Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9.

Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescence microscopy of fixed neurons confirmed the identical localization patterns of the tagged proteins to those of endogenous ones verified by immunohistochemistry. The system is also applicable for multiplexed labeling and live-cell imaging. Live imaging with total internal reflection fluorescence microscopy of a single dendritic process of a neuron double-labeled with Syp-mCherry and PSD-95-EGFP revealed the previously undescribed dynamic localization of the proteins synchronously moving along dendritic shafts. Our convenient and versatile strategy is potent for analysis of proteins whose ectopic expressions perturb cellular functions.

Optimized CRISPR/Cas9-mediated in vivo genome engineering applicable to monitoring dynamics of endogenous proteins in the mouse neural tissues.

To analyze the expression, localization, and functional dynamics of target proteins in situ, especially in living cells, it is important to develop a convenient, versatile, and efficient method to precisely introduce exogenous genes into the genome, which is applicable for labeling and engineering of the endogenous proteins of interest. By combining the CRISPR/Cas9 genome editing technology with an electroporation technique, we succeeded in creating knock-in alleles, from which GFP (RFP)-tagged endogenous proteins are produced, in neurons and glial cells in vivo in the developing mouse retina and brain. Correct gene targeting was confirmed by single-cell genotyping and Western blot analysis. Several gene loci were successfully targeted with high efficiency. Moreover, we succeeded in engineering the mouse genome to express foreign genes from the endogenous gene loci using a self-cleaving 2A peptide. Our method could be used to monitor the physiological changes in localization of endogenous proteins and expression levels of both mRNA and protein at a single cell resolution. This work discloses a powerful and widely applicable approach for visualization and manipulation of endogenous proteins in neural tissues.

Dynamic spatiotemporal patterns of alternative splicing of an F-actin scaffold protein, afadin, during murine development.

An F-actin scaffold protein, afadin, comprises two splice variants called l-afadin (a long isoform) and s-afadin (a short isoform). It is known that in adult tissues, l-afadin is ubiquitously expressed while s-afadin is restrictedly expressed in brain. In cultured cortical neurons, l-afadin potentiates axonal branching whereas s-afadin blocks axonal branching by functioning as a naturally occurring dominant-negative isoform that forms a heterodimer with l-afadin. However, the temporal and spatial expression pattern of s-afadin during development or across multiple tissues and organs has not been fully understood. In this study, using Western blotting and RT-qPCR techniques and the fluorescent splicing reporters, we examined the detailed expression patterns of l- and s-afadin isoforms across various tissues and cell types, including rat organs at developmental stages, primary cultured neuronal and non-neuronal cells prepared from the developing rat brain, and in neurons in vitro generated from P19 embryonal carcinoma (EC) cells. Both mRNA and protein of s-afadin were abundantly expressed in various regions of rat neuronal tissues, and their expression dynamically changed during development in vivo. The expression of s-afadin was also detected in primary rat cortical neurons, but not in astrocytes or fibroblasts, and the neuronal expression increased during neuronal maturation in vitro. The dynamic alternative splicing event of afadin during development was successfully visualized with the newly developed fluorescent splicing reporter plasmids at a single cell level. Moreover, s-afadin was undetectable in undifferentiated EC cells, and the expression was induced upon differentiation of the cells into neurons. These data suggest that spatiotemporal and dynamic alternative splicing produces differential expression patterns of afadin isoforms and that alternative splicing of afadin is controlled by the neuron-specific regulator(s) whose activity is triggered and dynamically altered during neuronal differentiation and maturation.

Differential regional and subcellular localization patterns of afadin splice variants in the mouse central nervous system.

AF6/afadin is an F-actin scaffold protein that plays essential roles in the organization of cell-cell junctions of polarized epithelia. Afadin comprises two major isoforms produced by alternative splicing - a longer isoform l-afadin, having the F-actin-binding domain, and a shorter isoform s-afadin, harboring the amino acid sequences unique to the isoform but lacking the F-actin-binding domain. We recently identified functional differences between l- and s-afadin isoforms in the regulation of axon branching in primary cultured cortical neurons; the former potentiates and the latter blocks axon branching. Previous biochemical reports indicate differences in tissue and cell-type distributions of isoforms, and it was shown that l-afadin is ubiquitously expressed in various tissues and cell-types, while s-afadin is predominantly expressed in neuronal tissues and cultured neurons. However, the spatial expression pattern of s-afadin across neuronal tissues or within neurons has not been revealed because no antibody specific for s-afadin is yet available. In this study, we report the generation and characterization of an antibody that specifically distinguishes s-afadin from l-afadin, and its application to investigate the expression profile of s-afadin in primary cultured neurons and tissue cryosections of adult mouse brain and retina. We describe differential regional and subcellular localization patterns of l- and s-afadin isoforms in the mouse central nervous system.

