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Whole-organ transcriptomic analyses have emerged as a common method for characterizing developmental transitions in mammalian organs. However, it is unclear if all cell types in an organ follow the whole-organ defined developmental trajectory. Recently, a postnatal two-stage developmental process was described for the mouse stomach. Here, using laser capture microdissection to obtain in situ transcriptomic data, we show that mouse gastric pit cells exhibit four postnatal developmental stages. Interestingly, early stages are characterized by the up-regulation of genes associated with metabolism, a functionality not typically associated with pit cells. Hence, beyond revealing that not all constituent cells develop according to the whole-organ determined pathway, these results broaden our understanding of the pit cell phenotypic landscape during stomach development.

The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue, as local interactions precipitate intra-cellular events that often lead to changes in expression. However, our understanding of these processes in tissues, whether healthy or diseased, is limited at present owing to the difficulty in acquiring comprehensive transcriptional programs of spatially- and phenotypically-defined cells in situ. Here we present a robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to acquire finely resolved transcriptional programs with as few as tens of cells from snap-frozen or RNAlater-treated clinical tissues sufficient to resolve even isoforms. The protocol is optimized to protect the RNA with a small molecule inhibitor, the ribonucleoside vanadyl complex (RVC), which thereby enables the typical time-consuming immunostaining and laser capture steps of this procedure during which RNA is usually severely degraded in existing approaches. The efficacy of this approach is exemplified by the characterization of differentially expressed genes between the mouse small intestine lacteal cells at the tip versus the main capillary body, including those that function in sensing and responding to local environmental cues to stimulate intra-cellular signalling. With the extensive repertoire of specific antibodies that are presently available, our method provides an unprecedented capability for the analysis of transcriptional networks and signalling pathways during development, pathogenesis, and aging of specific cell types within native tissues.

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