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.