The XLID protein PQBP1 and the GTPase Dynamin 2 define a signaling link that orchestrates ciliary morphogenesis in postmitotic neurons.

Intellectual disability (ID) is a prevalent developmental disorder of cognition that remains incurable. Here, we report that knockdown of the X-linked ID (XLID) protein polyglutamine-binding protein 1 (PQBP1) in neurons profoundly impairs the morphogenesis of the primary cilium, including in the mouse cerebral cortex in vivo. PQBP1 is localized at the base of the neuronal cilium, and targeting its WW effector domain to the cilium stimulates ciliary morphogenesis. We also find that PQBP1 interacts with Dynamin 2 and thereby inhibits its GTPase activity. Accordingly, Dynamin 2 knockdown in neurons stimulates ciliogenesis and suppresses the PQBP1 knockdown-induced ciliary phenotype. Strikingly, a mutation of the PQBP1 WW domain that causes XLID disrupts its ability to interact with and inhibit Dynamin 2 and to induce neuronal ciliogenesis. These findings define PQBP1 and Dynamin 2 as components of a signaling pathway that orchestrates neuronal ciliary morphogenesis in the brain.

Conditional expression of the TVA receptor allows clonal analysis of descendents from Cre-expressing progenitor cells.

An understanding of the number and types of progeny produced by progenitor cells during development provides a foundation for studies of when and where cell fate determination takes place. Lineal relationships can be revealed by the identification of descendents of cells that express a recombinase, such as Cre or Flp. This method provides data concerning gene expression history, but does not provide clonal resolution among the descendents. An alternative method employs retroviral labeling, which permits the identification of clones, but does not allow for the tracking of gene expression history. Here we report a combination of these methods to circumvent each method's limitations. By employing the specificity of Cre expression, and by selecting only a subset of cells with a Cre history for retroviral infection, clones with a gene expression history can be labeled. The method utilizes a conditional allele of the avian tumor virus receptor A (TVA), which allows infection of mouse cells following Cre activity, with mammalian retroviral vectors pseudotyped with the ASLV-A envelope glycoprotein (EnvA). We quantified the efficiency and specificity of this system in vivo and in vitro. We also generated a series of retroviral vectors encoding a variety of histochemical and fluorescent reporter genes that enable the tracking of mixtures of clones, thus enabling better resolution of clonal boundaries. This method and new vectors can be used to further our understanding of the gene expression patterns of progenitor cells that make particular daughter cells, as well as provide a platform for manipulating identified subsets of developing cells.

Dax1 binds to Oct3/4 and inhibits its transcriptional activity in embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Transcription factor Oct3/4 is an indispensable factor in the self-renewal of ES cells. In this study, we searched for a protein that would interact with Oct3/4 in ES cells and identified an orphan nuclear hormone receptor, Dax1. The association of Dax1 with Oct3/4 was mediated through the POU-specific domain of Oct3/4. Ectopic expression of Dax1 inhibited Oct3/4-mediated activation of an artificial Oct3/4-responsive promoter. Expression of Dax1 in ES cells also reduced the activities of Nanog and Rex1 promoters, while knockdown of Dax1 increased these activities. Pulldown and gel shift assays revealed that the interaction of Dax1 with Oct3/4 abolished the DNA binding activity of Oct3/4. Chromatin immunoprecipitation assay results showed that Dax1 inhibited Oct3/4 binding to the promoter/enhancer regions of Oct3/4 and Nanog. Furthermore, overexpression of Dax1 resulted in ES cell differentiation. Taken together, these data suggest that Dax1, a novel molecule interacting with Oct3/4, functions as a negative regulator of Oct3/4 in ES cells.

A core paired-type and POU homeodomain-containing transcription factor program drives retinal bipolar cell gene expression.

