Blue B. Lake

Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain

Single-nucleus gene expression Identifying the genes expressed at the level of a single cell nucleus can better help us understand the human brain. Blue et al. developed a single-nuclei sequencing technique, which they applied to cells in classically defined Brodmann areas from a postmortem brain. Clustering of gene expression showed concordance with the area of origin and defining 16 neuronal subtypes. Both excitatory and inhibitory neuronal subtypes show regional variations that define distinct cortical areas and exhibit how gene expression clusters may distinguish between distinct cortical areas. This method opens the door to widespread sampling of the genes expressed in a diseased brain and other tissues of interest. Science, this issue p. 1586 Individual neurons have specific transcriptomic signatures and transcriptomic heterogeneity. The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain.

Strabismus regulates asymmetric cell divisions and cell fate determination in the mouse brain

The planar cell polarity (PCP) pathway organizes the cytoskeleton and polarizes cells within embryonic tissue. We investigate the relationship between PCP signaling and cell fate determination during asymmetric division of neural progenitors (NPs) in mouse embryos. The cortex of Lp/Lp (Loop-tail) mice deficient in the essential PCP mediator Vangl2, homologue of Drosophila melanogaster Strabismus (Stbm), revealed precocious differentiation of neural progenitors into early-born neurons at the expense of late-born neurons and glia. Although Lp/Lp NPs were easily maintained in vitro, they showed premature differentiation and loss of asymmetric distribution of Leu-Gly-Asn–enriched protein (LGN)/partner of inscuteable (Pins), a regulator of mitotic spindle orientation. Furthermore, we observed a decreased frequency in asymmetric distribution of the LGN target nuclear mitotic apparatus protein (NuMa) in Lp/Lp cortical progenitors in vivo. This was accompanied by an increase in the number of vertical cleavage planes typically associated with equal daughter cell identities. These findings suggest that Stbm/Vangl2 functions to maintain cortical progenitors and regulates mitotic spindle orientation during asymmetric divisions in the vertebrate brain.

Antisense Suppression of Pygopus2 Results in Growth Arrest of Epithelial Ovarian Cancer

Purpose: The Pygopus proteins are critical elements of the canonical Wnt/β-catenin transcriptional complex. In epithelial ovarian cancer, constitutively active Wnt signaling is restricted to one (endometrioid) tumor subtype. The purpose of this study was to determine the level of expression and growth requirements of human Pygopus2 (hPygo2) protein in epithelial ovarian cancer. Experimental Design: Expression and subcellular localization of hPygo2 was determined in epithelial ovarian cancer cell lines and tumors using Northern blot, immunoblot, and immunofluorescence. Immunohistochemistry was done on 125 archived patient epithelial ovarian cancer tumors representing all epithelial ovarian cancer subtypes. T-cell factor–dependent transcription levels were determined in epithelial ovarian cancer cells using TOPflash/FOPflash in vivo assays. Phosphorothioated antisense oligonucleotides were transfected into cell lines and growth assayed by cell counting, anchorage-independent colony formation on soft agar, and xenografting into severe combined immunodeficient mice. Results: All six epithelial ovarian cancer cell lines and 82% of the patient samples overexpressed nuclear hPygo2 compared with control cells and benign disease. Depletion of hPygo2 by antisense oligonucleotides in both Wnt-active (TOV-112D) and Wnt-inactive serous (OVCAR-3, SKOV-3) and clear cell (TOV-21G) carcinoma cell lines halted growth, assessed using tissue culture, anchorage-independent, and xenograft assays. Conclusions: hPygo2 is unexpectedly widely expressed in, and required in the absence of, Wnt signaling for malignant growth of epithelial ovarian cancer, the deadliest gynecologic malignancy. These findings strongly suggest that inhibition of hPygo2 may be of therapeutic benefit for treating this disease.

