TL;DR
It is demonstrated that ciliary ARL13B is required to maintain normal ciliary composition and gene expression programs and underscores the value of multi-clone, rescue-based experimental designs for robust transcriptomic analyses.
Heterochromatin Protein 1γ, encoded by the Cbx3 gene, is a crucial epigenetic regulator that plays an essential role in mammalian meiotic progression. However, the functional divergence and conservation of this protein in teleosts—organisms possessing duplicated Cbx3 paralogs due to whole-genome duplication—remain to be elucidated. Building on previous research, we focused on cbx3a in Nile tilapia (Oreochromis niloticus), a significant aquaculture species and an excellent model for teleost reproductive studies, emphasizing its role in spermatogenesis. Expression analysis revealed that Cbx3a is localized to primordial germ cells and is sustained in spermatogonia, spermatocytes, and spermatids during spermatogenesis. CRISPR/Cas9-mediated knockout of cbx3a demonstrated that Cbx3a deficiency induces germ cell apoptosis, meiotic arrest, and sperm defects, including shortened tails and impaired motility, resulting in profound defects in sperm quantity and quality, strongly implying compromised male fertility. Transcriptomic analysis further identified dysregulated molecular pathways, including cytokine signaling and neuroactive ligand–receptor interactions. This provides novel mechanistic insights into HP1γ-mediated epigenetic regulation of meiosis. Notably, cbx3a mutants exhibited phenotypic bifurcation: a subset showed meiotic defects accompanied by sporadic germ cell apoptosis, whereas others underwent full meiotic arrest with pervasive germ cell apoptosis in adult gonads. Collectively, these findings clarify the essential and conserved role of Cbx3a/HP1γ in Nile tilapia spermatogenesis, thereby advancing the field of vertebrate reproductive epigenetics and providing a valuable theoretical basis for potential applications in reproductive management, such as improving sperm quality.
The primary cilium has been implicated in multiple developmental processes, such as cell migration and asymmetric cell division of stem- and progenitor cells. While most in vitro model systems examine ciliogenesis induced by serum starvation, it is not fully understood how de-and re-ciliation are regulated in proliferating stem- and progenitor cells. Here we employ the hierarchically organized C2C12 skeletal muscle cell line to examine how K-Ras4B participates in de- and re-ciliation processes of ciliated stem- and progenitor cells. We show that MAPK-pathway activation supports ciliogenesis through phosphorylation of centrosomal protein CEP55, which can then no longer stabilize the master regulator of de-ciliation Aurora kinase A. K-Ras4B localizes to the primary cilium aided by the ciliary trafficking chaperone PDE6D, which promotes ciliation. In line with this, depletion of components of the PDE6D machinery, RPGR and RPGRIP1L, decreases ciliation. Activation of the ciliary AMPK-PKG2-pathway increases S181-phosphorylation of K-Ras4B, which negatively regulates its binding to PDE6D, its ciliary abundance and promotes differentiation. Our work integrates a major mediator of mitogenic signaling into the regulation of ciliogenesis of proliferating muscle stem- and progenitor cells.
The small nuclear RNA U4atac is a core component of the minor spliceosome. In humans, homozygous or compound heterozygous point mutations in U4atac cause rare developmental disorders, such as Roifman syndrome, characterized by growth restriction, brain anomalies, and immune deficiency. To better define the pathophysiological role of U4atac mutations, we here establish a model of minor spliceosome dysfunction by generating a Drosophila melanogaster CRISPR/Cas9-induced U4atac mutant in the highly conserved stem II region. U4atac homozygous mutants exhibit growth and neurodevelopmental defects, immunodeficiency, and gastrointestinal symptoms. Using bulk RNA-sequencing and functional assays, we reveal that mutations in U4atac affect the splicing of a large set of transcripts involved in innate immunity, hematopoiesis, and intestinal cell functions, including the Drosophila Janus kinase (JAK) homolog hopscotch (hop). Importantly, U4atac deficiency reduces Hop expression and causes Hop-related hematopoietic defects at the embryonic and larval stages. Notably, we also observe reduced expression of Jak1 and attenuated activation of downstream signaling in patients with Roifman syndrome. Thus, our work identifies alterations of Jak signaling as part of the pathogenesis of RNU4atac-opathy.
Cohesin is a large multisubunit protein complex that plays essential roles in cell proliferation, genome organisation, and gene regulation in metazoans. Germline mutations in cohesin subunits or regulators cause a group of human developmental disorders collectively known as cohesinopathies. Increasing evidence indicates that individual cohesin subunits can confer distinct molecular functions to the complex; for example, STAG1 and STAG2 have both overlapping and non-overlapping roles in genome organisation. The zebrafish tailbud provides an excellent developmental model for investigating the coordination of cell proliferation and differentiation; processes in which cohesin has crucial functions. We previously demonstrated that loss of Stag2 disrupts Wnt signalling and mesoderm patterning in the zebrafish tailbud. Here, we show that, unlike mammals, zebrafish can tolerate complete loss of Stag1 from embryogenesis through to adulthood. In contrast to Stag2 deficiency, loss of Stag1 impairs cell cycle progression, activates p53 signalling, and induces a metabolic shift towards catabolism. BMP signalling is reduced in Stag1-deficient embryos and is accompanied by expansion of BMP antagonist chordin expression. Stag1 loss also alters chromatin accessibility at the chordin locus and affects accessibility at chromatin domain boundaries. We propose that modulation of growth and signalling pathways compensates for the absence of Stag1, allowing embryonic development to proceed correctly. Together, these findings reveal distinct contributions of Stag1 and Stag2 to cell-cycle regulation, chromatin architecture, and developmental signalling during vertebrate embryogenesis.
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