Yongping Chai

Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development.

Conditional gene knockout animals are valuable tools for studying the mechanisms underlying cell and developmental biology. We developed a conditional knockout strategy by spatiotemporally manipulating the expression of an RNA-guided DNA endonuclease, CRISPR-Cas9, in Caenorhabditis elegans somatic cell lineages. We showed that this somatic CRISPR-Cas9 technology provides a quick and efficient approach to generate conditional knockouts in various cell types at different developmental stages. Furthermore, we demonstrated that this method outperforms our recently developed somatic TALEN technique and enables the one-step generation of multiple conditional knockouts. By combining these techniques with live-cell imaging, we showed that an essential embryonic gene, Coronin, which is associated with human neurobehavioral dysfunction, regulates actin organization and cell morphology during C. elegans postembryonic neuroblast migration and neuritogenesis. We propose that the somatic CRISPR-Cas9 platform is uniquely suited for conditional gene editing-based biomedical research.

Conditional targeted genome editing using somatically expressed TALENs in C. elegans

We have developed a method for the generation of conditional knockouts in Caenorhabditis elegans by expressing transcription activator–like effector nucleases (TALENs) in somatic cells. Using germline transformation with plasmids encoding TALENs under the control of an inducible or tissue-specific promoter, we observed effective gene modifications and resulting phenotypes in specific developmental stages and tissues. We further used this method to bypass the embryonic requirement of cor-1, which encodes the homolog of human severe combined immunodeficiency (SCID) protein coronin, and we determined its essential role in cell migration in larval Q-cell lineages. Our results show that TALENs expressed in the somatic cells of model organisms provide a versatile tool for functional genomics.

Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development

Postembryonic development is an important process of organismal maturation after embryonic growth. Despite key progress in recent years in understanding embryonic development via fluorescence time-lapse microscopy, comparatively less live-cell imaging of postembryonic development has been done. Here we describe a protocol to image larval development in the nematode Caenorhabditis elegans. Our protocol describes the construction of fluorescent transgenic C. elegans, immobilization of worm larvae and time-lapse microscopy analysis. To improve the throughput of imaging, we developed a C. elegans triple-fluorescence imaging approach with a worm-optimized blue fluorescent protein (TagBFP), green fluorescent protein (GFP) and mCherry. This protocol has been previously applied to time-lapse imaging analysis of Q neuroblast asymmetric division, migration and apoptosis, and we show here that it can also be used to image neuritogenesis in the L1 larvae. Other applications are also possible. The protocol can be completed within 3 h and may provide insights into understanding postembryonic development.

Developmental stage-dependent transcriptional regulatory pathways control neuroblast lineage progression

Neuroblasts generate neurons with different functions by asymmetric cell division, cell cycle exit and differentiation. The underlying transcriptional regulatory pathways remain elusive. Here, we performed genetic screens in C. elegans and identified three evolutionarily conserved transcription factors (TFs) essential for Q neuroblast lineage progression. Through live cell imaging and genetic analysis, we showed that the storkhead TF HAM-1 regulates spindle positioning and myosin polarization during asymmetric cell division and that the PAR-1-like kinase PIG-1 is a transcriptional regulatory target of HAM-1. The TEAD TF EGL-44, in a physical association with the zinc-finger TF EGL-46, instructs cell cycle exit after the terminal division. Finally, the Sox domain TF EGL-13 is necessary and sufficient to establish the correct neuronal fate. Genetic analysis further demonstrated that HAM-1, EGL-44/EGL-46 and EGL-13 form three transcriptional regulatory pathways. We have thus identified TFs that function at distinct developmental stages to ensure appropriate neuroblast lineage progression and suggest that their vertebrate homologs might similarly regulate neural development.

Apoptotic regulators promote cytokinetic midbody degradation in C. elegans.

Independent of their role in apoptosis, cell engulfment proteins are essential for midbody internalization and degradation after cell division.

A proneural gene controls C. elegans neuroblast asymmetric division and migration

Proneural genes control the generation of neuroblasts from the neuroepithelium, but their functions in neuroblast asymmetric division and migration remain elusive. Here, we identified C aenorhabditis elegans mutants of a proneural transcription factor (TF) lin‐32, in which Q neuroblasts are produced. We showed that LIN‐32 functions in parallel with a storkhead TF, HAM‐1, to regulate Q neuroblast asymmetric division, and that Q neuroblast migration is inhibited in lin‐32 alleles. Consistently, lin‐32 is expressed throughout Q neuroblast lineage, suggesting that LIN‐32 may promote different target gene expression. Our studies thus uncovered previously unknown functions of a proneural gene in neuroblast development.

