no child left behind encodes a novel chromatin factor required for germline stem cell maintenance in males but not females

Abstract

Male and female germ cells follow distinct developmental paths with respect to germline stem cell (GSC) production and the types of differentiated progeny they produce (sperm versus egg). An essential aspect of germline development is how sexual identity is used to differentially regulate the male and female germ cell genomes to allow for these distinct outcomes. Here, we identify a gene, no child left behind (nclb), that plays very different roles in the male versus female germline in Drosophila. In particular, nclb is required for GSC maintenance in males, but not in females. Male GSCs mutant for nclb are rapidly lost from the niche, and begin to differentiate but cannot complete spermatogenesis. We further find that nclb encodes a member of a new family of conserved chromatin-associated proteins. NCLB interacts with chromatin in a specific manner and is associated with sites of active transcription. Thus, NCLB appears to be a novel chromatin regulator that exhibits very different effects on the male and female germ cell genomes.

Fig. 1.

nclb is required for male GSC maintenance. (A,B) In situ hybridization in stage 17 embryos reveals that nclb is expressed in male gonads and not female gonads. Staining in A is paternal X marker. Gonads are circled in B. (C) nclb mutants exhibit a reduced number of germline cells proximal to the hub compared with wild type (all wild type in this figure are a precise excision allele of EY10712). (D-G) Five-day-old adult testes immunostained for germ cells (VASA, red), hub cells (NCAD, green) and DNA (DAPI, blue). Note that nclb mutant testes exhibit germline loss relative to wild type that is more severe in stronger loss-of-function nclb mutants (e.g. G). (H,I) Newly eclosed adult testes immunostained for germ cells (VASA, red), hub cells (NCAD, blue) and STAT92E (anti-STAT92E, green). Germ cells adjacent to the hub (arrows) in nclb mutant testes do not exhibit anti-STAT 92E immunoreactivity. (J) Adult nclb mutant testis labeled by in situ hybridization for upd. Outline indicates apical end of testis. (K) nclb mutant adult testis immunostained for DE-cadherin (green). Note that the hub is also enlarged in nclb mutants. (L) nclb mutant adult testis immunolabeled for cyst stem cells (anti-ZFH-1, red, arrow), late somatic cyst cells (anti-EYA, green) and the hub (anti-NCAD, blue). Arrow indicates cyst stem cell. (M) Immunostaining of STAT92E (outline) is restricted to GSC in larval nclb mutant testis. (N,O) Centrosomes visualized with γ-tubulin antibody (green, arrows) are properly oriented in nclb mutant L2 larval testis (O), similar to wild-type adult (N) and larvae (not shown). Scale bars: 10 μm.

Fig. 2.

nclb mutant male germ cells initiate differentiation but do not progress in spermatogenesis. Genotypes and antibodies used are as indicated on panels; all wild type in this figure are a precise excision allele of EY10712. (A,B) Adult testes immunostained for the differentiation marker BAM (anti-BAM, green). Dotted outline indicates BAM expression in wild-type cysts (A) and in initial cysts in nclb mutants (B). (C,D) Adult testes immunostained to reveal the fusome (anti-spectrin, green). Note that small cysts in nclb mutants still produce branched fusomes characteristic of differentiating spermatogonia (dashed outline). Inset is a higher magnification of this region. Arrow in D indicates larger germ cells that do not have branched fusomes. (E,F) Larval testes immunostained with the differentiation marker BAM (anti-BAM, green). nclb-null mutant larval germ cells form initial cysts that express BAM similar to wild-type cysts (broken outlines). (G,H) Larval testes immunostained to reveal the fusome (anti-spectrin, green). Note that nclb-null larval testis form cysts with interconnected branched fusomes (arrow in H). Scale bars: 10 μm.

