Genetic, Cellular and Molecular Defects in Mouse Mutants with Severe Neural Tube Defects

Neural tube defects are one of the most common birth defects. This thesis includes genetic, molecular and cellular analysis of three mouse mutants with neural tube defects. chuzhoi was identified from an ENU G3 screen and exhibits craniorachischisis through failure to initiate neural tube closure. In this thesis, I show that chuzhoi carries a point mutation affecting a splice-site in the Ptk7 gene, leading to addition of three extra amino acids in the protein. Through phenotypic analysis I show that chuzhoi has a wider midline and a smaller length to width ratio, suggesting a defect in convergent extension. Previous work has shown that the Planar Cell Polarity (PCP) signalling pathway is required for the initiation of neural tube closure. Through genetic crosses between chuzhoi and mutants of PCP signalling, I show that Ptk7 can influence the PCP pathway without being a direct component of the pathway. Carrying a mutation in Scribble, Circletail is another mutant displaying craniorachischisis. Scribble in Drosophila is required for the establishment of apical-basal polarity and to control the rate of cell proliferation. I show that mouse Scribble is not required for the establishment of apical-basal polarity nor to control proliferation, during neural tube closure. Previous work in zebrafish has shown that the axis elongation of an embryo requires PCP-dependent orientation of cell division. Here, I show that the orientation of cell division is random in mice during the shaping of the neural plate prior to the initiation of the neural tube closure. Mouse mutants of Tulp3 exhibit spina bifida and exencephaly. The molecular role of Tulp3 is largely unknown; however, domains present in the Tulp3 protein suggest that it may act as a transcription factor and/or be involved in protein-protein interactions. I provide evidence to suggest that Tulp3 is not likely to function as a transcription factor but may participate in many protein-protein interactions to deliver its role during mouse embryogenesis

The novel mouse mutant, chuzhoi, has disruption of Ptk7 protein and exhibits defects in neural tube, heart and lung development and abnormal planar cell polarity in the ear

Background The planar cell polarity (PCP) signalling pathway is fundamental to a number of key developmental events, including initiation of neural tube closure. Disruption of the PCP pathway causes the severe neural tube defect of craniorachischisis, in which almost the entire brain and spinal cord fails to close. Identification of mouse mutants with craniorachischisis has proven a powerful way of identifying molecules that are components or regulators of the PCP pathway. In addition, identification of an allelic series of mutants, including hypomorphs and neomorphs in addition to complete nulls, can provide novel genetic tools to help elucidate the function of the PCP proteins. Results We report the identification of a new N-ethyl-N-nitrosourea (ENU)-induced mutant with craniorachischisis, which we have named chuzhoi (chz). We demonstrate that chuzhoi mutant embryos fail to undergo initiation of neural tube closure, and have characteristics consistent with defective convergent extension. These characteristics include a broadened midline and reduced rate of increase of their length-to-width ratio. In addition, we demonstrate disruption in the orientation of outer hair cells in the inner ear, and defects in heart and lung development in chuzhoi mutants. We demonstrate a genetic interaction between chuzhoi mutants and both Vangl2Lp and Celsr1Crsh mutants, strengthening the hypothesis that chuzhoi is involved in regulating the PCP pathway. We demonstrate that chuzhoi maps to Chromosome 17 and carries a splice site mutation in Ptk7. This mutation results in the insertion of three amino acids into the Ptk7 protein and causes disruption of Ptk7 protein expression in chuzhoi mutants. Conclusions The chuzhoi mutant provides an additional genetic resource to help investigate the developmental basis of several congenital abnormalities including neural tube, heart and lung defects and their relationship to disruption of PCP. The chuzhoi mutation differentially affects the expression levels of the two Ptk7 protein isoforms and, while some Ptk7 protein can still be detected at the membrane, chuzhoi mutants demonstrate a significant reduction in membrane localization of Ptk7 protein. This mutant provides a useful tool to allow future studies aimed at understanding the molecular function of Ptk7

The mouse Wnt/PCP protein Vangl2 is necessary for migration of facial branchiomotor neurons, and functions independently of Dishevelled

