Abstract
Synaptotagmin-13 (Syt13) is an atypical member of the vesicle trafficking synaptotagmin protein family. The expression pattern and the biological function of this Ca2+-independent protein are not well resolved. Here, we have generated a novel Syt13-Venus fusion (Syt13-VF) fluorescence reporter allele to track and isolate tissues and cells expressing Syt13 protein. The reporter allele is regulated by endogenous cis-regulatory elements of Syt13 and the fusion protein follows an identical expression pattern of the endogenous Syt13 protein. The homozygous reporter mice are viable and fertile. We identify the expression of the Syt13-VF reporter in different regions of the brain with high expression in tyrosine hydroxylase (TH)-expressing and oxytocin-producing neuroendocrine cells. Moreover, Syt13-VF is highly restricted to all enteroendocrine cells in the adult intestine that can be traced in live imaging. Finally, Syt13-VF protein is expressed in the pancreatic endocrine lineage, allowing their specific isolation by flow sorting. These findings demonstrate high expression levels of Syt13 in the endocrine lineages in three major organs harboring these secretory cells. Collectively, the Syt13-VF reporter mouse line provides a unique and reliable tool to dissect the spatio-temporal expression pattern of Syt13 and enables isolation of Syt13-expressing cells that will aid in deciphering the molecular functions of this protein in the neuroendocrine system.
Highlights
Synaptotagmins (SYTs) are membrane trafficking proteins that regulate intracellular vesicle movement and exocytosis
To provide a reliable and efficient tool for tracking and isolating cells expressing Syt13 protein, we applied CRISPR/Cas9-mediated double strand breaks and homologous recombination to generate a mouse line, in which Syt13 is fused with the fluorescence protein
We found the colocalization of Venus with the enteroendocrine cells (EECs) marker, Chromogranin A (ChgA) (Figure 3F)
Summary
Synaptotagmins (SYTs) are membrane trafficking proteins that regulate intracellular vesicle movement and exocytosis. In mammals, this protein family comprises 17 isoforms that are structurally characterized by an extracellular N-terminus region, a transmembrane (TM) domain and two tandem cytoplasmic (C2) domains at the C-terminus [1,2,3]. SYTs are mainly expressed in neurons and cell types that possess regulatory secretory pathways. Among these are neuroendocrine cells, which produce and secrete hormones into the blood circulation to regulate different systematic processes such as metabolism [8,9]. The Syt13-VF mouse line offers a unique tool to explore the expression and molecular action of Syt in different cell types such as endocrine lineage
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