Summary Dynamic coupling of microtubule ends to kinetochores, built on the centromeres of chromosomes, directs chromosome segregation during cell division. Here, we report that the evolutionarily ancient kinetochore-microtubule coupling machine, the KMN (Knl1/Mis12/Ndc80-complex) network, plays a critical role in neuronal morphogenesis. We show that the KMN network concentrates in microtubule-rich dendrites of developing sensory neurons that collectively extend in a multicellular morphogenetic event that occurs during C. elegans embryogenesis. Post-mitotic degradation of KMN components in sensory neurons disrupts dendritic extension, leading to patterning and functional defects in the sensory nervous system. Structure-guided mutations revealed that the molecular interface that couples kinetochores to spindle microtubules also functions in neuronal development. These results identify a cell-division-independent function for the chromosome-segregation machinery and define a microtubule-coupling-dependent event in sensory nervous system morphogenesis.

Abstract Single molecule RNA fluorescence in situ hybridization (smFISH) has become the standard tool for high spatial resolution analysis of gene expression in the context of tissue organization. This article describes protocols to perform smFISH on whole-mount mouse embryonic organs, where tissue organization can be compared to RNA expression by co-immunostaining of known protein markers. An enzymatic labeling strategy is also introduced to produce low-cost smFISH probes. Important considerations and practical guidelines for imaging smFISH samples using fluorescence confocal microscopy are described. Finally, a suite of custom-written ImageJ macros is included with detailed instructions to enable semi-automated smFISH image analysis of both 2D and 3D images. © 2018 by John Wiley & Sons, Inc.

Throughout metazoans, germ cells undergo incomplete cytokinesis to form syncytia connected by intercellular bridges. Gamete formation ultimately requires bridge closure, yet how bridges are reactivated to close is not known. The most conserved bridge component is centralspindlin, a complex of the Rho family GTPase-activating protein (GAP) CYK-4/MgcRacGAP and the microtubule motor ZEN-4/kinesin-6. Here, we show that oocyte production by the syncytial \textitCaenorhabditis elegans germline requires CYK-4 but not ZEN-4, which contrasts with cytokinesis, where both are essential. Longitudinal imaging after conditional inactivation revealed that CYK-4 activity is important for oocyte cellularization, but not for the cytokinesis-like events that generate syncytial compartments. CYK-4’s lipid-binding C1 domain and the GTPase-binding interface of its GAP domain were both required to target CYK-4 to intercellular bridges and to cellularize oocytes. These results suggest that the conserved C1-GAP region of CYK-4 constitutes a targeting module required for closure of intercellular bridges in germline syncytia.

Cell invasion across basement membrane barriers is important in both normal development and cancer metastasis. In this issue of Developmental Cell, Naegeli et al. (2017) identify a mechanism for breaching basement membranes. Localized lysosome exocytosis fuels generation of large, invasive cellular protrusions that expand tiny basement membrane openings.

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues – the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons – where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance.

Many embryonic organs undergo branching morphogenesis to maximize their functional epithelial surface area. Branching morphogenesis requires the coordinated interplay of multiple types of cells with the extracellular matrix (ECM). During branching morphogenesis, new branches form by “budding” or “clefting.” Cell migration, proliferation, rearrangement, deformation, and ECM dynamics have varied roles in driving budding versus clefting in different organs. Elongation of the newly formed branch and final maturation of the tip involve cellular mechanisms that include cell elongation, intercalation, convergent extension, proliferation, and differentiation. New methodologies such as high-resolution live imaging, tension sensors, and force-mapping techniques are providing exciting new opportunities for future research into branching morphogenesis.

Summary Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here, we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by the density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to the emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition.

C. elegans embryonic elongation is a morphogenetic event driven by actomyosin contractility and muscle-induced tension transmitted through hemidesmosomes. A role for the microtubule cytoskeleton has also been proposed, but its contribution remains poorly characterized. Here, we investigate the organization of the non-centrosomal microtubule arrays present in the epidermis and assess their function in elongation. We show that the microtubule regulators γ-tubulin and NOCA-1 are recruited to hemidesmosomes and adherens junctions early in elongation. Several parallel approaches suggest that microtubule nucleation occurs from these sites. Disrupting the epidermal microtubule array by overexpressing the microtubule-severing protein Spastin or by inhibiting the C. elegans ninein homolog NOCA-1 in the epidermis mildly affected elongation. However, microtubules were essential for elongation when hemidesmosomes or the activity of the Rho kinase LET-502/ROCK were partially compromised. Imaging of junctional components and genetic analyses suggest that epidermal microtubules function together with Rho kinase to promote the transport of E-cadherin to adherens junctions and myotactin to hemidesmosomes. Our results indicate that the role of LET-502 in junctional remodeling is likely to be independent of its established function as a myosin II activator, but requires a microtubule-dependent pathway involving the syntaxin SYX-5. Hence, we propose that non-centrosomal microtubules organized by epidermal junctions contribute to elongation by transporting junction remodeling factors, rather than having a mechanical role.

Non-centrosomal microtubule arrays assemble in differentiated tissues to perform mechanical and transport-based functions. In this study, we identify \textitCaenorhabditis elegans NOCA-1 as a protein with homology to vertebrate ninein. NOCA-1 contributes to the assembly of non-centrosomal microtubule arrays in multiple tissues. In the larval epidermis, NOCA-1 functions redundantly with the minus end protection factor Patronin/PTRN-1 to assemble a circumferential microtubule array essential for worm growth and morphogenesis. Controlled degradation of a γ-tubulin complex subunit in this tissue revealed that γ-tubulin acts with NOCA-1 in parallel to Patronin/PTRN-1. In the germline, NOCA-1 and γ-tubulin co-localize at the cell surface, and inhibiting either leads to a microtubule assembly defect. γ-tubulin targets independently of NOCA-1, but NOCA-1 targeting requires γ-tubulin when a non-essential putatively palmitoylated cysteine is mutated. These results show that NOCA-1 acts with γ-tubulin to assemble non-centrosomal arrays in multiple tissues and highlight functional overlap between the ninein and Patronin protein families.