AbstractDuring development, facial branchiomotor (FBM) neurons, which innervate muscles in the vertebrate head, migrate caudally and radially within the brainstem to form a motor nucleus at the pial surface. Several components of the Wnt/planar cell polarity (PCP) pathway, including the transmembrane protein Vangl2, regulate caudal migration of FBM neurons in zebrafish, but their roles in neuronal migration in mouse have not been investigated in detail. Therefore, we analyzed FBM neuron migration in mouse looptail (Lp) mutants, in which Vangl2 is inactivated. In Vangl2Lp/+ and Vangl2 Lp/Lp embryos, FBM neurons failed to migrate caudally from rhombomere (r) 4 into r6. Although caudal migration was largely blocked, many FBM neurons underwent normal radial migration to the pial surface of the neural tube. In addition, hindbrain patterning and FBM progenitor specification were intact, and FBM neurons did not transfate into other non-migratory neuron types, indicating a specific effect on caudal migration.Since loss-of-function in some zebrafish Wnt/PCP genes does not affect caudal migration of FBM neurons, we tested whether this was also the case in mouse. Embryos null for Ptk7, a regulator of PCP signaling, had severe defects in caudal migration of FBM neurons. However, FBM neurons migrated normally in Dishevelled (Dvl) 1/2 double mutants, and in zebrafish embryos with disrupted Dvl signaling, suggesting that Dvl function is essentially dispensable for FBM neuron caudal migration. Consistent with this, loss of Dvl2 function in Vangl2Lp/+ embryos did not exacerbate the Vangl2Lp/+ neuronal migration phenotype. These data indicate that caudal migration of FBM neurons is regulated by multiple components of the Wnt/PCP pathway, but, importantly, may not require Dishevelled function. Interestingly, genetic-interaction experiments suggest that rostral FBM neuron migration, which is normally suppressed, depends upon Dvl function

Cinacalcet corrects hypercalcemia in mice with an inactivating Gα11 mutation

Loss-of-function mutations of GNA11, which encodes G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in familial hypocalciuric hypercalcemia type 2 (FHH2). FHH2 is characterized by hypercalcemia, inappropriately normal or raised parathyroid hormone (PTH) concentrations, and normal or low urinary calcium excretion. A mouse model for FHH2 that would facilitate investigations of the in vivo role of Gα11 and the evaluation of calcimimetic drugs, which are CaSR allosteric activators, is not available. We therefore screened DNA from > 10,000 mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for GNA11 mutations and identified a Gα11 variant, Asp195Gly (D195G), which downregulated CaSR-mediated intracellular calcium signaling in vitro, consistent with it being a loss-of-function mutation. Treatment with the calcimimetic cinacalcet rectified these signaling responses. In vivo studies showed mutant heterozygous (Gna11+/195G) and homozygous (Gna11195G/195G) mice to be hypercalcemic with normal or increased plasma PTH concentrations and normal urinary calcium excretion. Cinacalcet (30mg/kg orally) significantly reduced plasma albumin-adjusted calcium and PTH concentrations in Gna11+/195G and Gna11195G/195G mice. Thus, our studies have established a mouse model with a germline loss-of-function Gα11 mutation that is representative for FHH2 in humans and demonstrated that cinacalcet can correct the associated abnormalities of plasma calcium and PTH

Gα11 mutation in mice causes hypocalcemia rectifiable by calcilytic therapy

Heterozygous germline gain-of-function mutations of G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesised that a previously reported dark skin mouse mutant (Dsk7), which has a germline hypermorphic Gα11 mutation, Ile62Val, may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant Dsk7/+ and Dsk7/Dsk7 mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα11 to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain-offunction. Treatment with NPS-2143, a calcilytic compound, normalised these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in Dsk7/Dsk7 and Dsk7/+ mice, which became normocalcemic. Thus, these studies have established Dsk7 mice, which harbor a germline gain-of-function Gα11 mutation, as a model for ADH2; and demonstrated calcilytics as a potential targeted therapy

Mouse hitchhiker mutants have spina bifida, dorso-ventral patterning defects and polydactyly: identification of Tulp3 as a novel negative regulator of the Sonic hedgehog pathway

