Dual in Utero Electroporation in Mice to Manipulate Two Specific Neuronal Populations in the Developing Cortex

Precise regulation of gene expression during development in cortical neurons is essential for the establishment and maintenance of neuronal connectivity and higher-order cognition. Dual in utero electroporation provides a precise and effective tool to label and manipulate gene expression in multiple neuronal populations within a circuit in a spatially and temporally regulated manner. In addition, this technique allows for morphophysiological investigations into neuronal development and connectivity following cell-specific gene manipulations. Here, we detail the dual in utero electroporation protocol.

Diversified expression of gamma-protocadherins regulates synaptic specificity in the mouse neocortex

Synaptic specificity is the basis of forming neural microcircuits. However, how a neuron chooses which neurons out of many potentials to form synapses remains largely unknown. Here we identified that the diversified expression of clustered protocadherin γs (cPCDHγs) plays an essential role in regulating such specificity. Our 5-prime end single-cell sequencing data revealed the diversified expression pattern of cPCDHγs in neocortical neurons. Whole-cell recording of neuron pairs in developing mouse brain slices showed that knocking out PCDHγs significantly increased the local connection rate of nearby pyramidal neurons. By contrast, neurons overexpressing the same group of clustered PCDHγ isoforms through in utero electroporation dramatically decreased their synaptic connectivity. Finally and more importantly, decreasing the similarity level of PCDHγ isoforms over-expressed in neuron pairs through sequential in utero electroporation led to a progressive elevation of synaptic connectivity. Our observations provide strong evidence to support that the existence of diversely expressed cPCDHγs allows a neuron to choose which neurons not to form a synapse, rather than choosing which neurons to make synapses.

Role of Nrp1 in controlling cortical interhemispheric circuits

Callosal projections establish topographically organized maps between cortical areas. Neuropilin-1 (Nrp1) cortical gradient induces an early segregation of developing callosal axons. We investigated later roles of Nrp1 on the development of callosal projections from layer (L) 2/3 of the primary (S1) and secondary (S2) somatosensory (SS) areas, which express higher and lower levels of Nrp1, respectively. We used in utero electroporation to knock down or overexpress Nrp1 combined with retrograde tracers, to map connections at postnatal day 16 and 30. High levels of Nrp1 blocked contralateral S2 innervation while promoted the late postnatal growth of homotopic S1L2/3 and heterotopic S2L2/3 branches into S1. Conversely, knocking down Nrp1 increased the growth of heterotopic S1L2/3 projections into S2, and the overall refinement of S2L2/3 branches, thereby diminishing the number of P30 S2L2/3 callosally projecting neurons. Thus, the Nrp1 gradient determines homotopic SSL2/3 callosal connectivity by regulating late postnatal branching and refinement in a topographic manner.

Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation

Glial cells such as astrocytes and oligodendrocytes play crucial roles in the central nervous system. To investigate the molecular mechanisms underlying the development and the biological functions of glial cells, simple and rapid techniques for glial cell-specific genetic manipulation in the mouse cerebrum would be valuable. Here we uncovered that the Gfa2 promoter is suitable for selective gene expression in astrocytes when used with the piggyBac system and in utero electroporation. In contrast, the Blbp promoter, which has been used to induce astrocyte-specific gene expression in transgenic mice, did not result in astrocyte-specific gene expression. We also identified the Plp1 and Mbp promoters could be used with the piggyBac system and in utero electroporation to induce selective gene expression in oligodendrocytes. Furthermore, using our technique, neuron-astrocyte or neuron-oligodendrocyte interactions can be visualized by labeling neurons, astrocytes and oligodendrocytes differentially. Our study provides a fundamental basis for specific transgene expression in astrocytes and/or oligodendrocytes in the mouse cerebrum.

EPEN-30. C11ORF95-RELA FUSION PROTEIN ENGAGES NOVEL GENOMIC LOCI TO DRIVE MURINE EPENDYMOMA GROWTH

RATIONALE Over 70% of supratentorial (ST) ependymoma are characterized by an oncogenic fusion between C11ORF95 and RELA. C11ORF95-RELA fusion is frequently the sole genetic driver detected in ST ependymoma, thus ranking this genomic event as a lead target for therapeutic investigation. RELA is a transcription factor (TF) central to mediating NF-kB pathway activation in processes such as inflammation, cellular metabolism, and chemotaxis. HYPOTHESIS: We posited that C11ORF95-RELA acts as an oncogenic TF that aberrantly shapes the tumor epigenome to drive aberrant transcription. Approach: To this end we developed an in utero electroporation (IUE) mouse model of ependymoma to express C11ORF95-RELA during embryonic development. Our IUE approach allowed us to develop C11ORF95-RELA driven tumor models and cell lines. We comprehensively characterized the epigenome and transcriptome of C11ORF95-RELA fusion driven mouse cells by H3K27ac ChIP-seq, ATAC-seq, and RNA-seq. RESULTS This data revealed that: 1) C11ORF95-RELA directly engages ‘open’ chromatin and is enriched at regions with known RELA TF binding sites as well as novel genomic loci/motifs, 2) C11ORF95-RELA preferentially binds to both H3K27ac (active) enhancers and promoters, and 3) Bound C11ORF95-RELA promoter loci are associated with increased transcription of genes shared with human ependymoma. CONCLUSION Our findings shed light on the transcriptional mechanisms of C11ORF95-RELA, and reveal downstream targets that may represent cancer dependency genes and molecular targets.