Role of Rab11 in planar cell polarity and apical constriction during vertebrate neural tube closure

Epithelial folding is a critical process underlying many morphogenetic events including vertebrate neural tube closure, however, its spatial regulation is largely unknown. Here we show that during neural tube formation Rab11-positive recycling endosomes acquire bilaterally symmetric distribution in the Xenopus neural plate, being enriched at medial apical cell junctions. This mediolateral polarization was under the control of planar cell polarity (PCP) signaling, was necessary for neural plate folding, and was accompanied by the polarization of the exocyst component Sec15. Our further experiments demonstrate that similar PCP-dependent polarization of Rab11 is essential for ectopic apical constriction driven by the actin-binding protein Shroom and during embryonic wound repair. We propose that anisotropic membrane trafficking plays key roles in diverse morphogenetic behaviors of individual cells and propagates in a tissue by a common mechanism that involves PCP.

Pygopus is required for embryonic brain patterning in Xenopus

We have identified two Xenopus mRNAs that encode proteins homologous to a component of the Wnt/beta-catenin transcriptional machinery known as Pygopus. The predicted proteins encoded by both mRNAs share the same structural properties with human Pygo-2, but with Xpygo-2alpha having an additional 21 N-terminal residues. Xpygo-2alpha messages accumulate in the prospective anterior neural plate after gastrulation and then are localized to the nervous system, rostral to and including the hindbrain. Xpygo-2beta mRNA is expressed in oocytes and early embryos but declines in level before and during gastrulation. In late neurula, Xpygo-2beta mRNA is restricted to the retinal field, including eye primordia and prospective forebrain. A C-terminal truncated mutant of Xpygo-2 containing the N-terminal Homology Domain (NHD) caused both axis duplication when injected at the 2-cell stage and inhibition of anterior neural development when injected in the prospective head, mimicking the previously described effects of Wnt-signaling activators. Inhibition of Xpygo-2alpha and Xpygo-2beta by injection of gene-specific antisense morpholino oligonucleotides into prospective anterior neurectoderm caused brain defects that were prevented by coinjection of Xpygo-2 mRNA. Both Xpygo-2alpha and Xpygo-2beta morpholinos reduced the eye and forebrain markers Xrx-1, Xpax-6, and XBF-1, while the Xpygo-2alpha morpholino also eliminated expression of the mid-hindbrain marker En-2. The differential expression and regulatory activities of Xpygo-2alpha/beta in rostral neural tissue indicate that they represent essential components of a novel mechanism for Wnt signaling in regionalization of the brain.

p53 induces skin aging by depleting Blimp1+ sebaceous gland cells

p53 is an important inducer of organismal aging. However, its roles in the aging of skin remain unclear. Here we show that mice with chronic activation of p53 develop an aging phenotype in the skin associated with a reduction of subcutaneous fat and loss of sebaceous gland (SG). The reduction in the fat layer may result from the decrease of mammalian TOR complex 1 (mTORC1) activity accompanied by elevated expression of energy expenditure genes, and possibly as compensatory effects, leading to the elevation of peroxisome proliferator-activated receptor (PPAR)γ, an inducer of sebocyte differentiation. In addition, Blimp1+ sebocytes become depleted concomitantly with an increase in cellular senescence, which can be reversed by PPARγ antagonist (BADGE) treatment. Therefore, our results indicate that p53-mediated aging of the skin involves not only thinning through the loss of subdermal fat, but also xerosis or drying of the skin through declining sebaceous gland activity.

Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain

Detailed characterization of the cell types in the human brain requires scalable experimental approaches to examine multiple aspects of the molecular state of individual cells, as well as computational integration of the data to produce unified cell-state annotations. Here we report improved high-throughput methods for single-nucleus droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive site sequencing (scTHS-seq). We used each method to acquire nuclear transcriptomic and DNA accessibility maps for >60,000 single cells from human adult visual cortex, frontal cortex, and cerebellum. Integration of these data revealed regulatory elements and transcription factors that underlie cell-type distinctions, providing a basis for the study of complex processes in the brain, such as genetic programs that coordinate adult remyelination. We also mapped disease-associated risk variants to specific cellular populations, which provided insights into normal and pathogenic cellular processes in the human brain. This integrative multi-omics approach permits more detailed single-cell interrogation of complex organs and tissues.

