A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice

Tuberous sclerosis (TS) is characterized by the development of hamartomas in various organs and is caused by a germ-line mutation in either TSC1 or TSC2 tumor suppressor genes. From the symptomatic resemblance among TS patients, involvement of TSC1 and TSC2 products in a common pathway has been suggested. Here, to analyze the function of the Tsc1 product, we established a line of Tsc1 (TSC1 homologue) knockout mouse by gene targeting. Heterozygous Tsc1 mutant (Tsc1+/−) mice developed renal and extra-renal tumors such as hepatic hemangiomas. In these tumors, loss of wild-type Tsc1 allele was observed. Homozygous Tsc1 mutants died around embryonic days 10.5–11.5, frequently associated with neural tube unclosure. As a whole, phenotypes of Tsc1 knockout mice resembled those of Tsc2 knockout mice previously reported, suggesting that the presumptive common pathway for Tsc1 and Tsc2 products may also exist in mice. Notably, however, development of renal tumors in Tsc1+/− mice was apparently slower than that in Tsc2+/− mice. The Tsc1 knockout mouse described here will be a useful model to elucidate the function of Tsc1 and Tsc2 products as well as pathogenesis of TS.

Combinations of closely situated cis-acting elements determine tissue-specific patterns and anterior extent of early Hoxc8 expression.

We have used a transgene mutation approach to study how expression domains of Hoxc8 are established during mouse embryogenesis. A cis-regulatory region located 3 kb upstream from the Hoxc8 translational start site directs the early phase of expression. Four elements, termed A, B, C, and D, were previously shown to direct expression to the neural tube. Here we report that a fifth element, E, located immediately downstream of D directs expression to mesoderm in combination with the other four elements. These elements are interdependent and partially redundant. Different combinations of elements determine expression in different posterior regions of the embryo. Neural tube expression is determined minimally by ABC, ABD, or ACD; somite expression by ACDE; and lateral plate mesoderm expression by DE. Neural tube and lateral plate mesoderm enhancers can be separated, but independent somite expression has not been achieved. Furthermore, mutations within these elements result in posteriorization of the reporter gene expression. Thus, the anterior extent of expression is determined by the combined action of these elements. We propose that the early phase of Hoxc8 expression is directed by two separate mechanisms: one that determines tissue specificity and another that determines anterior extent of expression.

Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis.

Presomitic and 3- to 12-somite pair cultured mouse embryos were deprived of retinoic acid (RA) by yolk-sac injections of antisense oligodeoxynucleotides for retinol binding protein (RBP). Inhibition of yolk-sac RBP synthesis was verified by immunohistochemistry, and the loss of activity of a lacZ-coupled RA-sensitive promoter demonstrated that embryos rapidly became RA-deficient. This deficiency resulted in malformations of the vitelline vessels, cranial neural tube, and eye, depending upon the stage of embryonic development at the time of antisense injection. Addition of RA to the culture medium at the time of antisense injection restored normal development implicating the role of RBP in embryonic RA synthesis. Furthermore, the induced RA deficiency resulted in early down-regulation of developmentally important genes including TGF-beta1 and Shh.

A long-range regulatory element of Hoxc8 identified by using the pClasper vector.

Hox genes are located in highly conserved clusters. The significance of this organization is unclear, but one possibility is that regulatory regions for individual genes are dispersed throughout the cluster and shared with other Hox genes. This hypothesis is supported by studies on several Hox genes in which even large genomic regions immediately surrounding the gene fail to direct the complete expression pattern in transgenic mice. In particular, previous studies have identified proximal regulatory regions that are primarily responsible for early phases of mouse Hoxc8 expression. To locate additional regulatory regions governing expression during the later periods of development, a yeast homologous recombination-based strategy utilizing the pClasper vector was employed. Using homologous recombination into pClasper, we cloned a 27-kb region around the Hoxc8 gene from a yeast artificial chromosome. A reporter gene was introduced into the coding region of the isolated gene by homologous recombination in yeast. This large fragment recapitulates critical aspects of Hoxc8 expression in transgenic mice. We show that the regulatory elements that maintain the anterior boundaries of expression in the neural tube and paraxial mesoderm are located between 11 and 19 kb downstream of the gene.

Reproduction and sera embryotoxicity after immunization of monkeys with the laminin peptides YIGSR, RGD, and IKVAV.

