Progress Toward Zygotic and Germline Gene Drives in Mice.

CRISPR-based synthetic gene drives have the potential to deliver a more effective and humane method of invasive vertebrate pest control than current strategies. Relatively efficient CRISPR gene drive systems have been developed in insects and yeast but not in mammals. Here, we investigated the efficiency of CRISPR-Cas9-based gene drives in by constructing "split drive" systems where gRNA expression occurs on a separate chromosome to Cas9, which is under the control of either a zygotic (CAG) or germline (Vasa) promoter.

SOX3 promotes generation of committed spermatogonia in postnatal mouse testes.

SOX3 is a transcription factor expressed within the developing and adult nervous system where it mostly functions to help maintain neural precursors. Sox3 is also expressed in other locations, notably within the spermatogonial stem/progenitor cell population in postnatal testis. Independent studies have shown that Sox3 null mice exhibit a spermatogenic block as young adults, the mechanism of which remains poorly understood.

Functional screening of GATOR1 complex variants reveals a role for mTORC1 deregulation in FCD and focal epilepsy.

Mutations in the GAP activity toward RAGs 1 (GATOR1) complex genes (DEPDC5, NPRL2 and NPRL3) have been associated with focal epilepsy and focal cortical dysplasia (FCD). GATOR1 functions as an inhibitor of the mTORC1 signalling pathway, indicating that the downstream effects of mTORC1 deregulation underpin the disease. However, the vast majority of putative disease-causing variants have not been functionally assessed for mTORC1 repression activity.

Abnormal Cell Sorting Underlies the Unique X-Linked Inheritance of PCDH19 Epilepsy.

X-linked diseases typically exhibit more severe phenotypes in males than females. In contrast, protocadherin 19 (PCDH19) mutations cause epilepsy in heterozygous females but spare hemizygous males. The cellular mechanism responsible for this unique pattern of X-linked inheritance is unknown.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

Background: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients.

Comparison of KP1019 and NAMI-A in tumour-mimetic environments.

NAMI-A and KP1019 are Ru(III)-based anti-metastatic and cytotoxic anti-cancer drugs, respectively, and have been proposed to be activated by reduction to Ru(II). The potential reduction of NAMI-A and KP1019 in the hypoxic environment of a tumour model of neuroblastoma was examined. Normoxic, hypoxic and necrotic tumour tissues were modelled by multicellular spheroids of SH-SY5Y human neuroblastoma cells of various diameters (50-800 μm).

Pcdh19 Loss-of-Function Increases Neuronal Migration In Vitro but is Dispensable for Brain Development in Mice.

Protocadherin 19 (Pcdh19) is an X-linked gene belonging to the protocadherin superfamily, whose members are predominantly expressed in the central nervous system and have been implicated in cell-cell adhesion, axon guidance and dendrite self-avoidance. Heterozygous loss-of-function mutations in humans result in the childhood epilepsy disorder PCDH19 Girls Clustering Epilepsy (PCDH19 GCE) indicating that PCDH19 is required for brain development. However, understanding PCDH19 function in vivo has proven challenging and has not been studied in mammalian models.

Identification of an IGSF1-specific deletion in a five-generation pedigree with X-linked Central Hypothyroidism without macroorchidism.

Objectives: IGSF1 deficiency syndrome (IDS) is a recently described X-linked congenital central hypothyroidism disorder characterized by loss-of-function mutations in the immunoglobulin superfamily member 1 (IGSF1) gene. The phenotypic spectrum and intrafamilial variability associated with IDS remain unclear due to a paucity of large, well-characterized pedigrees. Here, we present phenotypic analysis and molecular characterization of a five-generation pedigree with IGSF1 deficiency containing 10 affected males.

Polyalanine expansions drive a shift into α-helical clusters without amyloid-fibril formation.

Polyglutamine (polyGln) expansions in nine human proteins result in neurological diseases and induce the proteins' tendency to form β-rich amyloid fibrils and intracellular deposits. Less well known are at least nine other human diseases caused by polyalanine (polyAla)-expansion mutations in different proteins. The mechanisms of how polyAla aggregates under physiological conditions remain unclear and controversial.

Regulation of pluripotent cell differentiation by a small molecule, staurosporine.