Fig. 3.

nclb is autonomously required in the germline. (A,B,D,E) Immunostaining of adult testes. Antibodies used and genotypes are indicated on each panel. (A,B) Five-day-old nclb mutant testes show complete rescue of germline defects when UAS-nclb is expressed specifically in the (A) germline (nanos-GAL4) or (B) broadly (tubulin-GAL4). Compare with Fig. 1G. Arrow in A indicates elongated sperm. (C) FLP/FRT induction of wild-type versus nclb homozygous mutant GSCs. Graph indicates the percentage of testes with labeled wild-type or nclb mutant GSCs at the indicated times ACI. nclb mutant GSCs are lost at a higher frequency than wild-type GSCs. (D,E) FLP/FRT induced nclb-null mutant germ cells, as indicated by loss of β-Galactosidase expression (anti-β-gal, blue). (D) nclb-null mutant GSCs (broken outline and arrow) still exhibit increased STAT immunoreactivity (green) similar to wild-type GSCs. (E) nclb-null mutant germline cysts (broken outline and arrows) still exhibit BAM expression characteristic of differentiating cysts. Scale bars: 10 μm.

Fig. 4.

nclb affects only late stages of germ cell differentiation in females. (A-G) Five-day-old mated female ovaries immunostained for germ cells (anti-VASA, red) and DNA (green). Genotypes as indicated in individual panels. All wild type in this figure are a precise excision allele of EY10712. (A-D) nclb mutant ovaries show ample germline and normal early cyst and egg chamber development. nclb mutant egg chambers arrest with a `five blob' chromatin structure (dashed outline, D). (E-G) nclb mutants expressing UAS-nclb in combination with various Gal4 `drivers'. The egg chamber arrest phenotype is rescued only when nclb is expressed strongly in both the germline and soma (da-Gal4, G). (H,I) nclb homozygous null mutant cells generated using the FLP/FRT system. (H) nclb-null mutant germ cells (dashed outline) are able to form normal cysts and early egg chambers, but arrest with the same `five blob' chromatin phenotype as hypomorphic mutants. (I) nclb null mutant follicle cells contribute to the follicular epithelium (dashed outlines), but large clones of mutant follicle cells indicative of mutant follicle stem cells are not observed. Scale bars: 10 μm.

Fig. 5.

NCLB is a chromatin-associated protein. (A) The NCLB predicted protein contains WD40 repeats (blue) and a nuclear localization signal (green), and shows extensive homology to human (endonuclein) and yeast (PWP1). (B-F′) Immunostaining using a polyclonal anti-NCLB antibody (green). Genotypes and other antibodies used are as shown on individual panels. (B) NCLB immunoreactivity is observed in the nucleus of many embryonic cell types, including the germ cells (anti-VASA, red). (C) Anti-NCLB immunoreactivity is greatly reduced in nclb-null mutant embryos, indicating the specificity of the antisera. Residual staining may reflect maternal protein. (D-E′) Anti-NCLB immunoreactivity is observed in the germline of the adult testis (D,D′), and in both the germline and the soma in the ovary (E,E′). (F,F′) anti-NCLB immunostaining of third instar larval polytene chromosomes. Arrow indicates a `puff' or area of active transcription. Scale bars: 10 μm.

Fig. 6.

NCLB colocalizes with active RNA polymerase and facilitates active transcription. (A,B) Third instar larval polytene chromosomes immunostained to reveal NCLB (anti-NCLB, red) and active forms of RNA polymerase II (green). There are many positions where NCLB colocalizes with RNA polymerase II phosphorylated at serine 5 (A) and RNA polymerase II phosphorylated at serine 2 (B). Insets show the boxed region at a higher magnification, and with the channels merged and split to reveal opposing sides of the polytene chromosome. (C,D) Third instar larval polytene chromosomes labeled by fluorescent in situ hybridization to reveal the Hsp70 loci (green) and anti-NCLB (red). Insets show a higher magnification of the boxed region, arrows indicate the Hsp70 87C locus. NCLB is not observed at the Hsp70 loci prior to heat shock (C), but is present at the puffed Hsp70 loci after heat shock (D). Scale bars: 10 μm. (E) Western blot of third instar larval protein extracts probed with antibodies that recognize all Histone H3, or Histone H3 phosphorylated on serine 10.