The PDE4 inhibitor apremilast modulates ethanol responses in Gabrb1-S409A knock-in mice via PKA-dependent and independent mechanisms.

We previously showed that the PDE4 inhibitor apremilast reduces ethanol consumption in mice by protein kinase A (PKA) and GABAergic mechanisms. Preventing PKA phosphorylation of GABA β3 subunits partially blocked apremilast-mediated decreases in drinking. Here, we produced Gabrb1-S409A mice to render GABA β1 subunits resistant to PKA-mediated phosphorylation.

Generation and Validation of Tissue-Specific Knockout Strains for Toxicology Research.

Tissue-specific knockout mice are widely used throughout scientific research. A principle method for generating tissue-specific knockout mice is the Cre-loxP system. Here, we give a detailed description of the steps required to generate and validate tissue-specific knockout mice using the Cre-loxP system.

SLC30A10 transporter in the digestive system regulates brain manganese under basal conditions while brain SLC30A10 protects against neurotoxicity.

The essential metal manganese becomes neurotoxic at elevated levels. Yet, the mechanisms by which brain manganese homeostasis is regulated are unclear. Loss-of-function mutations in SLC30A10, a cell surface-localized manganese efflux transporter in the brain and liver, induce familial manganese neurotoxicity.

Hypothyroidism induced by loss of the manganese efflux transporter SLC30A10 may be explained by reduced thyroxine production.

SLC30A10 and SLC39A14 are manganese efflux and influx transporters, respectively. Loss-of-function mutations in genes encoding either transporter induce hereditary manganese toxicity. Patients have elevated manganese in the blood and brain and develop neurotoxicity.

Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.

Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging.

Rfx2 is required for spermatogenesis in the mouse.

RFX transcription factors are key regulators of ciliogenesis in vertebrates. In Xenopus and zebrafish embryos, knockdown of Rfx2 causes defects in neural tube closure and in left-right axis patterning. To determine the essential role of the Rfx2 gene in mammalian development, we generated Rfx2-deficient mice using an embryonic stem cell clone containing a lacZ gene trap reporter inserted into the first intron of the Rfx2 gene.

Sfrp5 is not essential for axis formation in the mouse.

Secreted frizzled related protein (Sfrp) genes encode extracellular factors that can modulate Wnt signaling. During early post-implantation mouse development Sfrp5 is expressed in the anterior visceral endoderm (AVE) and the ventral foregut endoderm. The AVE is important in anterior-posterior axis formation and the ventral foregut endoderm contributes to multiple gut tissues.

Lim 1 is required for nephric duct extension and ureteric bud morphogenesis.

The nephric duct plays a central role in orchestrating the development of the mammalian urogenital system. Lim 1 is a homeobox gene required for head and urogenital development in the mouse but most Lim 1-deficient embryos die by embryonic day 10. To determine the role of Lim 1 in the development of the nephric duct, we conditionally removed Lim 1 in the nephric epithelium just after the nephric duct begins to form using a floxed allele of Lim 1 and Pax2-cre transgenic mice.

Ssdp1 regulates head morphogenesis of mouse embryos by activating the Lim1-Ldb1 complex.

The transcriptional activity of LIM-homeodomain (LIM-HD) proteins is regulated by their interactions with various factors that bind to the LIM domain. We show that reduced expression of single-stranded DNA-binding protein 1 (Ssdp1), which encodes a co-factor of LIM domain interacting protein 1 (Ldb1), in the mouse mutant headshrinker (hsk) disrupts anterior head development by partially mimicking Lim1 mutants. Although the anterior visceral endoderm and the anterior definitive endoderm, which together comprise the head organizer, were able to form normally in Ssdp1(hsk/hsk) mutants, development of the prechordal plate was compromised.

The mammalian twisted gastrulation gene functions in foregut and craniofacial development.

Extracellular modulators of cell-cell signaling control numerous aspects of organismal development. The Twisted gastrulation (Twsg1) gene product is a small, secreted cysteine-rich protein that has the unusual property of being able to either enhance or inhibit signaling by the bone morphogenetic protein (BMP) subfamily of TGF-beta type factors in a context-dependent manner. In this report, we characterize the early embryonic and skeletal phenotypes associated with loss of Twsg1 function in mice.

Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo.

Patterning of the mouse embryo along the anteroposterior axis during body plan development requires migration of the distal visceral endoderm (DVE) towards the future anterior side by a mechanism that has remained unknown. Here we show that Nodal signalling and the regionalization of its antagonists are required for normal migration of the DVE. Whereas Nodal signalling provides the driving force for DVE migration by stimulating the proliferation of visceral endoderm cells, the antagonists Lefty1 and Cerl determine the direction of migration by asymmetrically inhibiting Nodal activity on the future anterior side.

Requirement of Lim1 for female reproductive tract development.

Lim1 encodes a LIM-class homeodomain transcription factor that is essential for head and kidney development. In the developing urogenital system, Lim1 expression has been documented in the Wolffian (mesonephric) duct, the mesonephros, metanephros and fetal gonads. Using, a Lim1 lacZ knock-in allele in mice, we identified a previously unreported urogenital tissue for Lim1 expression, the epithelium of the developing Müllerian duct that gives rise to the oviduct, uterus and upper region of the vagina of the female reproductive tract.