The mammalian Sonic hedgehog (Shh) signalling pathway is essential for embryonic development and the patterning of multiple organs. Disruption or activation of Shh signalling leads to multiple birth defects, including holoprosencephaly, neural tube defects and polydactyly, and in adults results in tumours of the skin or central nervous system. Genetic approaches with model organisms continue to identify novel components of the pathway, including key molecules that function as positive or negative regulators of Shh signalling. Data presented here define Tulp3 as a novel negative regulator of the Shh pathway. We have identified a new mouse mutant that is a strongly hypomorphic allele of Tulp3 and which exhibits expansion of ventral markers in the caudal spinal cord, as well as neural tube defects and preaxial polydactyly, consistent with increased Shh signalling. We demonstrate that Tulp3 acts genetically downstream of Shh and Smoothened (Smo) in neural tube patterning and exhibits a genetic interaction with Gli3 in limb development. We show that Tulp3 does not appear to alter expression or processing of Gli3, and we demonstrate that transcriptional regulation of other negative regulators (Rab23, Fkbp8, Thm1, Sufu and PKA) is not affected. We discuss the possible mechanism of action of Tulp3 in Shh-mediated signalling in light of these new data

Transition to professional practice: Perspectives of new nursing graduates of Nepal

Abstract Background Internationally, the transition from student nurse to practicing nurse is recognized as being the most stressful period. Yet very little is known about how new nursing graduates perceive this transition in Nepal. The study aimed to explore new nursing graduates’ perceptions of the transition to professional practice. Methods A qualitative descriptive methodology was used. In-depth semi-structured interviews were conducted with 10 purposively recruited participants from two private hospitals in Nepal. Data were analyzed using inductive thematic analysis. The Consolidated Criteria for Reporting Qualitative Research (COREQ) guidelines were used to report the findings of this study. Results New nursing graduates perceived the transition to professional practice as an intense experience. Inductive thematic analysis yielded four intrinsically linked themes that encompassed new nursing graduates’ transition experiences: ‘getting hit by reality’, ‘losing confidence’, ‘feeling unsupported’, and ‘gathering strengths.’ The theme ‘getting hit by reality’ included three sub-themes: ‘gap between theory and practice,’ ‘no protective shield,’ and ‘plethora of responsibilities’, which explains nurses’ initial encounter with real-world practice. The theme ‘losing confidence’ contained three sub-themes: ‘being fearful,’ ‘being ignored,’ and ‘being accused,’ which describes how nurses started losing confidence as they confronted the real side of the profession. The theme ‘feeling unsupported’ included two sub-themes: ‘left without guidance,’ and ‘limited support from seniors,’ which explains how nurses perceived their work environment. The theme ‘gathering strength’ contained two sub-themes: ‘reflecting’ and ‘asking for help,’ which describes how nurses coped with the challenges related to the transition. Conclusion To facilitate the transition to practice, educational institutions must impart to students a realistic understanding of the transition process, address the theory-practice gap, and collaborate with hospitals. Similarly, hospitals should have realistic expectations from new nurses, assign work according to their capabilities, and allow them sufficient time for role integration. Likewise, well-conceived detailed orientation, mentorship or preceptorship programs, and regular professional development programs are vital to easing the transition. Furthermore, establishing and maintaining a supportive work culture, which promotes equity, respect, and safety among employees, is crucial for positive transition experiences

Modulation of stretch-induced myocyte remodeling and gene expression by nitric oxide: a novel role for lipoma preferred partner in myofibrillogenesis

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signalling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors which convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain, 1Hz for 48 hours to mimic in vivo mechanical stress. Cells were also treated with and without L-NAME, a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres which was rescued by LNAME (P<0.05, n≥5 cultures). We hypothesized that the mechanism was through NOinduced alteration of myocyte gene expression. L-NAME up-regulated the mechanosensing proteins Muscle LIM protein (MLP (by 100%, p<0.05, n=4 cultures)) and lipoma preferred partner, a novel cardiac protein (LPP (by 80%, p<0.05, n=4 cultures)). L-NAME also significantly altered the subcellular localisation of LPP and MLP in a manner that favoured growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between iNOS and nNOS isoforms regulate gene expression. LPP knockdown by siRNA led to formation of α-actinin aggregates and Z-bodies showing that myofibrillogenesis was impaired. There was an up-regulation of E3 ubiquitin ligase (MUL1) by 75% (P<0.05, n=5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure. Keywords: Mechanotransduction, heart failure, stretch, heart, hypertroph