Rab11 regulates planar polarity and migratory behavior of multiciliated cells in Xenopus embryonic epidermis

Background: Xenopus embryonic skin is composed of the superficial layer with defined apicobasal polarity and the inner layer lacking the apical domain. Multiciliated cells (MCCs) originate in the inner layer of the epidermal ectoderm and subsequently migrate to the surface. How MCCs acquire the apicobasal polarity and intercalate into the superficial layer during neurulation is largely unknown. As Rab11‐dependent vesicle trafficking has been implicated in ciliary membrane assembly and in apical domain formation in epithelial cells, we assessed the involvement of Rab11 in MCC development. Results: Here we report that Rab11 is specifically enriched and becomes apically polarized in skin MCCs. Interference with Rab11 function by overexpression of a dominant negative mutant or injection of a specific morpholino oligonucleotide inhibited MCC intercalation into the superficial layer. Dominant negative Rab11‐expressing MCC precursors revealed intrinsic apicobasal polarity, characterized by the apical domain, which is not normally observed in inner layer cells. Despite the presence of the apical domain, the cells with inhibited Rab11 function were randomly oriented relative to the plane of the tissue, thereby demonstrating a defect in planar polarity. Conclusions: These results establish a requirement for Rab11 in MCC development and support a two‐step model, in which the initial polarization of MCC precursors is critical for their integration into the superficial cell layer. Developmental Dynamics 241:1385–1395, 2012.

Context-dependent enhancement of induced pluripotent stem cell reprogramming by silencing Puma.

Reprogramming of the somatic state to pluripotency can be induced by a defined set of transcription factors including Oct3/4, Sox2, Klf4, and c‐Myc [Cell 2006;126:663‐676]. These induced pluripotent stem cells (iPSCs) hold great promise in human therapy and disease modeling. However, tumor suppressive activities of p53, which are necessary to prevent persistence of DNA damage in mammalian cells, have proven a serious impediment to formation of iPSCs [Nat Methods 2011;8:409‐412]. We examined the requirement for downstream p53 activities in suppressing efficiency of reprogramming as well as preventing persistence of DNA damage into the early iPSCs. We discovered that the majority of the p53 activation occurred through early reprogramming‐induced DNA damage with the activated expression of the apoptotic inducer Puma and the cell cycle inhibitor p21. While Puma deficiency increases reprogramming efficiency only in the absence of c‐Myc, double deficiency of Puma and p21 has achieved a level of efficiency that exceeded that of p53 deficiency alone. We further demonstrated that, in both the presence and absence of p21, Puma deficiency was able to prevent any increase in persistent DNA damage in early iPSCs. This may be due to a compensatory cellular senescent response to reprogramming‐induced DNA damage in pre‐iPSCs. Therefore, our findings provide a potentially safe approach to enhance iPSC derivation by transiently silencing Puma and p21 without compromising genomic integrity. STEM CELLS 2012;30:888–897

A comparative strategy for single-nucleus and single-cell transcriptomes confirms accuracy in predicted cell-type expression from nuclear RNA

Significant heterogeneities in gene expression among individual cells are typically interrogated using single whole cell approaches. However, tissues that have highly interconnected processes, such as in the brain, present unique challenges. Single-nucleus RNA sequencing (SNS) has emerged as an alternative method of assessing a cell’s transcriptome through the use of isolated nuclei. However, studies directly comparing expression data between nuclei and whole cells are lacking. Here, we have characterized nuclear and whole cell transcriptomes in mouse single neurons and provided a normalization strategy to reduce method-specific differences related to the length of genic regions. We confirmed a high concordance between nuclear and whole cell transcriptomes in the expression of cell type and metabolic modeling markers, but less so for a subset of genes associated with mitochondrial respiration. Therefore, our results indicate that single-nucleus transcriptome sequencing provides an effective means to profile cell type expression dynamics in previously inaccessible tissues.