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Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues., Summary: A novel targeted knock-in technique based on CRISPR/Cas9 and in utero electroporation enables detection of homozygous knock-in based on dual fluorescent reporters in both mouse and ferret.

The precise control of neuronal migration and morphological changes during differentiation is essential for neocortical development. We hypothesized that the transition of progenitors through progressive stages of differentiation involves dynamic changes in mitochondrial reactive oxygen species (mtROS) levels, depending on cell requirements. We found that progenitors had higher levels of mtROS, but that these levels were significantly decreased with differentiation. The Prdm16 gene was identified as a candidate modulator of mtROS using microarray analysis, and was specifically expressed by progenitors in the ventricular zone. However, Prdm16 expression declined during the transition into NeuroD1-positive multipolar cells. Subsequently, repression of Prdm16 expression by NeuroD1 on the periphery of ventricular zone was crucial for appropriate progression of the multipolar phase and was required for normal cellular development. Furthermore, time-lapse imaging experiments revealed abnormal migration and morphological changes in Prdm16-overexpressing and -knockdown cells. Reporter assays and mtROS determinations demonstrated that PGC-1α is a major downstream effector of Prdm16 and NeuroD1, and is required for regulation of the multipolar phase and characteristic modes of migration. Taken together, these data suggest that Prdm16 plays an important role in dynamic cellular redox changes in developing neocortex during neural differentiation.

Cellular diversity of the brain is largely attributed to the spatial and temporal heterogeneity of progenitor cells. In mammalian cerebral development,it has been difficult to determine how heterogeneous the neural progenitor cells are, owing to dynamic changes in their nuclear position and gene expression. To address this issue, we systematically analyzed the cDNA profiles of a large number of single progenitor cells at the mid-embryonic stage in mouse. By cluster analysis and in situ hybridization, we have identified a set of genes that distinguishes between the apical and basal progenitors. Despite their relatively homogeneous global gene expression profiles, the apical progenitors exhibit highly variable expression patterns of Notch signaling components, raising the possibility that this causes the heterogeneous division patterns of these cells. Furthermore, we successfully captured the nascent state of basal progenitor cells. These cells are generated shortly after birth from the division of the apical progenitors, and show strong expression of the major Notch ligand delta-like 1, which soon fades away as the cells migrate in the ventricular zone. We also demonstrated that attenuation of Notch signals immediately induces differentiation of apical progenitors into nascent basal progenitors. Thus, a Notch-dependent feedback loop is likely to be in operation to maintain both progenitor populations.

The RIKEN Centre for Developmental Biology recently hosted a joint UK-Asian Pacific Developmental Biology Network meeting called `Development and Emergence of Function in the Nervous System'. The meeting's program, which was organized by James Briscoe and Krishnaswamy VijayRaghavan, covered a spectrum of processes and mechanisms in neurodevelopment, ranging from the patterning of neural tissue to the initiation of a functional nervous system. One idea to have emerged during this meeting is that `form underlies function'. Here we discuss some of the themes that were addressed and provide a broad impression of what was a highly stimulating and successful conference.

Like other tissues and organs in vertebrates, multipotential stem cells serve as the origin of diverse cell types during genesis of the mammalian central nervous system (CNS). During early development, stem cells self-renew and increase their total cell numbers without overt differentiation. At later stages, the cells withdraw from this self-renewal mode, and are fated to differentiate into neurons and glia in a spatially and temporally regulated manner. However, the molecular mechanisms underlying this important step in cell differentiation remain poorly understood. In this study, we present evidence that the expression and function of the neural-specific transcription factors Mash-1 and Prox-1 are involved in this process. In vivo, Mash-1- and Prox-1-expressing cells were defined as a transient proliferating population that was molecularly distinct from self-renewing stem cells. By taking advantage of in vitro culture systems, we showed that induction of Mash-1 and Prox-1 coincided with an initial step of differentiation of stem cells. Furthermore, forced expression of Mash-1 led to the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells, and up-regulation of Prox-1, suggesting that Mash-1 positively regulates cell differentiation. In support of these observations in vitro, we found specific defects in cellular differentiation and loss of expression of Prox-1 in the developing brain of Mash-1 mutant mice in vivo. Thus, we propose that induction of Mash-1 and Prox-1 is one of the critical molecular events that control early development of the CNS.

When neuroblasts divide, prospero protein and mRNA segregate asymmetrically into the daughter neuroblast and sibling ganglion mother cell. miranda is known to localize prospero protein to the basal cell cortex of neuroblasts while the staufen RNA-binding protein mediates prospero mRNA localization. Here we show that miranda is required for asymmetric staufen localization in neuroblasts. Analyses using miranda mutants reveal that prospero and staufen interact with miranda under the same cell-cycle-dependent control. miranda thus acts to partition both prospero protein and mRNA. Furthermore, miranda localizes prospero and staufen to the basolateral cortex in dividing epithelial cells, which express the three proteins prior to neurogenesis. Our observations suggest that the epithelial cell and neuroblast (both of epithelial origin) share the same molecular machinery for creating cellular asymmetry.