The diversity of cell types found within the vertebrate CNS arises in part from action of complex transcriptional programs. In the retina, the programs driving diversification of various cell types have not been completely elucidated. To investigate gene regulatory networks that underlie formation and function of one retinal circuit component, the bipolar cell, transcriptional regulation of three bipolar cell-enriched genes was analyzed. Using in vivo retinal DNA transfection and reporter gene constructs, a 200 bp Grm6 enhancer sequence, a 445 bp Cabp5 promoter sequence, and a 164 bp Chx10 enhancer sequence, were defined, each driving reporter expression specifically in distinct but overlapping bipolar cell subtypes. Bioinformatic analysis of sequences revealed the presence of potential paired-type and POU homeodomain-containing transcription factor binding sites, which were shown to be critical for reporter expression through deletion studies. The paired-type homeodomain transcription factors (TFs) Crx and Otx2 and the POU homeodomain factor Brn2 are expressed in bipolar cells and interacted with the predicted binding sequences as assessed by electrophoretic mobility shift assay. Grm6, Cabp5, and Chx10 reporter activity was reduced in Otx2 loss-of-function retinas. Endogenous gene expression of bipolar cell molecular markers was also dependent on paired-type homeodomain-containing TFs, as assessed by RNA in situ hybridization and reverse transcription-PCR in mutant retinas. Cabp5 and Chx10 reporter expression was reduced in dominant-negative Brn2-transfected retinas. The paired-type and POU homeodomain-containing TFs Otx2 and Brn2 together appear to play a common role in regulating gene expression in retinal bipolar cells.

Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3.

BACKGROUND: The transcription factor STAT3 is a downstream target of the LIF signalling cascade. LIF signalling or activation is sufficient to maintain embryonic stem (ES) cells in an undifferentiated and pluripotent state. To further investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells. RESULTS: Transgenic STAT3-MER blastocysts yielded pluripotent germline-competent ES cells at a high frequency in the absence of LIF when established in tamoxifen-containing medium. Expression profiling of tamoxifen-induced transgenic FVB ES cell lines revealed a set of 26 genes that were markedly up- or down-regulated when compared with wild type cells. The expression of four of the up-regulated genes (Hexokinase II, Lefty2, Pramel7, PP1rs15B) was shown to be restricted to the inner cell mass (ICM) of the blastocysts. These differentially expressed genes represent potential candidates for the maintenance of pluripotency of ES cells. We finally overexpressed two candidate genes, Pem/Rhox5 and Pramel7, in ES cells and demonstrated that their overexpression is sufficient for the maintenance of expression of ES cell markers as well as of the typical morphology of pluripotent ES cells in absence of LIF. CONCLUSION: Overexpression of STAT3-MER in the inner cell mass of blastocyst facilitates the establishment of ES cells and induces the upregulation of potential candidate genes involved in the maintenance of pluripotency. Two of them, Pem/Rhox5 and Pramel7, when overexpressed in ES cells are able to maintain the embryonic stem cells in a pluripotent state in a LIF independent manner as STAT3 or Nanog.

Analysis of gene function in the retina.

The Retina is a good model system for studies of neural development and disease because of its simplicity and accessibility. To analyze gene function rapidly and conveniently, we developed an electroporation technique in mice and rats for use in vivo and in vitro. The efficiency of electroporation into the neonatal retina is quite good, and transgene expression persists for more than a month. With this technique, various types of DNA constructs, including RNA interference (RNAi) vectors, are readily introduced into the retina without DNA size limitation. In addition, more than two different DNA constructs can be introduced into the retina at once, with very high cotransfection efficiency. In vivo and in vitro electroporation will provide a powerful method to analyze the molecular mechanisms of retinal development and disease.

alpha-Aminoadipate induces progenitor cell properties of Müller glia in adult mice.

