Biology subjects

Kanzler, C. R.

Publications and source records attributed to Kanzler, C. R..

3 recordsLinked to original sources

bioRxiv2023

Functional significance of PUF partnerships in C. elegans germline stem cells

PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal of C. elegans germline stem cells together with two intrinsically disordered proteins, LST-1 and SYGL-1. Based on yeast two-hybrid results, we proposed a composite self-renewal hub in the stem cell regulatory network, with eight PUF partnerships and extensive redundancy. Here, we investigate LST-1-PUF and SYGL-1-PUF partnerships and their molecular activities in their natural context - nematode stem cells. We confirm LST-1-PUF partnerships and their specificity to self-renewal PUFs by co-immunoprecipitation and show that an LST-1(AmBm) mutant defective for PUF-interacting motifs does not complex with PUFs in nematodes. LST-1(AmBm) is used to explore the functional significance of the LST-1-PUF partnership. Tethered LST-1 requires the partnership to repress expression of a reporter RNA, and LST-1 requires the partnership to co-immunoprecipitate with NTL-1/Not1 of the CCR4-NOT complex. We suggest that the partnership provides multiple molecular interactions that work together to form an effector complex on PUF target RNAs. Comparison of PUF-LST-1 and Pumilio-Nanos reveals fundamental molecular differences, making PUF-LST-1 a distinct paradigm for PUF partnerships. Summary statementPartnerships between PUF RNA-binding proteins and intrinsically disordered proteins are essential for stem cell maintenance and RNA repression.

developmental biology↗

bioRxiv2020

Systemic paralogy and function of retinal determination network homologs in arachnids

Arachnids are important components of cave ecosystems and display many examples of troglomorphisms, such as blindness, depigmentation, and elongate appendages. Little is known about how the eyes of arachnids are specified genetically, let alone the mechanisms for eye reduction and loss in troglomorphic arachnids. Additionally, paralogy of Retinal Determination Gene Network (RDGN) homologs in spiders has convoluted functional inferences extrapolated from single-copy homologs in pancrustacean models. Here, we investigated a sister species pair of Israeli cave whip spiders (Arachnopulmonata, Amblypygi, Charinus) of which one species has reduced eyes. We generated the first embryonic transcriptomes for Amblypygi, and discovered that several RDGN homologs exhibit duplications. We show that paralogy of RDGN homologs is systemic across arachnopulmonates (arachnid orders that bear book lungs), rather than being a spider-specific phenomenon. A differential gene expression (DGE) analysis comparing the expression of RDGN genes in field-collected embryos of both species identified candidate RDGN genes involved in the formation and reduction of eyes in whip spiders. To ground bioinformatic inference of expression patterns with functional experiments, we interrogated the function of three candidate RDGN genes identified from DGE in a spider, using RNAi in the spider Parasteatoda tepidariorum. We provide functional evidence that one of these paralogs, sine oculis/Six1 A (soA), is necessary for the development of all arachnid eye types. Our results support the conservation of at least one RDGN component across Arthropoda and establish a framework for investigating the role of gene duplications in arachnid eye diversity.

developmental biology↗

bioRxiv2019

A PUF hub drives self-renewal in C. elegans germline stem cells

Stem cell regulation relies on extrinsic signaling from a niche plus intrinsic factors that respond and drive self-renewal within stem cells. A priori, loss of niche signaling and loss of the intrinsic self-renewal factors might be expected to have equivalent stem cell defects. Yet this simple prediction has not been borne out for most stem cells, including C. elegans germline stem cells (GSCs). The central regulators of C. elegans GSCs include extrinsically-acting GLP-1/Notch signaling from the niche, intrinsically-acting RNA binding proteins in the PUF family, termed FBF-1 and FBF-2 (collectively FBF), and intrinsically-acting PUF partner proteins that are direct Notch targets. Abrogation of either GLP-1/Notch signaling or its targets yields an earlier and more severe GSC defect than loss of FBF-1 and FBF-2, suggesting that additional intrinsic regulators must exist. Here, we report that those missing regulators are two additional PUF proteins, PUF-3 and PUF-11. Remarkably, an fbf-1 fbf-2; puf-3 puf-11 quadruple null mutant has a GSC defect virtually identical to that of a glp-1/Notch null mutant. PUF-3 and PUF-11 both affect GSC maintenance; both are expressed in GSCs; and epistasis experiments place them at the same position as FBF within the network. Therefore, action of PUF-3 and PUF-11 explains the milder GSC defect in fbf-1 fbf-2 mutants. We conclude that a \"PUF hub\", comprising four PUF proteins and two PUF partners, constitutes the intrinsic self-renewal node of the C. elegans GSC RNA regulatory network. Discovery of this hub underscores the significance of PUF RNA-binding proteins as key regulators of stem cell maintenance.

developmental biology↗