Integrating EM and Patch-seq data: Synaptic connectivity and target specificity of predicted Sst transcriptomic types.

Neural circuit function is shaped both by the cell types that comprise the circuit and the connections between those cell types 1 . Neural cell types have previously been defined by morphology 2, 3 , electrophysiology 4, 5 , transcriptomic expression 6-8 , connectivity 9-13 , or even a combination of such modalities 14-16 . More recently, the Patch-seq technique has enabled the characterization of morphology (M), electrophysiology (E), and transcriptomic (T) properties from individual cells 17-20 . Using this technique, these properties were integrated to define 28, inhibitory multimodal, MET-types in mouse primary visual cortex 21 . It is unknown how these MET-types connect within the broader cortical circuitry however. Here we show that we can predict the MET-type identity of inhibitory cells within a large-scale electron microscopy (EM) dataset and these MET-types have distinct ultrastructural features and synapse connectivity patterns. We found that EM Martinotti cells, a well defined morphological cell type 22, 23 known to be Somatostatin positive (Sst+) 24, 25 , were successfully predicted to belong to Sst+ MET-types. Each identified MET-type had distinct axon myelination patterns and synapsed onto specific excitatory targets. Our results demonstrate that morphological features can be used to link cell type identities across imaging modalities, which enables further comparison of connectivity in relation to transcriptomic or electrophysiological properties. Furthermore, our results show that MET-types have distinct connectivity patterns, supporting the use of MET-types and connectivity to meaningfully define cell types.

Subconjunctival gene delivery of the transcription factor GA-binding protein delays corneal neovascularization in a mouse model.

Corneal neovascularization can reduce visual acuity. GA-binding protein (GABP) is a transcription factor that regulates the expression of target genes including vascular endothelial growth factor (VEGF) and roundabout4 (Robo4), which participate in pathologic angiogenesis. We assessed whether intraocular injection of the GABP gene affects the growth of new corneal blood vessels in a mouse ocular neovascularization model. Transfection of human GABPalpha and GABPbeta gene (GABPalpha/beta) into human conjunctival epithelial cells resulted in decreased VEGF and Robo4 expression. Three groups of mice underwent chemical and mechanical denudation of the corneal epithelium. Subsequently, two groups were administered subconjunctival injection of lipoplexes carrying plasmid DNA encoding for human GABPalpha/beta or an empty plasmid DNA at 1-week intervals. The third group served as an experimental control. In vivo delivery of human GABPalpha/beta into mouse neovascularized cornea reduced VEGF and Robo4 gene expression. Biomicroscopic examination showed that, at 1 week after one or two injections, GABPalpha/beta-treated eyes had significantly less neovascularized corneal area than did eyes treated with the empty vector. Histologic examination showed significantly less vascularized area and fewer blood vessels in the GABP-treated group at 1 week after injections. However, these angiosuppressive effects were weakened at 2 weeks after injections. Our results indicate that subconjunctival GABP gene delivery delays corneal neovascularization for up to 2 weeks in a mouse model of deliberate corneal injury.

KITENIN recruits Dishevelled/PKC delta to form a functional complex and controls the migration and invasiveness of colorectal cancer cells.

BACKGROUND AND AIMS: KITENIN was previously reported to promote metastasis in mouse colon tumour models; however, the signalling mechanism of KITENIN at the cellular level was unknown. Here the functional role of KITENIN with respect to colorectal cancer (CRC) cell invasion and its expression in CRC tissues were investigated. METHODS: The effect of KITENIN on cell motility was analysed in a migration and invasion assay upon its overexpression and knockdown. Immunoprecipitation was used to elucidate binding partners, and immunohistochemistry was used to study expression levels. RESULTS: KITENIN overexpression enhanced the migration of rat intestinal epithelial cells, whereas a loss of invasiveness was observed in CRC cells after KITENIN knockdown. Mechanically, KITENIN served as a scaffolding molecule that simultaneously recruited both Dishevelled (Dvl) and protein kinase C delta (PKC delta) through the membrane-spanning C-terminal region to form a complex that stimulated extracellular signal-regulated kinase (ERK)/activating protein-1 (AP-1) via a PKC delta component but also organised the actin filament via a Dvl component. The KITENIN complex controlled the invasiveness of CRC cells aetiologically harbouring various mutations in APC, beta-catenin or K-ras, in which AP-1 activation is redundant but the organisation of the actin filament is indispensable for cell motility. Clinically, KITENIN expression was significantly higher in colon cancer tissues from advanced stage (III, IV) than that of stage I CRC and also in corresponding metastatic tissues. CONCLUSIONS: The functional KITENIN complex acts as an executor with regard to cell motility and thereby controls CRC cell invasion, which may contribute to promoting metastasis.