PURPOSE: Retinal Müller glia in higher vertebrates have been reported to possess progenitor cell properties and the ability to generate new neurons after injury. This study was conducted to determine the signals that can activate this dormant capacity of Müller glia in adult mice, by studying their behavior during glutamate stimulation. METHODS: Various concentrations of glutamate and its analogue alpha-aminoadipate, which specifically binds Müller glia, were injected subretinally in adult mice. Proliferating retinal cells were labeled by subretinal injection of 5'-bromo-2'-deoxyuridine (BrdU) followed by immunohistochemistry. Müller cell fates were analyzed in retinal sections by using double immunolabeling with primary antibodies against Müller and other retina-specific cell markers. The effects of glutamate and alpha-aminoadipate were also determined in purified Müller cell cultures. RESULTS: Although high levels of glutamate induce retinal damage, subtoxic levels of glutamate directly stimulate Müller glia to re-enter the cell cycle and induce neurogenesis in vivo and in purified Müller cell cultures. alpha-Aminoadipate, which selectively target glial cells, also induced expression of progenitor cell markers by Müller cells in vitro or stimulated Müller cell migration to the outer nuclear layer (ONL) and to differentiate into photoreceptors in vivo. CONCLUSIONS: Mature Müller glia in adult mice can be induced to dedifferentiate, migrate, and generate new retinal neurons and photoreceptor cells by alpha-aminoadipate or glutamate signaling. The results of this study suggest a novel potential strategy for treating retinal neurodegeneration, including retinitis pigmentosa and age-related macular degeneration, without transplanting exogenous cells.

STAT3 and Oct-3/4 control histone modification through induction of Eed in embryonic stem cells.

Mouse embryonic stem (ES) cells can self-renew in the presence of leukemia inhibitory factor (LIF). Several essential transcription factors have been identified for the self-renewal of mouse ES cells, including STAT3, Oct-3/4, and Nanog. The molecular mechanism of ES cell self-renewal, however, is not fully understood. In the present study, we identified Eed, a core component of Polycomb repressive complex 2, as a downstream molecule of STAT3 and Oct-3/4. Artificial activation of STAT3 resulted in increased expression of Eed, whereas expression of a dominant negative mutant of STAT3 or suppression of Oct-3/4 expression led to down-regulation of Eed. Reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays revealed that STAT3 and Oct-3/4 directly bind to the promoter region of Eed, suggesting that Eed is a common target molecule of STAT3 and Oct-3/4. We also found that suppression of STAT3, Oct-3/4, or Eed causes induction of differentiation-associated genes as well as loss of Lys(27)-trimethylated histone H3 at the promoter regions of the differentiation-associated genes. Suppression of STAT3 and Oct-3/4 also resulted in the absence of Eed at the promoter regions. These results suggest that STAT3 and Oct-3/4 maintain silencing of differentiation-associated genes through up-regulation of Eed in self-renewing ES cells.

Controlled expression of transgenes introduced by in vivo electroporation.

In vivo electroporation is a powerful technique for the introduction of genes into organisms. Temporal and spatial regulation of expression of introduced genes, or of RNAi, would further enhance the utility of this method. Here we demonstrate conditional regulation of gene expression from electroporated plasmids in the postnatal rat retina and the embryonic mouse brain. For temporal regulation, Cre/loxP-mediated inducible expression vectors were used in combination with a vector expressing a conditionally active form of Cre recombinase, which is activated by 4-hydroxytamoxifen. Onset of gene expression was regulated by the timing of 4-hydroxytamoxifen administration. For spatial regulation, transgenes were expressed by using promoters specific for rod photoreceptors, bipolar cells, amacrine cells, Müller glia or progenitor cells. Combinations of these constructs will facilitate a variety of experiments, including cell-type-specific gene misexpression, conditional RNAi, and fate mapping of progenitor and precursor cells.

Identification of Zfp-57 as a downstream molecule of STAT3 and Oct-3/4 in embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Transcription factor STAT3 is essential for the self-renewal of ES cells. In this study, we searched for downstream molecules of STAT3 in ES cells. Using DNA chip analysis, we obtained zinc finger protein (Zfp)-57. The expression of Zfp-57 was restricted to undifferentiated ES cells and activation of STAT3 led to expression of Zfp-57. We also found that forced expression of a dominant-negative mutant of STAT3 or repression of Oct-3/4 expression led to down-regulation of Zfp-57. Targeted disruption of Zfp-57 resulted in no gross phenotypical defects, including expression of undifferentiated-state-specific genes. These data suggest that Zfp-57 is a downstream molecule of STAT3 and Oct-3/4 in ES cells, although dispensable for their self-renewal.

Production of N-lauroylated G protein alpha-subunit in Sf9 insect cells: the type of N-acyl group of Galpha influences G protein-mediated signal transduction.