Research in the embryo and in culture has resulted in a sophisticated understanding of many regulators of pluripotent cell differentiation. As a consequence, protocols for the differentiation of pluripotent cells generally rely on a combination of exogenous growth factors and endogenous signalling. Little consideration has been given to manipulating other pathways to achieve pluripotent cell differentiation.

Molecular pathology of polyalanine expansion disorders: new perspectives from mouse models.

Disease-causing polyalanine (PA) expansion mutations have been identified in nine genes, eight of which encode transcription factors (TFs) with important roles in development. In vitro and cell overexpression studies have shown that expanded PA tracts result in protein misfolding and the formation of aggregates. This feature of PA proteins is reminiscent of the related polyglutamine (PQ) disease proteins, which have been shown to cause disease via a gain-of-function (GOF) mechanism.

Mutations in DEPDC5 cause familial focal epilepsy with variable foci.

The majority of epilepsies are focal in origin, with seizures emanating from one brain region. Although focal epilepsies often arise from structural brain lesions, many affected individuals have normal brain imaging. The etiology is unknown in the majority of individuals, although genetic factors are increasingly recognized.

A system to enrich for primitive streak-derivatives, definitive endoderm and mesoderm, from pluripotent cells in culture.

Two lineages of endoderm develop during mammalian embryogenesis, the primitive endoderm in the pre-implantation blastocyst and the definitive endoderm at gastrulation. This complexity of endoderm cell populations is mirrored during pluripotent cell differentiation in vitro and has hindered the identification and purification of the definitive endoderm for use as a substrate for further differentiation. The aggregation and differentiation of early primitive ectoderm-like (EPL) cells, resulting in the formation of EPL-cell derived embryoid bodies (EPLEBs), is a model of gastrulation that progresses through the sequential formation of primitive streak-like intermediates to nascent mesoderm and more differentiated mesoderm populations.

PRRT2 mutations cause benign familial infantile epilepsy and infantile convulsions with choreoathetosis syndrome.

Benign familial infantile epilepsy (BFIE) is a self-limited seizure disorder that occurs in infancy and has autosomal-dominant inheritance. We have identified heterozygous mutations in PRRT2, which encodes proline-rich transmembrane protein 2, in 14 of 17 families (82%) affected by BFIE, indicating that PRRT2 mutations are the most frequent cause of this disorder. We also report PRRT2 mutations in five of six (83%) families affected by infantile convulsions and choreoathetosis (ICCA) syndrome, a familial syndrome in which infantile seizures and an adolescent-onset movement disorder, paroxysmal kinesigenic choreoathetosis (PKC), co-occur.

Response to BMP4 signalling during ES cell differentiation defines intermediates of the ectoderm lineage.

The formation and differentiation of multipotent precursors underlies the generation of cell diversity during mammalian development. Recognition and analysis of these transient cell populations has been hampered by technical difficulties in accessing them in vivo. In vitro model systems, based on the differentiation of embryonic stem (ES) cells, provide an alternative means of identifying and characterizing these populations.

A novel role for gamma-secretase in the formation of primitive streak-like intermediates from ES cells in culture.

gamma-Secretase is a membrane-associated protease with multiple intracellular targets, a number of which have been shown to influence embryonic development and embryonic stem (ES) cell differentiation. This paper describes the use of the gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) to evaluate the role of gamma-secretase in the differentiation of pluripotent stem cells to the germ lineages. The addition of DAPT did not prevent the formation of primitive ectoderm-like cells from ES cells in culture.

Manipulation of cell:cell contacts and mesoderm suppressing activity direct lineage choice from pluripotent primitive ectoderm-like cells in culture.

In the mammal, the pluripotent cells of embryo differentiate and commit to either the mesoderm/endoderm lineages or the ectoderm lineage during gastrulation. In culture, the ability to direct lineage choice from pluripotent cells into the mesoderm/endoderm or ectoderm lineages will enable the development of technologies for the formation of highly enriched or homogenous populations of cells. Here we show that manipulation of cell:cell contact and a mesoderm suppressing activity in culture affects the outcome of pluripotent cell differentiation and when both variables are manipulated appropriately they can direct differentiation to either the mesoderm or ectoderm lineage.