Early dorsomedial tissue interactions regulate gyrification of distal neocortex.

The extent of neocortical gyrification is an important determinant of a species' cognitive abilities, yet the mechanisms regulating cortical gyrification are poorly understood. We uncover long-range regulation of this process originating at the telencephalic dorsal midline, where levels of secreted Bmps are maintained by factors in both the neuroepithelium and the overlying mesenchyme. In the mouse, the combined loss of transcription factors Lmx1a and Lmx1b, selectively expressed in the midline neuroepithelium and the mesenchyme respectively, causes dorsal midline Bmp signaling to drop at early neural tube stages. This alters the spatial and temporal Wnt signaling profile of the dorsal midline cortical hem, which in turn causes gyrification of the distal neocortex. Our study uncovers early mesenchymal-neuroepithelial interactions that have long-range effects on neocortical gyrification and shows that lissencephaly in mice is actively maintained via redundant genetic regulation of dorsal midline development and signaling.

Roof Plate-Derived Radial Glial-like Cells Support Developmental Growth of Rapidly Adapting Mechanoreceptor Ascending Axons.

Spinal cord longitudinal axons comprise some of the longest axons in our body. However, mechanisms that drive this extra long-distance axonal growth are largely unclear. We found that ascending axons of rapidly adapting (RA) mechanoreceptors closely abut a previously undescribed population of roof plate-derived radial glial-like cells (RGLCs) in the spinal cord dorsal column, which form a network of processes enriched with growth-promoting factors. In dreher mutant mice that lack RGLCs, the lengths of ascending RA mechanoreceptor axon branches are specifically reduced, whereas their descending and collateral branches, and other dorsal column and sensory pathways, are largely unaffected. Because the number and intrinsic growth ability of RA mechanoreceptors are normal in dreher mice, our data suggest that RGLCs provide critical non-cell autonomous growth support for the ascending axons of RA mechanoreceptors. Together, our work identifies a developmental mechanism specifically required for long-range spinal cord longitudinal axons.

A Designed Inhibitor of p53 Aggregation Rescues p53 Tumor Suppression in Ovarian Carcinomas.

Half of all human cancers lose p53 function by missense mutations, with an unknown fraction of these containing p53 in a self-aggregated amyloid-like state. Here we show that a cell-penetrating peptide, ReACp53, designed to inhibit p53 amyloid formation, rescues p53 function in cancer cell lines and in organoids derived from high-grade serous ovarian carcinomas (HGSOC), an aggressive cancer characterized by ubiquitous p53 mutations. Rescued p53 behaves similarly to its wild-type counterpart in regulating target genes, reducing cell proliferation and increasing cell death. Intraperitoneal administration decreases tumor proliferation and shrinks xenografts in vivo. Our data show the effectiveness of targeting a specific aggregation defect of p53 and its potential applicability to HGSOCs.

ETV2 expression increases the efficiency of primitive endothelial cell derivation from human embryonic stem cells.

BACKGROUND: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal. FINDINGS: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression. CONCLUSIONS: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.

Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2.

Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.

Hydrodynamic stretching of single cells for large population mechanical phenotyping.

Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.

Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells.

The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright) cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright) iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright) iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells.

Pluripotent stem cells hold significant promise in regenerative medicine due to their unlimited capacity for self-renewal and potential to differentiate into any cell type of the body. In this study, we demonstrate that proper mitochondrial function is essential for proliferation of undifferentiated ESCs. Attenuating mitochondrial function under self-renewing conditions makes these cells more glycolytic-dependent, and it is associated with an increase in the mRNA reserves of Nanog, Oct4, and Sox2. In contrast, attenuating mitochondrial function during the first 7 days of differentiation results in normal repression of Oct4, Nanog, and Sox2. However, differentiation potential is compromised as revealed by abnormal transcription of multiple Hox genes. Furthermore, under differentiating conditions in which mitochondrial function is attenuated, tumorigenic cells continue to persist. Our results, therefore establish the importance of normal mitochondrial function in ESC proliferation, regulating differentiation, and preventing the emergence of tumorigenic cells during the process of differentiation.

Loss of Pten causes tumor initiation following differentiation of murine pluripotent stem cells due to failed repression of Nanog.

