AFQ056, a new mGluR5 antagonist for treatment of fragile X syndrome.

Fragile X syndrome, the most common form of inherited intellectual disability, is caused by a lack of FMRP, which is the product of the Fmr1 gene. FMRP is an RNA-binding protein and a component of RNA-granules found in the dendrites of neurons. At the synapse, FMRP is involved in regulation of translation of specific target mRNAs upon stimulation of mGluR5 receptors. In this study, we test the effects of a new mGluR5 antagonist (AFQ056) on the prepulse inhibition of startle response in mice. We show that Fmr1 KO mice have a deficit in inhibition of the startle response after a prepulse and that AFQ056 can rescue this phenotype. We also studied the effect of AFQ056 on cultured Fmr1 KO hippocampal neurons; untreated neurons showed elongated spines and treatment resulted in shortened spines. These results suggest that AFQ056 might be a potent mGluR5 antagonist to rescue various aspects of the fragile X phenotype.

Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system.

The vertebrate urogenital system forms due to inductive interactions between the Wolffian duct, its derivative the ureteric bud, and their adjacent mesenchymes. These establish epithelial primordia within the mesonephric (embryonic) and metanephric (adult) kidneys and the Müllerian duct, the anlage of much of the female reproductive tract. We show that Wnt9b is expressed in the inductive epithelia and is essential for the development of mesonephric and metanephric tubules and caudal extension of the Müllerian duct. Wnt9b is required for the earliest inductive response in metanephric mesenchyme. Further, Wnt9b-expressing cells can functionally substitute for the ureteric bud in these interactions. Wnt9b acts upstream of another Wnt, Wnt4, in this process, and our data implicate canonical Wnt signaling as one of the major pathways in the organization of the mammalian urogenital system. Together these findings suggest that Wnt9b is a common organizing signal regulating diverse components of the mammalian urogenital system.

Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches.

To directly test the requirement for hedgehog signaling in the telencephalon from early neurogenesis, we examined conditional null alleles of both the Sonic hedgehog and Smoothened genes. While the removal of Shh signaling in these animals resulted in only minor patterning abnormalities, the number of neural progenitors in both the postnatal subventricular zone and hippocampus was dramatically reduced. In the subventricular zone, this was partially attributable to a marked increase in programmed cell death. Consistent with Hedgehog signaling being required for the maintenance of stem cell niches in the adult brain, progenitors from the subventricular zone of floxed Smo animals formed significantly fewer neurospheres. The loss of hedgehog signaling also resulted in abnormalities in the dentate gyrus and olfactory bulb. Furthermore, stimulation of the hedgehog pathway in the mature brain resulted in elevated proliferation in telencephalic progenitors. These results suggest that hedgehog signaling is required to maintain progenitor cells in the postnatal telencephalon.

Wnt3 signaling in the epiblast is required for proper orientation of the anteroposterior axis.

The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.

Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain.

We have generated a transgenic line that expresses the Cre gene product under the regulation of a 12.5 kb upstream regulatory sequence from the Sox2 gene. Using a R26R reporter line, we show that this transgenic line induces recombination in all epiblast cells by embryonic day (E) 6.5 but little or no activity in other extraembryonic cell types at this time. When crossed to a conditional allele of the Sonic hedgehog gene (Shhc), all Sox2Cre;Shhn/Shhc embryos displayed a phenotype indistinguishable from that of the Shh null mutant. Sox2Cre functioned more efficiently in epiblast-mediated recombination than the Mox2Cre (MORE) transgenic line, which has also been shown to drive Cre-mediated recombination exclusively in the embryonic component of the early mouse embryo. Although most MORE; shhh/shhc embryos have a shh hull phenotype, 33% displayed a milder skeletal phenotype, most likely result of incomplete recombination at egg cylinder stages. In agreement with these findings, Sox2Cre was active earlier and Sox2Cre-mediated recombination was more advanced than MORE-mediated recombination at early gastrulation stages. The Sox2Cre line is likely to be more effective in generating complete, epiblast-specific removal of gene activity, and the mosaic activity of the MORE line will be helpful in generating partial loss-of-function phenotypes in the embryo-proper.

Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain.

