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[
Cell,
2016]
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.
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[
J Cell Biol,
2019]
Fam20C is a secreted protein kinase mutated in Raine syndrome, a human skeletal disorder. In vertebrates, bone and enamel proteins are major Fam20C substrates. However, Fam20 kinases are conserved in invertebrates lacking bone and enamel, suggesting other ancestral functions. We show that FAMK-1, the <i>Caenorhabditis elegans</i> Fam20C orthologue, contributes to fertility, embryogenesis, and development. These functions are not fulfilled when FAMK-1 is retained in the early secretory pathway. During embryogenesis, FAMK-1 maintains intercellular partitions and prevents multinucleation; notably, temperature elevation or lowering cortical stiffness reduces requirement for FAMK-1 in these contexts. FAMK-1 is expressed in multiple adult tissues that undergo repeated mechanical strain, and selective expression in the spermatheca restores fertility. Informatic, biochemical, and functional analysis implicate lectins as FAMK-1 substrates. These findings suggest that FAMK-1 phosphorylation of substrates, including lectins, in the late secretory pathway is important in embryonic and tissue contexts where cells are subjected to mechanical strain.
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[
Cell,
2016]
CSR-1 is a germline-expressed C. elegans Argonaute protein essential for viability. In this issue of Cell, Gerson-Gurwitz et al. now demonstrate a role for CSR-1 and its slicer activity in downregulating the levels of maternally deposited mRNAs to fine-tune the expression of proteins with critical roles in embryonic cell divisions.
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[
Mol Biol Cell,
2021]
Centromeres are epigenetically defined by the centromere-specific histone H3 variant CENP-A. Specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, with CENP-A-dependent centromeres. Here, we show that the extended N-terminal tail of <i>C. elegans</i> CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly; removal of this region prevents CENP-A loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-Tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-Tail containing the predicted structured region binds to KNL-2, a conserved SANTA and Myb domain-containing protein (referred to as M18BP1 in vertebrates) specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode <i>C. briggsae</i>, despite divergence of the N-Tail and KNL-2 primary sequences. Thus, the extended N-Tail of CENP-A is essential for CENP-A chromatin assembly in <i>C. elegans</i> and partially substitutes for the function of Scm3/HJURP, in that it mediates a direct interaction between CENP-A and KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A-specific chaperone/targeting factor of the Scm3/HJURP family.
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[
J Vis Exp,
2019]
C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80-100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.
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Ochoa SD, Green RA, Desai A, Hendel JM, Wang S, Oegema K, Zhao Z, Gerson-Gurwitz A, Chisholm AD, Biggs R, Khaliullin RN
[
Development,
2019]
The <i>C. elegans</i> embryo is an important model for analyzing mechanisms of cell fate specification and tissue morphogenesis. Sophisticated lineaging approaches for analyzing embryogenesis have been developed but are labor-intensive and do not naturally integrate morphogenetic readouts. To enable the rapid classification of developmental phenotypes, we developed a high-content method that employs two custom strains: a Germ Layer strain expressing nuclear markers in the ectoderm, mesoderm and endoderm/pharynx, and a Morphogenesis strain expressing markers labeling epidermal cell junctions and the neuronal cell surface. We describe a procedure that allows simultaneous live imaging of development in 80-100 embryos and provide a custom program that generates cropped, oriented image stacks of individual embryos to facilitate analysis. We demonstrate the utility of our method by perturbing 40 previously characterized developmental genes in variants of the two strains containing RNAi-sensitizing mutations. The resulting datasets yielded distinct, reproducible signature phenotypes for a broad spectrum of genes involved in cell fate specification and morphogenesis. Our analysis additionally provides new <i>in vivo</i> evidence for MBK-2 function in mesoderm fate specification and LET-381 function in elongation.
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Chow TL, Green R, Cheerambathur DK, Kim T, Gerson-Gurwitz A, Hattersley N, Wang S, Prevo B, Desai A, Zhao Z, Oegema K
[
Dev Cell,
2019]
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-microtubulecoupling 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.
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Green, R.A.*, Biggs, R., Hendel, J., Desai, A., Oegema, K.*, Wang, S., Gerson-Gurwitz, A., Ochoa, S., Khaliullin, R., Zhao, Z.
[
International Worm Meeting,
2017]
An important challenge is to systematically define the contributions of the ~2600 genes that control embryogenesis to specify cell fate and position and drive the formation and morphogenesis of multi-cellular tissues. To address this challenge, we developed methods to film embryogenesis following RNAi-based gene knockdown in two engineered strains expressing fluorescent markers that: (1) mark nuclei in cells in the three germ layers (endoderm, ectoderm and mesoderm) in three different colors, and (2) mark epithelial cell junctions and the surface of a subset of neurons in green and red (morphogenesis strain). Developing an automated method to analyze this 4D data and compare the complex phenotypes resulting from gene knockdowns presents a significant challenge. To tackle this problem, we manually scored ~7000 individual embryo time-lapse datasets for a pilot set of 500 genes to generate a data set that identified the spectrum of embryonic developmental defects and served as a guide for the development of custom automated algorithms. Automated algorithms were developed that: (1) monitor the increase in the number of nuclei in each of the three germ layers over time and (2) measure the distribution of nuclei in the germ layer strain and the fluorescent signals in the morphogenesis strain over time by measuring their relative center of mass and moment of inertia around orthogonal axes bisecting the embryo. To evaluate the phenotypic similarity between RNAi conditions, we measure the distance between phenotypes in n-dimensional space, where n is the number of measured parameters. To correct for the fact that the Euclidean distance between genes increases non-uniformly as phenotypes become more severe, we measure the angle between the average phenotypes for the two conditions in n-dimensional space (phenotypic angle of deviation; PAD). This automated method is highly effective in identifying groups of genes that yield similar phenotypes, suggesting that they function together in specific developmental pathways. Our library of developmental data and predicted functional groupings for each essential gene will be made publically available as a resource to the scientific community. This work represents the first fully automated high-content screen of an intact developing organism and is the most complex morphological profiling effort to date.
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Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO
[
Sci Rep,
2021]
The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).
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[
Worm Breeder's Gazette,
2003]
Wormgenes is a new resource for C.elegans offering a detailed summary about each gene and a powerful query system.