Pluripotent stem cells (PSCs) hold significant promise in regenerative medicine due to their unlimited capacity for self-renewal and potential to differentiate into every cell type in the body. One major barrier to the use of PSCs is their potential risk for tumor initiation following differentiation and transplantation in vivo. In the current study we sought to evaluate the role of the tumor suppressor Pten in murine PSC neoplastic progression. Using eight functional assays that have previously been used to indicate PSC adaptation or transformation, Pten null embryonic stem cells (ESCs) failed to rate as significant in five of them. Instead, our data demonstrate that the loss of Pten causes the emergence of a small number of aggressive, teratoma-initiating embryonic carcinoma cells (ECCs) during differentiation in vitro, while the remaining 90-95% of differentiated cells are non-tumorigenic. Furthermore, our data show that the mechanism by which Pten null ECCs emerge in vitro and cause tumors in vivo is through increased survival and self-renewal, due to failed repression of the transcription factor Nanog.

Lmx1a regulates fates and location of cells originating from the cerebellar rhombic lip and telencephalic cortical hem.

The cerebellar rhombic lip and telencephalic cortical hem are dorsally located germinal zones which contribute substantially to neuronal diversity in the CNS, but the mechanisms that drive neurogenesis within these zones are ill defined. Using genetic fate mapping in wild-type and Lmx1a(-/-) mice, we demonstrate that Lmx1a is a critical regulator of cell-fate decisions within both these germinal zones. In the developing cerebellum, Lmx1a is expressed in the roof plate, where it is required to segregate the roof plate lineage from neuronal rhombic lip derivatives. In addition, Lmx1a is expressed in a subset of rhombic lip progenitors which produce granule cells that are predominantly restricted to the cerebellar posterior vermis. In the absence of Lmx1a, these cells precociously exit the rhombic lip and overmigrate into the anterior vermis. This overmigration is associated with premature regression of the rhombic lip and posterior vermis hypoplasia in Lmx1a(-/-) mice. These data reveal molecular organization of the cerebellar rhombic lip and introduce Lmx1a as an important regulator of rhombic lip cell-fate decisions, which are critical for maintenance of the entire rhombic lip and normal cerebellar morphogenesis. In the developing telencephalon Lmx1a is expressed in the cortical hem, and in its absence cortical hem progenitors contribute excessively to the adjacent hippocampus instead of producing Cajal-Retzius neurons. Thus, Lmx1a activity is critical for proper production of cells originating from both the cerebellar rhombic lip and the telencephalic cortical hem.

Overlapping function of Lmx1a and Lmx1b in anterior hindbrain roof plate formation and cerebellar growth.

The roof plate is an organizing center in the dorsal CNS that controls specification and differentiation of adjacent neurons through secretion of the BMP and WNT signaling molecules. Lmx1a, a member of the LIM-homeodomain (LIM-HD) transcription factor family, is expressed in the roof plate and its progenitors at all axial levels of the CNS and is necessary and sufficient for roof plate formation in the spinal cord. In the anterior CNS, however, a residual roof plate develops in the absence of Lmx1a. Lmx1b, another member of the LIM-HD transcription factor family which is highly related to Lmx1a, is expressed in the roof plate in the anterior CNS. Although Lmx1b-null mice do not show a substantial deficiency in hindbrain roof plate formation, Lmx1a/Lmx1b compound-null mutants fail to generate hindbrain roof plate. This observation indicates that both genes act in concert to direct normal hindbrain roof plate formation. Since the requirement of Lmx1b function for normal isthmic organizer at the mid-hindbrain boundary complicates analysis of a distinct dorsal patterning role of this gene, we also used a conditional knock-out strategy to specifically delete dorsal midline Lmx1b expression. Phenotypic analysis of single and compound conditional mutants confirmed overlapping roles for Lmx1 genes in regulating hindbrain roof plate formation and growth and also revealed roles in regulating adjacent cerebellar morphogenesis. Our data provides the first evidence of overlapping function of the Lmx1 genes during embryonic CNS development.

The roof plate regulates cerebellar cell-type specification and proliferation.

During embryogenesis, the isthmic organizer, a well-described signaling center at the junction of the mid-hindbrain, establishes the cerebellar territory along the anterior/posterior axis of the neural tube. Mechanisms specifying distinct populations within the early cerebellar anlage are less defined. Using a newly developed gene expression map of the early cerebellar anlage, we demonstrate that secreted signals from the rhombomere 1 roof plate are both necessary and sufficient for specification of the adjacent cerebellar rhombic lip and its derivative fates. Surprisingly, we show that the roof plate is not absolutely required for initial specification of more distal cerebellar cell fates, but rather regulates progenitor proliferation and cell position within the cerebellar anlage. Thus, in addition to the isthmus, the roof plate represents an important signaling center controlling multiple aspects of cerebellar patterning.