We have generated a transgenic line that expresses the Cre gene product under the regulation of a 12.5 kb upstream regulatory sequence from the Sox2 gene. Using a R26R reporter line, we show that this transgenic line induces recombination in all epiblast cells by embryonic day (E) 6.5 but little or no activity in other extraembryonic cell types at this time. When crossed to a conditional allele of the Sonic hedgehog gene (Shh(c)), all Sox2Cre;Shh(n)/Shh(c) embryos displayed a phenotype indistinguishable from that of the Shh null mutant. Sox2Cre functioned more efficiently in epiblast-mediated recombination than the Mox2Cre (MORE) transgenic line, which has also been shown to drive Cre-mediated recombination exclusively in the embryonic component of the early mouse embryo. Although most MORE; shh(h)/shh(c) embryos have a shh hull phenotype, 33% displayed a milder skeletal phenotype, most likely result of incomplete recombination at egg cylinder stages. In agreement with these findings, Sox2Cre was active earlier and Sox2Cre-mediated recombination was more advanced than MORE-mediated recombination at early gastrulation stages. The Sox2Cre line is likely to be more effective in generating complete, epiblast-specific removal of gene activity, and the mosaic activity of the MORE line will be helpful in generating partial loss-of-function phenotypes in the embryo-proper.

Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.

In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ER) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. In the current work, we have generated a transgenic mouse line in which Cre-ER is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration.

Maternal inheritance of Cre activity in a Sox2Cre deleter strain.

The Sox2 gene is expressed in several undifferentiated cell types. In an earlier study we described a Sox2Cre transgene that mediates epiblast-specific Cre-mediated modification of gene activity in the embryo. Here we report that this transgene is active in the female germline. Consequently, all offspring that arise from female mice heterozygous for the Sox2Cre transgene have demonstrable Cre activity irrespective of whether they inherit the transgene itself. Maternal inheritance of Cre activity allows the efficient modification of gene activity for functional analysis.

Cell fate decisions within the mouse organizer are governed by graded Nodal signals.

It is well known that cell fate decisions in the mouse organizer region during gastrulation ultimately govern gut formation and patterning, left-right axis determination, and development of the central nervous system. Previous studies suggest that signaling pathways activated by Nodal, bone morphogenetic protein (BMP), and Wnt ligands coordinately regulate patterning of the streak and the formation of midline organizing tissues, but the specific contributions of these molecules within discrete cell lineages are poorly defined. Here we removed Smad2 activity in the epiblast, using a conditional inactivation strategy. Abrogation of Smad2 does not compromise primitive streak (PS) formation or gastrulation movements, but rather results in a failure to correctly specify the anterior definitive endoderm (ADE) and prechordal plate (PCP) progenitors. To selectively lower Nodal activity in the posterior epiblast, we generated a novel allele lacking the proximal epiblast enhancer (PEE) governing Nodal expression in the PS. As for conditional inactivation of Smad2, germ-line deletion of the PEE selectively disrupts development of the anterior streak. In striking contrast, the node and its midline derivatives, the notochord and floor plate, develop normally in both categories of mutant embryos. Finally, we show that removal of one copy of Smad3 in the context of a Smad2-deficient epiblast results in a failure to specify all axial midline tissues. These findings conclusively demonstrate that graded Nodal/Smad2 signals govern allocation of the axial mesendoderm precursors that selectively give rise to the ADE and PCP mesoderm.

SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult.

Multipotent neural stem cells are present throughout the development of the central nervous system (CNS), persist into adulthood in defined locations and can be derived from more primitive embryonic stem cells. We show that SOX2, an HMG box transcription factor, is expressed in multipotent neural stem cells at all stages of mouse ontogeny. We have generated transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the endogenous locus-regulatory regions of the Sox2 gene to prospectively identify neural stem/progenitor cells in vivo and in vitro. Fluorescent cells coexpress SOX2 protein, and EGFP fluorescence is detected in proliferating neural progenitor cells of the entire anterior-posterior axis of the CNS from neural plate stages to adulthood. SOX2-EGFP cells can form neurospheres that can be passaged repeatedly and can differentiate into neurons, astrocytes and oligodendrocytes. Moreover, prospective clonal analysis of SOX2-EGFP-positive cells shows that all neurospheres, whether isolated from the embryonic CNS or the adult CNS, express SOX2-EGFP. In contrast, the pattern of SOX2-EGFP expression using randomly integrated Sox2 promoter/reporter construct differs, and neurospheres are heterogeneous for EGFP expression. These studies demonstrate that SOX2 may meet the requirements of a universal neural stem cell marker and provides a means to identify cells which fulfill the basic criteria of a stem cell: self-renewal and multipotent differentiation.