CED-10-WASP-Arp2/3 signaling axis regulates apoptotic cell corpse engulfment in C. elegans

Efficient clearance of apoptotic cells is essential for tissue homeostasis in metazoans. Genetic studies in Caenorhabditis elegans have identified signaling cascades that activate CED-10/Rac1 GTPase and promote actin cytoskeletal rearrangement during apoptotic cell engulfment. However, the molecular connection between CED-10 activation and actin reorganization remains elusive. Here, we provide evidence that CED-10 binds to the Arp2/3 nucleation promoting factor WASP; CED-10 recruits WASP and Arp2/3 to apoptotic cell corpses in the phagocytes. The loss of WASP and Arp2/3 impaired cell corpse engulfment. Furthermore, we uncover that a WASP-activating factor SEM-5/GRB2 functions in the phagocytes to promote cell corpse clearance. Together, our results suggest CED-10 reorganizes the actin cytoskeleton by recruiting the WASP-Arp2/3 actin nucleation factors during apoptotic cell engulfment.

Migration of Q Cells in Caenorhabditis elegans

During C. elegans larval development, the Q neuroblasts produce their lineage by three rounds of divisions along with continuous cell migrations. Their neuronal progeny is dispersed from the pharynx to the anus. This in vivo system to study cell migration is appealing for several reasons. The lineage development is stereotyped; functional analysis and genomic screens are rendered easy and powerful thanks to powerful tools; transgenic manipulations and genome engineering are efficient and can be conveniently combined with live-cell imaging. Here we describe a series of protocols in Q cell migration studies, including quantifications of progeny position, genetic screening strategies, preparation of migration mutants or transgenic worms expressing related fluorescent proteins, multipositional time-lapse tracking of Q cell migration using confocal microscopy and image analyses of single cell movements and dynamics.

Hippo kinases maintain polarity during directional cell migration in Caenorhabditis elegans

Precise positioning of cells is crucial for metazoan development. Despite immense progress in the elucidation of the attractive cues of cell migration, the repulsive mechanisms that prevent the formation of secondary leading edges remain less investigated. Here, we demonstrate that Caenorhabditis elegans Hippo kinases promote cell migration along the anterior–posterior body axis via the inhibition of dorsal–ventral (DV) migration. Ectopic DV polarization was also demonstrated in gain‐of‐function mutant animals for C. elegans RhoG MIG‐2. We identified serine 139 of MIG‐2 as a novel conserved Hippo kinase phosphorylation site and demonstrated that purified Hippo kinases directly phosphorylate MIG‐2S139. Live imaging analysis of genome‐edited animals indicates that MIG‐2S139 phosphorylation impedes actin assembly in migrating cells. Intriguingly, Hippo kinases are excluded from the leading edge in wild‐type cells, while MIG‐2 loss induces uniform distribution of Hippo kinases. We provide evidence that Hippo kinases inhibit RhoG activity locally and are in turn restricted to the cell body by RhoG‐mediated polarization. Therefore, we propose that the Hippo–RhoG feedback regulation maintains cell polarity during directional cell motility.

Analysis of postcytokinetic roles of cytokinetic components in Caenorhabditis elegans

Cytokinesis requires the interplay between the cytoskeleton and plasma membrane. Emerging evidence indicates that some cytokinetic components are essential for the postcytokinetic events such as epithelium organization and neural development. We have recently developed live cell imaging and conditional knockout techniques to visualize cytokinetic proteins in Caenorhabditis elegans Q neuroblasts and separate their postcytokinetic functions from cytokinetic ones. Here we describe how the fluorescent reporter strains and conditional knockout C. elegans are generated and how live cell imaging of Q neuroblast development are performed in our laboratory. Using these protocols, we uncovered a novel role of Anillin in stabilizing the actin network during neuronal migration and neurite outgrowth, and the postcytokinetic fate of midbody, which is released into the extracellular space and degraded by the adjacent macrophage using an apoptotic mimicry. These protocols could also be applicable to study other cellular processes in C. elegans or adapted to other model organisms, to provide better insights into their developmental basis.