The mouse secreted frizzled-related protein 5 gene is expressed in the anterior visceral endoderm and foregut endoderm during early post-implantation development.

The anterior visceral endoderm (AVE) plays an important role in anterior-posterior axis formation in the mouse. The AVE functions in part by expressing secreted factors that antagonize growth factor signaling in the proximal epiblast. Here we report that the Secreted frizzled-related protein 5 (Sfrp5) gene, which encodes a secreted factor that can antagonize Wnt signaling, is expressed in the AVE and foregut endoderm during early mouse development.

Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks.

The anterior visceral endoderm plays a pivotal role in establishing anterior-posterior polarity of the mouse embryo, but the molecular nature of the signals required remains to be determined. Here, we demonstrate that Cerberus-like(-/-);Lefty1(-/-) compound mutants can develop a primitive streak ectopically in the embryo. This defect is not rescued in chimeras containing wild-type embryonic, and Cerberus-like(-/-);Lefty1(-/-) extraembryonic, cells but is rescued in Cerberus-like(-/-); Lefty1(-/-) embryos after removal of one copy of the Nodal gene.

Lim1 activity is required for intermediate mesoderm differentiation in the mouse embryo.

During gastrulation and early organogenesis, Lim1 is expressed in the visceral endoderm, the anterior mesendoderm, and the lateral mesoderm that comprises the lateral plate and intermediate mesoderm. A previous study has reported that kidneys and gonads are missing in the Lim1 null mutants (W. Shawlot and R.

The cerberus-related gene, Cerr1, is not essential for mouse head formation.

The Xenopus cerberus gene encodes a secreted factor expressed in the Spemann organizer that can cause ectopic head formation when its mRNA is injected into Xenopus embryos. In mouse, the cerberus-related gene, Cerr1, is expressed in the anterior mesendoderm that underlies the presumptive anterior neural plate and its expression is downregulated in Lim1 headless embryos. To determine whether Cerr1 is required for head formation we generated a null mutation in Cerr1 by gene targeting in mouse embryonic stem cells.

Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation in the mouse.

Lim1 is a homeobox gene expressed in the extraembryonic anterior visceral endoderm and in primitive streak-derived tissues of early mouse embryos. Mice homozygous for a targeted mutation of Lim1 lack head structures anterior to rhombomere 3 in the hindbrain. To determine in which tissues Lim1 is required for head formation and its mode of action, we have generated chimeric mouse embryos and performed tissue layer recombination explant assays.

HNF3beta and Lim1 interact in the visceral endoderm to regulate primitive streak formation and anterior-posterior polarity in the mouse embryo.

Recent embryological and genetic experiments have suggested that the anterior visceral endoderm and the anterior primitive streak of the early mouse gastrula function as head- and trunk-organising centers, respectively. Here, we report that HNF3beta and Lim1 are coexpressed in both organising centers suggesting synergistic roles of these genes in regulating organiser functions and hence axis development in the mouse embryo. To investigate this possibility, we generated compound HNF3beta and Lim1 mutant embryos.

Sequence and genomic organization of the mouse Lim1 gene.

The sequence and genomic organization of the mouse Lim1 gene were determined. The mouse Lim1 gene has five coding exons. The Lim1 transcription initiation start site was determined by 5' RACE.

YAC rescue of downless locus mutations in mice.

Mice with mutations at the downless (dl) locus have defects in hair follicle, tooth, sweat gland, preputial gland, Meibomian gland, and tail development. The dl phenotype is analogous to the human genetic disorder termed autosomal hypohidrotic (or anhidrotic) ectodermal dysplasia (HED). On the basis of the identification of two related transgenic insertional mutations in the downless gene, yeast artificial chromosomes (YACs) were identified that map to the critical region of mouse Chromosome (Chr) 10.

Restricted beta-galactosidase expression of a hygromycin-lacZ gene targeted to the beta-actin locus and embryonic lethality of beta-actin mutant mice.

beta-actin is a cytoskeletal protein that is ubiquitously expressed. To exploit the regulation the beta-actin gene, a promoterless hygromycin-lacZ fusion gene with a splice acceptor was introduced into the first intron of the beta-actin locus by homologous recombination in mouse embryonic stem (ES) cells. The targeted ES cells were hygromycin resistant and expressed beta-galactosidase (beta-gal) activity.

Expression of the mouse cerberus-related gene, Cerr1, suggests a role in anterior neural induction and somitogenesis.

The Xenopus cerberus gene encodes a secreted factor that is expressed in the anterior endomesoderm of gastrula stage embryos and can induce the formation of ectopic heads when its mRNA is injected into Xenopus embryos [Bouwmeester, T., Kim, S., Lu, B.

Sequential roles for Otx2 in visceral endoderm and neuroectoderm for forebrain and midbrain induction and specification.

The homeobox gene Otx2 is a mouse cognate of the Drosophila orthodenticle gene, which is required for development of the brain, rostral to rhombomere three. We have investigated the mechanisms involved in this neural function and specifically the requirement for Otx2 in the visceral endoderm and the neuroectoderm using chimeric analysis in mice and explant recombination assay. Analyses of chimeric embryos composed of more than 90% of Otx2-/- ES cells identified an essential function for Otx2 in the visceral endoderm for induction of the forebrain and midbrain.