeled with germinal histone H4 (gH4) in magenta to detect mitotic spermatogonia and tektin-1 (tkn-1) in cyan to mark meiotic spermatocytes. boule1(RNAi) animals show absence of meiotic labeling, but expansion of spermatogonia. The spermatogonial layer is reduced in boule2(RNAi) animals, whereas the spermatocyte population is comparable to controls. (E) Animals fixed following four feedings of dsRNA spaced 4–5 d apart. boule1(RNAi) testes contain clusters of SSCs and spermatogonia; meiotic and postmeiotic male germ cells are absent. boule2(RNAi) animals show a loss of all male germ cells. The remaining gH4 label coincides with neoblasts (somatic stem cells). Left in D and E show whole-mount images. (Scale bars, 1 mm.) Middle and Right in D and E show high magnification view of testis lobes. (Scale bars, 50 μm.)
Fig. 2.
Fig. 2.
boule2 is required for maintenance of early male germ cells but not required for respecification of SSCs. (A) Experimental scheme for testing the requirement of a gene for de novo respecification of SSCs. Animals are fed control/boule1/boule2 dsRNA three times and amputated anterior to the ovaries. Head fragments, lacking reproductive structures, are allowed to regenerate. Tail fragments are also maintained for knockdown validation. At 14 d following amputation, head fragments are fixed for nanos FISH and RNA is extracted from the tail fragment to ensure that test mRNA levels are reduced. nanos labels planarian SSCs. (B) Control (RNAi), boule1(RNAi), and boule2(RNAi) animals all show respecification of nanos+ SSCs. (Scale bars, 100 μm.) (C) Sexual hatchlings (<48 h old) are fed liver containing dsRNA until control animals are sexually mature (∼10–12 feedings over ∼2 mo). SSCs in control (RNAi) animals differentiate and form mature testes. boule1(RNAi) animals have testis lobes with only SSCs (nanos+) and spermatogonia (gH4+). boule2(RNAi) animals lack male germ cells; remnant gH4 signal is due to neoblasts. (Scale bars, 50 μm.)
Fig. 3.
Fig. 3.
Planarian boule genes play a role in the ovaries. (A) Following 4 feedings of dsRNA, boule1(RNAi) and boule2(RNAi) female gonads appear similar to controls. Oocytes are marked using Contig2621 (magenta) (26), and gH4 (green) labels early female germ cells. (B) Following 10 feedings of dsRNA, boule1(RNAi) and boule2(RNAi) ovaries have early female germ cells, but lack oocytes. (C) A similar absence of oocytes was seen when sexual hatchlings were fed dsRNA over a period of 2 mo. Dashed circles outline the ovaries. (Scale bars, 50 μm.)
Fig. 4.
Fig. 4.
Homologs of vertebrate DAZ-associated proteins are expressed and function in the testes of S. mediterranea. (A) DAZAP1, DAZAP2, and DZIP (Smed-iguana) transcripts are detected in the testes by ISH. (Scale bars, 1 mm.) (B) Adults fed control dsRNA in homeostasis show robust spermiogenesis. Thin, threadlike nuclei of mature sperm in the lumen of testis lobes are labeled with DAPI (marked by red asterisk). Both (C) DAZAP1(RNAi) and (D) iguana(RNAi) animals lack mature sperm, but have spermatogonia and spermatocytes similar to control (RNAi) animals. (E) Sexually immature regenerates fed control dsRNA regenerate their testes, whereas (F) DAZAP2 dsRNA-fed regenerates have small testes containing only SSCs and spermatogonia. (Scale bars, 50 μm.)
Fig. 5.
Fig. 5.
Knockdown of putative DAZ family targets phenocopies boule2(RNAi). (A) SDAD1, CDC25-1, and CDC25-2 are enriched in planarian testes. (Scale bars, 1 mm.) (B–E) Animals fed control, SDAD1, CDC25-1, and CDC25-2 dsRNA (three feedings spaced 4–5 d apart) labeled with gH4 and tkn-1. Similar to boule2(RNAi), RNAi knockdown of these putative targets results in animals having fewer spermatogonia, and the spermatocyte layer remains intact. Continued SDAD1(RNAi) and CDC25-1(RNAi) results in lysis, whereas (F) CDC25-2(RNAi) animals lose all male germ cells over time. (Scale bars, 50 μm.)
Fig. 6.
Fig. 6.
Phylogenetic analysis reveals independent origins of planarian Boule2 and vertebrate Dazl. Phylogenetic tree topology of DAZ gene family from ML and BI analysis. Numbers above nodes indicate ML bootstrap resampling frequencies (500 replicates). Numbers below nodes indicate Bayesian posterior probability values.
Fig. 7.
Fig. 7.
Summary of DAZ family functions in planarians and other systems. (Center) Different stages of spermatogenesis. (Upper) Known functions of the DAZ family in male germ cell development in other systems. (Lower) Summary of the functions of planarian boule genes, their putative-associated proteins, and targets.
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