Brainstem and autonomic circuitry, though understudied in neurodevelopmental disorders, are implicated in pathophysiology and co-occurring medical conditions, such as gastrointestinal disturbances (GID). The goal of this R21 project is to fill this knowledge gap, based on significant preliminary data. Experiments will determine the molecular profiles and developmental mechanisms that govern the development of subpopulations of autonomic brainstem neurons that are delimited by specific expression of the MET receptor tyrosine kinase, a pleiotropic signaling receptor that can regulate neuronal migration, axon guidance and synapse development. The analysis of MET as a critical neurodevelopmental gene in the brainstem will lead to determining the molecular networks that are involved in disrupted central regulation of autonomic and visceral functions. For brainstem autonomic nuclei, this is largely unknown with regard to any neurodevelopmental disorder (e.g. Rett Syndrome, Fragile X, ASD). Several lines of evidence suggest that MET may serve as an important translational link with autonomic brainstem development. We know that a functional promoter variant of the MET gene is enriched in children with ASD who also have GIDs. We also know that the promoter variant, which reduced MET gene expression, has functional and structural impact on human cortical circuits. MET expression is reduced in the temporal neocortex in both ASD and Rett subjects, and MeCP2 is a transcriptional regulator of MET. MET is part of PI3 Kinase/ERK signaling networks that play a significant role in syndromic neurodevelopmental disorders. We propose experiments to address the specific hypothesis that one point of convergence for neurodevelopmental disorders and certain co-occurring medical phenotypes is atypical development of autonomic and visceral brainstem circuits. The R21 will launch a new research program for testing this hypothesis. Preliminary studies support the promise of this experimental effort in mice: 1) Mapping of Met/MET expression prenatally reveals a highly selective developmental expression pattern of Met in autonomic brainstem neurons, including subpopulations in dorsal motor vagal nucleus (DMV) and nucleus Ambiguus (nA);and 2) conditional deletion of Met in motor neurons using Islet1Cre disrupts nA development.
In Aim 1, a unique genetic reporter scheme will be used to label differentially and then sort MET+ (GFPON/tdTOMATOON) motor neurons from MET- neurons (GFPOFF/tdTOMATOON) in nA and DMV for RNA-sequencing. This data will form the basis for the analysis in Aim 2, in which Met is deleted conditionally using Islet1Cre on a MetEGFP transgenic background. Altered histogenesis, migration and axon patterning of DMV and nA neurons will be measured. The influence of MET signaling on molecular differentiation will be examined by analysis of gene expression (Aim 1) in Met-subytpe neurons in DMV and nA following genetic deletion of Met. These data will establish a basis for future functional studies o understand genetic risk related to clinical phenotypes due to disrupted central autonomic circuitry.
There is well-known autonomic dysfunction in children with autism and other neurodevelopmental disorders, but there is a significant knowledge gap in understanding the mechanisms that underlie the development of brainstem regions implicated in functional problems, particularly related to ASD risk genes. The research proposal will examine, in mice, the relation between function of a well-known autism risk gene, Met, and development of autonomic motor neurons in nucleus Ambiguus and Dorsal Motor Vagal nucleus. The information will provide new insight into molecular and structural aspects of autonomic brainstem development, leading ultimately to measuring functional outcomes of developmental disturbances, as well as the design of new interventions for children who have co-occurring medical conditions and are at-risk for autism and other neurodevelopmental disorders.