Monkeys with excellent reproductive histories were immunized with the laminin peptides YIGSR, RGD, IKVAV, and YD, a control sequence with no known biological function. Sera from the YIGSR-immunized monkey became toxic, causing neural tube defects in whole rat embryo cultures, and this monkey experienced fetal loss after immunization. Sera from the RGD-immunized monkey also became embryotoxic in culture after immunization, but this monkey appeared to become infertile as she failed to initiate a pregnancy for at least 2 years after immunization. In contrast, embryos cultured on sera from the IKVAV- or YD-immunized monkeys were predominantly normal and both monkeys completed successful pregnancies. Antibody levels to the respective peptides or to laminin were not predictive of embryotoxicity, but antibody binding to homogenized yolk sacs as well as to yolk sacs of cultured embryos was associated with sera embryotoxicity and reproductive outcomes in vivo. These observations suggested that the laminin sequences YIGSR and RGD may play a role in immune-mediated reproductive failure by reacting directly with embryonic tissue and could provide a basis for identifying individuals at risk for both spontaneous abortion and infertility.

Critical relationship between sodium valproate dose and human teratogenicity: results of the Australian register of anti-epileptic drugs in pregnancy

To compare the incidence of foetal malformations (FMs) in pregnant women with epilepsy treated with different anti-epileptic drugs (AED) and doses, and the influence of seizures, family and personal history, and environmental factors. A prospective, observational, community-based cohort study. Methods. A voluntary, Australia-wide, telephone-interview-based register prospectively enrolling three groups of pregnant women: taking AEDs for epilepsy; with epilepsy not taking AEDs; taking AEDs for a non-epileptic indication. Four hundred and fifty eligible women were enrolled over 40 months. Three hundred and ninety six pregnancies had been completed, with 7 sets of twins, for a total of 403 pregnancy outcomes. Results. 354 (87.8%) pregnancy outcomes resulted in a healthy live birth, 26 (6.5%) had a FM, 4 (1%) a death in utero, 1 (0.2%) a premature labour with stillbirth, 14 (3.5%) a spontaneous abortion and 4 lost to follow-up. The FM rate was greater in pregnancies exposed to sodium valproate (VPA) in the first trimester (116.0%) compared with those exposed to all other AEDs (16.0% vs. 2.4%, P < 0.01) or no AEDs (16.0% vs. 3.1 %, P < 0.01). The mean daily dose of VPA taken in pregnancy with FMs was significantly greater than in those without (11975 vs: 1128 mg, P < 0.01). The incidence of FM with VPA doses greater than or equal to 1100 mg was 30.2% vs. 3.2% with doses < 1100 mg (P < 0.01). Conclusions. There is a dose-effect relationship for FM and exposure to VPA during the first trimester of pregnancy, with higher doses of VPA associated with a significantly greater risk than with lower doses or with other AEDs. These results highlight the need to limit, where possible, the dose of VPA in pregnancy. (C) 2004 Elsevier Ltd. All rights reserved.

BOC, brother of CDO, is a dorsoventral axon-guidance molecule in the embryonic vertebrate brain

The early axon scaffolding in the embryonic vertebrate brain consists of a series of ventrally projecting axon tracts that grow into a single major longitudinal pathway connected across the midline by commissures. We have investigated the role of Brother of CDO (BOC), an immunoglobulin (Ig) superfamily member distantly related to the Roundabout (Robo) family of axon-guidance receptors, in the development of this embryonic template of axon tracts in the zebrafish brain. A zebrafish homologue of BOC was isolated and shown to be expressed predominantly in the developing neural plate and later in the neural tube and developing brain. Zebrafish boc was initially highly localized to discrete bands in the mid- and hindbrain, but, as the major brain subdivisions emerged, it became more evenly expressed along the rostrocaudal axis, particularly in dorsal regions. The function of zebrafish boc was examined by a loss-of-function approach. Analysis of embryos injected with antisense morpholinos designed against boc revealed highly selective defects in the development of dorsoventrally projecting axon tracts. Loss of boc caused ventrally projecting axons, particularly those arising from the presumptive telencephalon, to follow aberrant trajectories. These data indicate that boc is an axon-guidance molecule playing a fundamental role in pathfinding during the early patterning of the axon scaffold in the embryonic vertebrate brain. (c) 2005 Wiley-Liss, Inc.

Folate: Methods of analysis

Folates and its derivatives occur as polyglutamates in nature. The multiplicity of forms and the generally low levels in foods makes quantitative analysis of folate a difficult task. The assay of folates from foods generally involves three steps: liberation of folates from the cellular matrix; deconjugation from the polyglutamate to the mono and di-glutamate forms; and the detection of the biological activity or chemical concentration of the resulting folates. The detection methods used are the microbiological assay relying on the turbidimetric bacterial growth of Lactobacillus rhamnosus which is by far the most commonly used method; the HPLC and LC/MS techniques and bio-specific procedures. This review attempts to describe the methods along with the merits and demerits of using each of these methods.

