Biology subjects

Kirby, E. D.

Publications and source records attributed to Kirby, E. D..

3 recordsLinked to original sources

bioRxiv2019

Stereological characterization of the major cell lineages in the mouse dorsal dentate gyrus

Adult neurogenesis in the dorsal dentate gyrus (DG) subregion of the mammalian hippocampus supports critical cognitive processes related to memory. Local DG cell populations form a neurogenic niche specialized to regulate adult neurogenesis. Recently, DG astrocytes, microglia, endothelia, and neural stem cells have been identified as sources of neurogenesismodulating secreted factors. Accurately estimating the size of these cell populations is useful for elucidating their relative contributions to niche physiology. Previous studies have characterized these cell types individually, but to our knowledge no comprehensive study of all these cell types exists. This is problematic because considerable variability in reported population size complicates comparisons across studies. Here, we apply consistent stereological methods within a single study to estimate cell density for neurogenesis-modulating secretory cell types in the dorsal DG of adult mice. We used immunohistochemical phenotypic markers to quantify cell density and found that stellate astrocytes were the most numerous followed by endothelia, intermediate progenitors, microglia, and neural stem cells. We did not observe any significant sex differences in cell density. We expect our data will facilitate efforts to elucidate the role of DG secretory cell populations in regulating adult neurogenesis.

neuroscience↗

bioRxiv2019

Poor concordance of floxed sequence recombination in single neural stem cells: Implications for cell autonomous studies

To manipulate target gene function in specific adult cell populations, tamoxifen-dependent CreERT2 is widely used to drive inducible, site-specific recombination of LoxP flanked sequences. In studies of cell autonomous target gene function, it is common practice to combine these CreERT2-lox systems with a ubiquitously-expressed stop-floxed fluorescent reporter gene to identify single cells supposedly undergoing target gene recombination. Here, we studied the reliability of using Cre-induced recombination of one gene to predict recombination in another gene at the single cell level in adult hippocampal neural stem and progenitor cells. Using two separate stop-floxed reporters plus a Nestin promoter-driven CreERT2, we found that, in individual cells, expression of one reporter was a poor predictor of expression of the other. These findings imply that use of stop-floxed reporters to investigate cell autonomous gene function is likely to lead to false conclusions because recombination in separate genes shows poor concordance in individual cells.

neuroscience↗

bioRxiv2019

Defining the adult hippocampal neural stem cell secretome: in vivo versus in vitro transcriptomic differences and their correlation to secreted protein levels

Recent evidence shows that adult hippocampal neural stem and progenitor cells (NSPCs) secrete a variety of proteins that affect tissue function. Though several individual NSPC-derived proteins have been shown to impact cellular processes like neuronal maturation and stem cell maintenance, a broad characterization of NSPC-secreted factors is lacking. Secretome profiling of low abundance stem cell populations is typically achieved via proteomic characterization of in vitro, isolated cells. Here, we analyzed the in vitro NSPC secretome using conditioned media from cultured adult mouse hippocampal NSPCs and detected over 200 different bioactive proteins with an antibody array. We next assessed the NSPC secretome on a transcriptional level with RNA sequencing (RNAseq) of cultured NSPCs. This comparison revealed that quantification of gene expression did not accurately predict relative protein abundance for several factors. Furthermore, comparing our transcriptional data with previously published single cell RNA sequencing datasets of freshly isolated hippocampal NSPCs, we found key differences in gene expression of secreted proteins between cultured and acutely isolated NSPCs. Understanding the components and functions of the NSPC secretome is essential to understanding how these cells may modulate the hippocampal neurogenic niche, as well as how they can be applied therapeutically. Cumulatively, our data emphasize the importance of using proteomic analysis in conjunction with transcriptomic studies and highlights the need for better methods of global unbiased secretome profiling.

neuroscience↗