The alpha-subunit of rod photoreceptor G protein transducin (G(t1)alpha) is heterogeneously modified at the N-terminus by a mixture of acyl groups, laurate (C12:0), myristate (C14:0), and two unsaturated fatty acids (C14:1 and C14:2). Although the N-fatty acylation of G(t1)alpha plays important roles in protein-protein and protein-membrane interactions in light signaling, the biological significance of the heterogeneous acylation remains unclear due to the difficulty in isolating each G(t1)alpha isoform from the retinal rod cells. Here we found that G(t1)alpha/G(i1)alpha chimera (G(t/i)alpha) expressed in Sf9 cells is also heterogeneously modified by myristate (approximately 90%) and laurate (approximately 10%), raising the possibility that the N-acyl group of recombinant G(t/i)alpha may be manipulated by modifying culture media. In fact, addition of myristic acid to the medium decreased the relative content of lauroylated G(t/i)alpha to an undetectable level, whereas exogenously added lauric acid significantly increased the relative content of lauroylated G(t/i)alpha in a concentration-dependent manner. By culturing the G(t/i)alpha-virus infected Sf9 cells with fatty acids, we obtained four different preparations of N-acylated G(t/i)alpha, in which the relative abundance of lauroylated isoform was 0%, 20%, 33% and approximately 70%, respectively. Functional analysis of these proteins showed that an increase in the relative content of the lauroylated isoform remarkably slowed down the steady-state GTP hydrolysis rate of G(t/i)alpha; the steady-state GTPase activity of the lauroylated isoform was estimated to be one order of magnitude lower than that of the myristoylated isoform. These results suggest that the retinal G(t1)alpha is composed of isoforms having functionally heterogeneous signaling properties.

Cdh1-APC controls axonal growth and patterning in the mammalian brain.

The anaphase-promoting complex (APC) is highly expressed in postmitotic neurons, but its function in the nervous system was previously unknown. We report that the inhibition of Cdh1-APC in primary neurons specifically enhanced axonal growth. Cdh1 knockdown in cerebellar slice overlay assays and in the developing rat cerebellum in vivo revealed cell-autonomous abnormalities in layer-specific growth of granule neuron axons and parallel fiber patterning. Cdh1 RNA interference in neurons was also found to override the inhibitory influence of myelin on axonal growth. Thus, Cdh1-APC appears to play a role in regulating axonal growth and patterning in the developing brain that may also limit the growth of injured axons in the adult brain.

Involvement of Ras in extraembryonic endoderm differentiation of embryonic stem cells.

Embryonic stem (ES) cells, derived from the inner cell mass of blastocyst can differentiate into multiple cell lineages. In this study, we examined the possible involvement of Ras in ES cell differentiation. We found that Ras was activated upon formation of embryoid bodies (EBs), an initial step in ES cell differentiation. When expressed during EB differentiation, a dominant-negative mutant of Ras suppressed induction of marker genes for extraembryonic endoderm differentiation, including GATA-4, GATA-6, alpha-fetoprotein, and hepatocyte nuclear factor 3beta, while an activated mutant promoted their induction. Expression of a Ras mutant that selectively activates the Raf/MEK/Erk pathway also enhanced induction of extraembryonic endoderm markers, and treatment with a MEK inhibitor resulted in their decreased expression. In addition, Ras stimulated downregulation of Nanog, a suppressor of endoderm differentiation in ES cells. These data suggest that Ras activation during EB differentiation plays a crucial role in initiation of extraembryonic endoderm differentiation.

Electroporation and RNA interference in the rodent retina in vivo and in vitro.

The large number of candidate genes made available by comprehensive genome analysis requires that relatively rapid techniques for the study of function be developed. Here, we report a rapid and convenient electroporation method for both gain- and loss-of-function studies in vivo and in vitro in the rodent retina. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups was taken up by a significant fraction of exposed cells after several pulses of high voltage. With this technique, GFP expression vectors were efficiently transfected into retinal cells with little damage to the operated pups. Transfected GFP allowed clear visualization of cell morphologies, and the expression persisted for at least 50 days. DNA-based RNA interference vectors directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knockout mice. Reporter constructs carrying retinal cell type-specific promoters were readily introduced into the retina in vivo, where they exhibited the appropriate expression patterns. Plasmid DNA was also efficiently transfected into retinal explants in vitro by high-voltage pulses.