Mihoko Kato

Functional transcriptomics of a migrating cell in Caenorhabditis elegans

In both metazoan development and metastatic cancer, migrating cells must carry out a detailed, complex program of sensing cues, binding substrates, and moving their cytoskeletons. The linker cell in Caenorhabditis elegans males undergoes a stereotyped migration that guides gonad organogenesis, occurs with precise timing, and requires the nuclear hormone receptor NHR-67. To better understand how this occurs, we performed RNA-seq of individually staged and dissected linker cells, comparing transcriptomes from linker cells of third-stage (L3) larvae, fourth-stage (L4) larvae, and nhr-67-RNAi–treated L4 larvae. We observed expression of 8,000–10,000 genes in the linker cell, 22–25% of which were up- or down-regulated 20-fold during development by NHR-67. Of genes that we tested by RNAi, 22% (45 of 204) were required for normal shape and migration, suggesting that many NHR-67–dependent, linker cell-enriched genes play roles in this migration. One unexpected class of genes up-regulated by NHR-67 was tandem pore potassium channels, which are required for normal linker-cell migration. We also found phenotypes for genes with human orthologs but no previously described migratory function. Our results provide an extensive catalog of genes that act in a migrating cell, identify unique molecular functions involved in nematode cell migration, and suggest similar functions in humans.

Wide and scalable field-of-view Talbot-grid-based fluorescence microscopy

Here we report a low-cost and simple wide field-of-view (FOV) on-chip fluorescence-imaging platform, termed fluorescence Talbot microscopy (FTM), which utilizes the Talbot self-imaging effect to enable efficient fluorescence imaging over a large and directly scalable FOV. The FTM prototype has a resolution of 1.2 μm and an FOV of 3.9 mm × 3.5 mm. We demonstrate the imaging capability of FTM on fluorescently labeled breast cancer cells (SK-BR-3) and human embryonic kidney 293 (HEK) cells expressing green fluorescent protein.

The C. elegans tailless/Tlx homolog nhr-67 regulates a stage-specific program of linker cell migration in male gonadogenesis

Cell migration is a common event during organogenesis, yet little is known about how migration is temporally coordinated with organ development. We are investigating stage-specific programs of cell migration using the linker cell (LC), a migratory cell crucial for male gonadogenesis of C. elegans. During the L3 and L4 larval stages of wild-type males, the LC undergoes changes in its position along the migratory route, in transcriptional regulation of the unc-5 netrin receptor and zmp-1 zinc matrix metalloprotease, and in cell morphology. We have identified the tailless homolog nhr-67 as a cell-autonomous, stage-specific regulator of timing in LC migration programs. In nhr-67-deficient animals, each of the L3 and L4 stage changes is either severely delayed or never occurs, yet LC development before the early L3 stage or after the mid-L4 stage occurs with normal timing. We propose that there is a basal migration program utilized throughout LC migration that is modified by stage-specific regulators such as nhr-67.

Mapping a multiplexed zoo of mRNA expression

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. Summary: Multiplexed in situ hybridisation chain reaction allows visualisation of multiple mRNAs in a single sample with subcellular resolution. This technology can be applied in many species.

LINKIN, a new transmembrane protein necessary for cell adhesion.

In epithelial collective migration, leader and follower cells migrate while maintaining cell–cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG–GAP domains in the extracellular domain, which potentially folds into a β-propeller structure resembling the α-integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and α-tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain. DOI: http://dx.doi.org/10.7554/eLife.04449.001

Abstract A46: Comparison of genes upregulated in metastasis with three C. elegans cell migrations

Cell migration is an important process occurring during normal animal development but also in the early stages of metastatic cancer when cells invasively migrate out of the primary tumor. It is therefore likely that some of the same genes function in both migrations. Although transcriptional profiling has identified many genes differentially expressed between metastases and primary tumors, we do not understand the function of most of these genes in either normal or disease states. Our goal has been to identify conserved genes used in migration of mammalian metastasis and normal C. elegans development to further characterize their function in both systems. From two published transcriptional profile datasets1,2, we selected genes that were upregulated in metastases compared to primary tumors, and then identified the C. elegans orthologs of those genes to generate a list of 107 genes. We further narrowed our selection to 87 genes based on the gene9s expression in the linker cell, a migratory C. elegans cell that we had previously transcriptionally profiled3. We used RNAi by feeding to reduce the function of these genes in three migratory cell types in C. elegans. The male linker cell and hermaphrodite distal tip cell are both somatic gonadal cells that migrate long distances while pulling along attached, non-motile gonadal cells, yet differ in their gender and in their migratory path. The sex myoblast migrates individually and for a short distance. We scored defects in migratory path, cell shape, and speed. 19 genes affected the migration of the linker cell and 16 genes that of the distal tip cell. Six genes overlapped between these two cell migrations, including SNTB2/syntrophin, an adaptor protein in the dystrophin pathway, and ATP6V0A1, a vacuolar proton-translocating ATPase. The sex myoblast screen is still ongoing, but only one of 25 genes affects its migration, showing even fewer overlapping genes with the other two cells. Among the genes that have penetrant but cell-specific defects are UTRN/utrophin, CAP1/ adenylate cyclase-associated protein, STXBP2/syntaxin binding protein, and MYL12A/myosin light chain regulatory subunit. Our results indicate that different cells use different genes for their migration, and suggest that different cancer cell types may also. This underscores the importance of characterizing the function of diverse genes to both understand their role in metastasis and identify cell type-specific drug targets. 1. Alonso, S.R. et al. (2007) A high-throughput study in melanoma identifies epithelial-mesenchymal transition as a major determinant of metastasis. Cancer Res. 67:3450-3460. 2. Patsialou, A. et al. (2012) Selective gene-expression profiling of migratory tumor cells in vivo predicts clinical outcome in breast cancer patients. Breast Cancer Res. 14:R139. 3. Schwarz, E.M.*, Kato, M*. Sternberg, P.W. (2012) Functional transcriptomics of a migrating cell in Caenorhabditis elegans. Proc. Natl. Acad. Sci.109: 16246-51. Citation Format: Mihoko Kato, Jonathan Liu, Olivia Box Power, Anand Upadhyaya, John Yim, Paul Sternberg. Comparison of genes upregulated in metastasis with three C. elegans cell migrations. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A46.