Thursday, April 3, 2025
4/3/2025
Determining the role of astrocyte BMP signaling in cortical astrocyte heterogeneity - PROJECT SUMMARY Astrocytes, like neurons, display regional and intra-regional heterogeneity in both function and transcriptional state. This heterogeneity impacts neighboring neurons, making it imperative to identify on a molecular level what drives regional specialization of astrocytes. This question is addressed in the cortex, where astrocytes in distinct cortical layers have distinct transcriptional profiles. The hypothesis that members of the BMP family of secreted morphogens are responsible for driving layer-specific gene expression is tested. This is based on BMP target genes such as ID3 being enriched in astrocytes in deep and superficial layers, and the secreted BMP inhibitor CHRDL1 being enriched in astrocytes in mid to upper layers. This proposal investigates the consequences of manipulating BMP signaling in astrocytes on establishment of cortical astrocyte heterogeneity and effects on neurons. Preliminary experiments show that genetic removal of the BMP-transcriptional mediator SMAD4 from cortical astrocytes throughout development alters their transcriptional profile, assessed through bulk RNA sequencing. This includes downregulation of BMP-target genes, as well as genes enriched in astrocytes in specific cortical layers. The goals of Aim 1 are to ask on a single-cell level how BMP signaling impacts astrocyte state and heterogeneity. To give insight into the spatial impact of removing BMP signaling from astrocytes, single nucleus RNA sequencing of cortical astrocytes and MERFISH spatial transcriptomics in SMAD4 cKO mice will be performed, with a prediction that effects will be greatest in deep and superficial layer astrocytes where BMP signaling is highest. SMAD4 is a downstream mediator of other members of the TGF beta superfamily, so to determine if effects are specific to the BMP pathway single nucleus RNA sequencing will be performed from astrocytes lacking the BMP receptor. To ask if production of the secreted BMP antagonist CHRDL1 by astrocytes in mid to upper layers is responsible for reducing BMP signaling in these cells, single nucleus RNA sequencing of astrocytes from CHRDL1 cKO mice will be performed. These experiments will determine how BMP signaling regulates heterogeneous astrocyte transcriptional state, and in Aim 2 the functional impact of these alterations will be investigated. Preliminary experiments using in vivo TurboID proximity labeling identified a downregulation of proteins that induce synapse maturation in astrocytes in SMAD4 cKO mice, suggesting removing BMP signaling makes astrocytes functionally immature. This will be assessed by analyzing astrocyte morphology and complexity, which is predicted to reflect an immature state. Impact on neurons will be determined by assaying excitatory and inhibitory neuron synapse number, function and maturation state using electrophysiology and immunohistochemistry. The prediction is that synapses will be immature in SMAD4 cKO mice, which will be rescued by delivery of synaptogenic cues that are downregulated in SMAD4 cKO. Determining what drives the heterogeneity of astrocytes within the cortex is an important unanswered question. This proposal will determine if BMP signaling is responsible for driving diversity, and the impact this has on the function of cortical circuits.
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