Foetal malformations and seizure control: 52 months data of the Australian Pregnancy Registry

The Australian Pregnancy Registry, affiliated European Register of Antiepileptic drugs in Pregnancy (EURAP), recruits informed consenting women with epilepsy on treatment with antiepileptic drugs (AEDs), those untreated, and women on AEDs for other indications. Enrolment is considered prospective if it has occurred before presence or absence of major foetal malformations (FMs) are known, or retrospective, if they had occurred after the birth of infant or detection of major FM. Telephone Interviews are conducted to ascertain pregnancy outcome and collect data about seizures. To date 630 women have been enrolled, with 565 known pregnancy outcomes. Valproate (VPA) above 1100 mg/day was associated with a significantly higher incidence of FMs than other AEDs (P < 0.05). This was independent of other AED use or potentially confounding factors on multivariate analysis (OR = 7.3, P < 0.0001). Lamotrigine (LTG) monotherapy (n = 65), has so far been free of malformations. Although seizure control was not a primary outcome, we noted that more patients on LTG than on VPA required dose adjustments to control seizures. Data indicate an increased risk of FM in women taking VPA in doses > 1100 mg/day compared with other AEDs. The choice of AED for pregnant women with epilepsy requires assessment of balance of risks between teratogenicity and seizure control.

Mitochondrial dysmorphology in the neuroepithelium of rat embryos following a single dose of maternal hyperthermia during gestation

Hyperthermia is teratogenic to human and animal embryos and induces mainly anomalies of the nervous system. However, the teratogenic mechanism is poorly understood. Mammalian embryos are known to switch from anaerobic to aerobic metabolism around the time of neural tube closure. This critical event might be sensitive to hyperthermia. The objective of the present study was to evaluate the ultrastructural changes of the mitochondria of the neuroepithelium (NE) of rat embryos following maternal exposure to hyperthermia. Pregnant rats were heat stressed for an hour on gestation day (GD) 9 and embryos were examined by electron microscopy on GD 10. NE presented extensive apoptosis. Intercellular junctions were weakened and copious cellular debris projected into the ventricle. The mitochondria were of diverse size and shape. Most of them were swollen and had short cristae and electron dense matrix. Hydropic changes were also observed in numerous mitochondria. Lipid-laden mitochondria were found in the apical portions of neuroblasts. The mesenchyme (ME) of heat-treated embryos showed paucity of cells and only as frequent apoptosis as the controls. Their mitochondria also showed changes similar to those of the NE. Additionally extensive lipid accumulation was observed in and in the vicinity of mitochondria, often surrounded by short strands of endoplasmic reticulum. Whereas mitochondrial pathology was associated with profound apoptosis in the NE, growth restriction and lipid accumulation accompanied mitochondrial changes in the ME. The results of this study indicate that the embryonic response to maternal heat shock is tissue-specific and morphologically distinct in this species.

Neogenin: A multi-functional receptor regulating diverse developmental processes

Neogenin, a close relative of the axon guidance receptor Deleted in Colorectal Cancer (DCC), has been shown to be a receptor for members of the Netrin and Repulsive Guidance Molecule (RGM) families. While Netrin-l-Neogenin interactions result in a chernoattractive axon guidance response, the interaction between Neogenin and RGMa induces a chemorepulsive response. Evidence is now accumulating that Neogenin is a multi-functional receptor regulating many diverse developmental processes, including neural tube and mammary gland formation, myogenesis and angiogenesis. Little is known of the function of Neogenin in the adult, however, a novel role in the regulation of iron homeostasis is now emerging. While the signal transduction pathways activated by Neogenin are poorly understood, it is clear that the functional outcome of Neogenin activation, at least in the embryo, depends on both the developmental context as well as the nature of the ligand. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular control of cell type diversity in the developing spinal cord

1, During embryonic development, a diverse array of neurons and glia are generated at specific positions along the dorsoventral and rostro-caudal axes of the spinal cord from a common pool of precursor cells. 2. This cell type diversity can be distinguished by the spatially and temporally coordinated expression of several transcription factors that are also linked to cell type specification at a very early stage of spinal cord development. 3, Recent studies have started to uncover that the generation of cell type diversity in the developing spinal cord. Moreover, distinct cell types in the spinal cord appear to be determined by the spatially and temporally coordinated expression of transcription factors. 4. The expression of these factors also appears to be controlled by gradients of factors expressed by ventral and dorsal midline cells, namely Sonic hedgehog and members of the transforming growth factor-beta family. 5, Changes in the competence of precursor cells and local cell interactions may also play important roles in cell type specification within the developing spinal cord.

Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model

Endothelin 3 (Edn3) is a ligand important to developing neural crest cells (NCC). Some NCC eventually migrate into the skin and give rise to the pigment-forming melanocytes found in hair follicles. Edn3's effects on NCC have been largely explored through spontaneous mutants and cell culture experiments. These studies have shown the Endothelin receptor B/Edn3 signaling pathway to be important in the proliferation/survival and differentiation of developing melanocytes. To supplement these investigations I have created doxycycline-responsive transgenic mice which conditionally over-express Edn3. These mice will help us clarify Edn3's role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. The transgene mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal keratinocytes of the skin. Relative to littermate controls, transgenics develop increased pigmentation on most areas of the skin. My doxycycline-based temporal studies have shown that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of my Edn3 transgenic mice may offer some insight into the genetics behind benign dermal pigmentation and offer clues about the time periods important in establishing these conditions. This apparently abnormal development is echoed in a benign condition of human skin. Cases of dermal melanocytosis, such as common freckles, Mongolian spotting, and nevus of Ito demonstrate histological and etiological characteristics similar to those of the transgenic mice generated in this study.

Melanocytes in the developing and adult atrioventricular valves of the murine heart

The Neural Crest (NC) is a multipotential group of cells that arises from the dorsal aspect of the neural tube early in development. It is well established that a group of NC cells named Cardiac Neural Crest (CNC) migrates to the heart and plays a critical role in the remodeling of the aortic arch arteries and septation of the outflow tract. In this study, using the mouse mutant Pax3sp/sp that has CNC deficits I have identified a putative novel role for the CNC in regulating apoptosis in the atrioventricular (AV) endocardial cushion. The AV endocardial cushion undergoes remodeling to give rise to the cardiac AV valves. Using a transgenic mouse that carries the LacZ reporter gene under the control of the Dopachrome tautomerase promoter (Dct-LacZ), I found that another NC derived population, melanocyte precursors, also contribute to the AV endocardial cushion and developing AV valves. The analysis of Dct-LacZ embryos at different stages showed that NC cells already committed to the melanocytic fate migrate to the heart along the same initial pathway taken by those that will populate the skin. Hypopigmented mice carrying mutations in the Kit and Endothelin receptor b genes, that are critical for the proper development of skin melanocytes, do not have cardiac melanocytes indicating that cardiac and skin melanocyte precursors share the same initial signaling requirements. The analysis of murine adult hearts showed that melanocytes are mostly found in the atrial sides of the tricuspid and mitral valve leaflets. The distribution of melanocytes in the AV valves corresponds exactly to areas of high Versican B expression, a proteoglycan essential for the process of AV valve remodeling. To evaluate a potential role for melanocytes in the AV valves, a nanoindentation analysis of the tricuspid valves of wild type, hypopigmented and hyperpigmented mice was performed. The storage modulus, a measure of stiffness, for the leaflets obtained from hyperpigmented mice was considerably higher (10.5GPa) than that for the leaflets from wild type (7.5GPa) and hypopigmented animals (between 3.5 and 5.5 GPa) suggesting that melanocytes may contribute to the mechanical properties of the AV valves.

Inducible Overexpression of Endothelin-3 in a Transgenic Mouse Leads to Hyperpigmentation That Decreases Over Time

Endothelin-3 (Edn3) has been shown to be an essential environmental cue in melanocyte development. Edn3 and its receptor, EdnrB, are allelic to mouse mutations occurring at the lethal spotting and piebald loci, respectively; these mutations result in hypopigmentation phenotypes. Mutations in the genes for both Edn3 and EdnrB are implicated in human pigmentation disorders such as Waardenburg-Shah syndrome, which is characterized by pigmentation defects, deafness, and megacolon. In this study, a tetracycline-inducible transgenic mouse model that overexpresses Edn3 under the control of the Keratin 5 promoter was shown to produce a hyperpigmentation phenotype that decreases over time. The expression pattern of transgenic Edn3 and its effects on the melanocyte population were examined in transgenic embryos, postnatal skin, and the skin of adult mice that exhibit faded hyperpigmentation. These studies suggest that overexpression of Edn3 in this model is restricted primarily to the roof plate of the neural tube and surface ectoderm in the developing embryo and to keratinocytes in the epidermis of postnatal mice. A decline in transgenic expression and a reduction in the dermal melanocytes and free melanin that characterize the phenotype in juvenile mice were shown to correlate with the fading of the hyperpigmentation phenotype. Transgenic mice in which transgenic expression was repressed (resulting in the disappearance of the hyperpigmentation phenotype) also exhibited a decrease in the dermal melanocyte population. The Edn3-overexpressing mice used in this study might be helpful m understanding human skin conditions characterized by dermal melanocytosis.