RESUMO
Single cell techniques permit the analysis of cellular properties that are obscured by studying the average behavior of cell populations. One way to determine how gene expression contributes to phenotypic differences among cells is to combine functional analysis with transcriptional profiling of single cells. Here we describe a microfluidic device for monitoring the responses of single cells to a ligand and then collecting cells of interest for transcriptional profiling or other assays. As a test, cells from the olfactory epithelium of zebrafish were screened by calcium imaging to identify sensory neurons that were responsive to the odorant L-lysine. Single cells were subsequently recovered for transcriptional profiling by qRT-PCR. Responsive cells all expressed TRPC2 but not OMP, consistent with known properties of amino-acid sensitive olfactory neurons. The device can be adapted for other areas in biology where there is a need to sort and analyze cells based on their signaling responses.
Assuntos
Rastreamento de Células/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Neurônios Receptores Olfatórios/metabolismo , Análise de Célula Única/instrumentação , Animais , Cálcio/química , Expressão Gênica/genética , Ligantes , Lisina/administração & dosagem , Técnicas Analíticas Microfluídicas/métodos , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/efeitos dos fármacos , Células Receptoras Sensoriais/efeitos dos fármacos , Análise de Célula Única/métodos , Peixe-ZebraRESUMO
Growth cones are guided to their final destination by intermediate targets. Here, we identify intermediate targets and signaling components acting on zebrafish habenula commissural axons. Live imaging establishes that axons pause at the medial habenula before and after crossing the roof plate. esrom mutants axons fail to advance beyond the ipsilateral medial habenula. Tsc2 function is reduced in mutant axons, indicating cell autonomous defects in signaling. Consistent with signaling properties changing outside the roof plate, EphB is surface localized on axon segments within a zone demarcated by the medial habenula. wnt4a is expressed in the medial habenula and morpholino knockdown causes loss of the commissure. Electroporation of truncated Ryk causes axons to reenter the midline after reaching the contralateral habenula. These data identify Esrom as a mediator of growth cone navigation at an intermediate target and underscore the importance of midline boundaries as signaling centers for commissure formation.
