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Abstract Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing and stimulus responses, which collectively define the thousands of unique cell types in the body1–3. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for these intended purposes has arisen naturally. Here we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell-type specificity. We take advantage of innovations in deep neural network modelling of CRE activity across three cell types, efficient in silico optimization and massively parallel reporter assays to design and empirically test thousands of CREs4–8. Through large-scale in vitro validation, we show that synthetic sequences are more effective at driving cell-type-specific expression in three cell lines compared with natural sequences from the human genome and achieve specificity in analogous tissues when tested in vivo. Synthetic sequences exhibit distinct motif vocabulary associated with activity in the on-target cell type and a simultaneous reduction in the activity of off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs from massively parallel reporter assay models and demonstrate the required literacy to write fit-for-purpose regulatory code.
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Dynamic proteomic and phosphoproteomic atlas of corticostriatal axons in neurodevelopment
Mammalian axonal development begins in embryonic stages and continues postnatally. After birth, axonal proteomic landscape changes rapidly, coordinated by transcription, protein turnover, and post-translational modifications. Comprehensive profiling of axonal proteomes across neurodevelopment is limited, with most studies lacking cell-type and neural circuit specificity, resulting in substantial information loss. We create a Cre-dependent APEX2 reporter mouse line and map cell-type-specific proteome of corticostriatal projections across postnatal development. We synthesize analysis frameworks to define temporal patterns of axonal proteome and phosphoproteome, identifying co-regulated proteins and phosphorylations associated with genetic risk for human brain disorders. We discover proline-directed kinases as major developmental regulators. APEX2 transgenic reporter proximity labeling offers flexible strategies for subcellular proteomics with cell type specificity in early neurodevelopment, a critical period for neuropsychiatric disease.
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- Award ID(s):
- 1846234
- PAR ID:
- 10462236
- Date Published:
- Journal Name:
- eLife
- Volume:
- 11
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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The cerebral cortex contains numerous neuronal cell types, distinguished by their molecular identity as well as their electrophysiological and morphological properties. Cortical function is reliant on stereotyped patterns of synaptic connectivity and synaptic function among these neuron types, but how these patterns are established during development remains poorly understood. Selective targeting not only of different cell types but also of distinct postsynaptic neuronal domains occurs in many brain circuits and is directed by multiple mechanisms. These mechanisms include the regulation of axonal and dendritic guidance and fine-scale morphogenesis of pre- and postsynaptic processes, lineage relationships, activity dependent mechanisms and intercellular molecular determinants such as transmembrane and secreted molecules, many of which have also been implicated in neurodevelopmental disorders. However, many studies of synaptic targeting have focused on circuits in which neuronal processes target different lamina, such that cell-type-biased connectivity may be confounded with mechanisms of laminar specificity. In the cerebral cortex, each cortical layer contains cell bodies and processes from intermingled neuronal cell types, an arrangement that presents a challenge for the development of target-selective synapse formation. Here, we address progress and future directions in the study of cell-type-biased synaptic targeting in the cerebral cortex. We highlight challenges to identifying developmental mechanisms generating stereotyped patterns of intracortical connectivity, recent developments in uncovering the determinants of synaptic target selection during cortical synapse formation, and current gaps in the understanding of cortical synapse specificity.more » « less
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Abstract The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes. Defining the cell-type specific neuroproteomes is important for understanding the development and functional organization of neural circuits. This task remains challenging in complex tissue, due to suboptimal protein isolation techniques that often result in loss of cell-type specific information and incomplete capture of subcellular compartments. Here, we develop a genetically targeted proximity labeling approach to identify cell-type specific subcellular proteomes in the mouse brain, confirmed by imaging, electron microscopy, and mass spectrometry. We virally express subcellular-localized APEX2 to map the proteome of direct and indirect pathway spiny projection neurons in the striatum. The workflow provides sufficient depth to uncover changes in the proteome of striatal neurons following chemogenetic activation of Gα q -coupled signaling cascades. This method enables flexible, cell-type specific quantitative profiling of subcellular proteome snapshots in the mouse brain.more » « less
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Abstract Harmful algal blooms (HABs) produce neurotoxins that affect human health. Developmental exposure of zebrafish embryos to the HAB toxin domoic acid (DomA) causes myelin defects, loss of reticulospinal neurons, and behavioral deficits. However, it is unclear whether DomA primarily targets myelin sheaths, leading to the loss of reticulospinal neurons, or reticulospinal neurons, causing myelin defects. Here, we show that while exposure to DomA at 2 dpf did not reduce the number of oligodendrocyte precursors prior to myelination, it led to fewer myelinating oligodendrocytes that produced shorter myelin sheaths and aberrantly wrapped neuron cell bodies. DomA-exposed larvae lacked Mauthner neurons prior to the onset of myelination, suggesting that axonal loss is not secondary to myelin defects. The loss of the axonal targets may have led oligodendrocytes to inappropriately myelinate neuronal cell bodies. Consistent with this, GANT61, a GLI1/2 inhibitor that reduces oligodendrocyte number, caused a reduction in aberrantly myelinated neuron cell bodies in DomA-exposed fish. Together, these results suggest that DomA initially alters reticulospinal neurons and the loss of axons causes aberrant myelination of nearby cell bodies. The identification of initial targets and perturbed cellular processes provides a mechanistic understanding of how DomA alters neurodevelopment, leading to structural and behavioral phenotypes.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.7554/eLife.78847
