Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii.

Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.

A gap-free genome assembly of Chlamydomonas reinhardtii and detection of translocations induced by CRISPR-mediated mutagenesis.

Genomic assemblies of the unicellular green alga Chlamydomonas reinhardtii have provided important resources for researchers. However, assembly errors, large gaps, and unplaced scaffolds as well as strain-specific variants currently impede many types of analysis. By combining PacBio HiFi and Oxford Nanopore long-read technologies, we generated a de novo genome assembly for strain CC-5816, derived from crosses of strains CC-125 and CC-124. Multiple methods of evaluating genome completeness and base-pair error rate suggest that the final telomere-to-telomere assembly is highly accurate. The CC-5816 assembly enabled previously difficult analyses that include characterization of the 17 centromeres, rDNA arrays on three chromosomes, and 56 insertions of organellar DNA into the nuclear genome. Using Nanopore sequencing, we identified sites of cytosine (CpG) methylation, which are enriched at centromeres. We analyzed CRISPR-Cas9 insertional mutants in the PF23 gene. Two of the three alleles produced progeny that displayed patterns of meiotic inviability that suggested the presence of a chromosomal aberration. Mapping Nanopore reads from pf23-2 and pf23-3 onto the CC-5816 genome showed that these two strains each carry a translocation that was initiated at the PF23 gene locus on chromosome 11 and joined with chromosomes 5 or 3, respectively. The translocations were verified by demonstrating linkage between loci on the two translocated chromosomes in meiotic progeny. The three pf23 alleles display the expected short-cilia phenotype, and immunoblotting showed that pf23-2 lacks the PF23 protein. Our CC-5816 genome assembly will undoubtedly provide an important tool for the Chlamydomonas research community.

Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors.

Testicular Leydig cells produce androgens essential for proper male reproductive development and fertility. Here, we describe a new Leydig cell ablation model based on Cre/Lox recombination of mouse Gata4 and Gata6, two genes implicated in the transcriptional regulation of steroidogenesis. The testicular interstitium of adult Gata4flox/flox ; Gata6flox/flox mice was injected with adenoviral vectors encoding Cre + GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-GFP). The vectors efficiently and selectively transduced Leydig cells, as evidenced by GFP reporter expression. Three days after Ad-Cre-IRES-GFP injection, expression of androgen biosynthetic genes (Hsd3b1, Cyp17a1 and Hsd17b3) was reduced, whereas expression of another Leydig cell marker, Insl3, was unchanged. Six days after Ad-Cre-IRES-GFP treatment, the testicular interstitium was devoid of Leydig cells, and there was a concomitant loss of all Leydig cell markers. Chromatin condensation, nuclear fragmentation, mitochondrial swelling, and other ultrastructural changes were evident in the degenerating Leydig cells. Liquid chromatography-tandem mass spectrometry demonstrated reduced levels of androstenedione and testosterone in testes from mice injected with Ad-Cre-IRES-GFP. Late effects of treatment included testicular atrophy, infertility and the accumulation of lymphoid cells in the testicular interstitium. We conclude that adenoviral-mediated gene delivery is an expeditious way to probe Leydig cell function in vivo Our findings reinforce the notion that GATA factors are key regulators of steroidogenesis and testicular somatic cell survival.Free Finnish abstract: A Finnish translation of this abstract is freely available at http://www.reproduction-online.org/content/154/4/455/suppl/DC2.