Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states1–3. However, the intermediates through which individual cells progress are largely undefined. Here we used single-cell RNA-seq4–7 at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts suggesting the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

The first major developmental transition in vertebrate embryos is the maternal-to-zygotic transition (MZT) when maternal mRNAs are degraded and zygotic transcription begins. During the MZT, the embryo takes charge of gene expression to control cell differentiation and further development. This spectacular organismal transition requires nuclear reprogramming and the initiation of RNAPII at thousands of promoters. Zygotic genome activation (ZGA) is mechanistically coordinated with other embryonic events including changes in the cell cycle, chromatin state, and nuclear-to-cytoplasmic component ratios. Here, we review progress in understanding vertebrate ZGA dynamics in frogs, fish, mice, and humans to explore differences and emphasize common features.

a b s t r a c tGene duplication provides genetic material required for functional diversification. An interesting example is the amyloid precursor protein (APP) protein family. The APP gene family has experienced both expansion and contraction during evolution. The three mammalian members have been studied quite extensively in combined knock out models. The underlying assumption is that APP, amyloid precursor like protein 1 and 2 (APLP1, APLP2) are functionally redundant. This assumption is primarily supported by the similarities in biochemical processing of APP and APLPs and on the fact that the different APP genes appear to genetically interact at the level of the phenotype in combined knockout mice. However, unique features in each member of the APP family possibly contribute to specification of their function. In the current review, we discuss the evolution and the biology of the APP protein family with special attention to the distinct properties of each homologue. We propose that the functions of APP, APLP1 and APLP2 have diverged after duplication to contribute distinctly to different neuronal events. Our analysis reveals that APLP2 is significantly diverged from APP and APLP1.

Highlightsd CRISPRd disrupts transcription factor DNA interactions using dCas9d Single TF binding sites can be targeted because sgRNAs are longer than binding sites d CRISPRd distinguishes between inhibitory and activating TF binding sites

Neuregulin 1 (NRG1) and the γ-secretase subunit APH1B have been previously implicated as genetic risk factors for schizophrenia and schizophrenia relevant deficits have been observed in rodent models with loss of function mutations in either gene. Here we show that the Aph1b-γ-secretase is selectively involved in Nrg1 intracellular signalling. We found that Aph1b-deficient mice display a decrease in excitatory synaptic markers. Electrophysiological recordings show that Aph1b is required for excitatory synaptic transmission and plasticity. Furthermore, gain and loss of function and genetic rescue experiments indicate that Nrg1 intracellular signalling promotes dendritic spine formation downstream of Aph1b-γ-secretase in vitro and in vivo. In conclusion, our study sheds light on the physiological role of Aph1b-γ-secretase in brain and provides a new mechanistic perspective on the relevance of NRG1 processing in schizophrenia.DOI: http://dx.doi.org/10.7554/eLife.02196.001

The expression pattern and regulatory functions of microRNAs (miRNAs) are intensively investigated in various tissues, cell types and disorders. Differential miRNA expression signatures have been revealed in healthy and unhealthy tissues using high-throughput profiling methods. For further analyses of miRNA signatures in biological samples, we describe here a simple and efficient method to detect multiple miRNAs simultaneously in total RNA. The size-coded ligation-mediated polymerase chain reaction (SL-PCR) method is based on size-coded DNA probe hybridization in solution, followed-by ligation, PCR amplification and gel fractionation. The new method shows quantitative and specific detection of miRNAs. We profiled miRNAs of the let-7 family in a number of organisms, tissues and cell types and the results correspond with their incidence in the genome and reported expression levels. Finally, SL-PCR detected let-7 expression changes in human embryonic stem cells as they differentiate to neuron and also in young and aged mice brain and bone marrow. We conclude that the method can efficiently reveal miRNA signatures in a range of biological samples.

Alzheimer's disease amyloid precursor protein (APP) has been implicated in many neurobiologic processes, but supporting evidence remains indirect. Studies are confounded by the existence of two partially redundant APP homologues, APLP1 and APLP2. APP/APLP1/APLP2 triple knockout (APP tKO) mice display cobblestone lissencephaly and are perinatally lethal. To circumvent this problem, we generated APP triple knockout embryonic stem (ES) cells and differentiated these to APP triple knockout neurons in vitro and in vivo. In comparison with wild-type (WT) ES cell-derived neurons, APP tKO neurons formed equally pure neuronal cultures, had unaltered in vitro migratory capacities, had a similar acquisition of polarity, and were capable of extending long neurites and forming active excitatory synapses. These data were confirmed in vivo in chimeric mice with APP tKO neurons expressing the enhanced green fluorescent protein (eGFP) present in a WT background brain. The results suggest that the loss of the APP family of proteins has no major effect on these critical neuronal processes and that the apparent multitude of functions in which APP has been implicated might be characterized by molecular redundancy. Our stem cell culture provides an excellent tool to circumvent the problem of lack of viability of APP/APLP triple knockout mice and will help to explore the function of this intriguing protein further in vitro and in vivo.

SummaryExpression of amyloid precursor protein (APP) and its two paralogues, APLP1 and APLP2 during brain development coincides with key cellular events such as neuronal differentiation and migration. However, genetic knockout and shRNA studies have led to contradictory conclusions about their role during embryonic brain development. To address this issue, we analysed in depth the role of APLP2 during neurogenesis by silencing APLP2 in vivo in an APP/APLP1 double knockout mouse background. We find that under these conditions cortical progenitors remain in their undifferentiated state much longer, displaying a higher number of mitotic cells. In addition, we show that neuron-specific APLP2 downregulation does not impact the speed or position of migrating excitatory cortical neurons. In summary, our data reveal that APLP2 is specifically required for proper cell cycle exit of neuronal progenitors, and thus has a distinct role in priming cortical progenitors for neuronal differentiation.