Identifying candidate markers of sub-populations of V2 interneurons in the adult mouse spinal cord

Details

Supervisors

Clotman, Frédéric

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

Research focusing on the nervous system is crucial for understanding the fundamental mechanisms underlying motor control, cognitive functions, and the complexity of neurological disorders. Current knowledge of neuronal populations is well established at the embryonic stage compared to what we know in the adult spinal cord (SC). However, due to the known complexity of motor outputs controlled by the SC, these populations defined in the embryo are likely to encompass a broad range of heterogeneous cell types in the adult mouse. My work aims at unravelling a small portion of the heterogeneity within the neuronal populations of the adult mouse SC, focusing on the subpopulations of V2 interneurons (INs). Arising from the p2 progenitor domain cells, the V2 INs differentiate into at least five molecularly distinct populations: V2a, V2b, V2c, V2d, and V2-Pax6. Research made it clear that these populations can be further subdivided into subpopulations. To gain insight into these subtypes of V2 INs, single-cell RNA sequencing (scRNA-seq) papers were used to identify potential markers for these subpopulations. After data analyses, clusters of V2 IN populations were predicted, dividing V2a INs into 3 subpopulations and V2b INs into 2 subpopulations, all designated by combinations of marker genes. For example, V2a INs were predicted to be divided into: cluster A expressing Chx10, Zfhx3, Zfhx4; cluster B with Chx10, Nfib, NeuroD2, Sp8; and cluster C expressing Chx10, Nfib, Prox1. Immunofluorescence (IF) was used to visualize the expression pattern of each potential marker protein within the embryonic and adult mouse SC. Experiments were conducted to improve the IF protocol for the adult SC and visualize the potential loss of known embryonic marker genes. The Hes2-iCre|tdTomato transgenic mice were used to label all V2a and V2b INs with tdTomato. The use of co-IF shows that for V2a INs, the predicted subpopulations seemed well defined. Indeed, the population of Chx10+ cells colocalizes with about 50% of tdTomato+ cells. Moreover, the tdTomato+ cells that are not marked by Chx10 are probably V2b type INs as, at the embryonic stage, this mouse line labels both V2a and V2b INs. Unfortunately, no marker for this second population worked in IF experiments. More precisely, cluster A seemed to cover 65% of Chx10+ cells. Further triple colocalization was performed to distinguish cluster A from clusters B and C, showing that Zfhx3 and Zfhx4 are almost never found in cells also labeled for Sp8, Nfib, or Prox1. These two subpopulations, B and C, seem to correspond to 10% of all V2a INs. These results also proved that variations exist along the SC, making it difficult to define subpopulations using only IF. This leads us to conclude that scRNA-seq is an excellent tool for investigating the heterogeneity of cells in a specific tissue, as we were able to predict subpopulations of V2a INs from a small amount of data. Ultimately, these findings were later used to define the belonging of cells of interest to subpopulation A of V2 INs.