Cloning and expression of the large zebrafish protocadherin gene, Fat

The cadherin superfamily members play an important role in mediating cell-cell contact and adhesion (Takeichi, M., 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455). A distinct subfamily, neither belonging to the classical or protocadherins includes Fat, the largest member of the cadherin super-family. Fat was originally identified in Drosophila. Subsequently, orthologues of Fat have been described in man (Dunne, J., Hanby, A. M., Poulsom, R., Jones, T. A., Sheer, D., Chin, W. G., Da, S. M., Zhao, Q., Beverley, P. C., Owen, M. J., 1995. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30, 207-223), rat (Ponassi, M., Jacques, T. S., Ciani, L., ffrench, C. C., 1999. Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech. Dev. 80, 207-212) and mouse (Cox, B., Hadjantonakis, A. K., Collins, J. E., Magee, A. I., 2000. Cloning and expression throughout mouse development of mfat 1, a homologue of the Drosophila tumour suppressor gene fat [In Process Citation]. Dev. Dyn. 217, 233-240). In Drosophila, Fat has been shown to play an important role in both planar cell polarity and cell boundary formation during development. In this study we describe the characterization of zebrafish Fat, the first non-mammalian, vertebrate Fat homologue to be identified. The Fat protein has 64% amino acid identity and 80% similarity to human FAT and an identical domain structure to other vertebrate Fat proteins. During embryogenesis fat mRNA is expressed in the developing brain, specialised epithelial surfaces the notochord, ears, eyes and digestive tract, a pattern similar but distinct to that found in mammals. (c) 2005 Elsevier B.V. All rights reserved.

Establishment of metanephros transplantation in mice highlights contributions by both nephrectomy and pregnancy to developmental progression

Background: It has been demonstrated that embryonic kidneys (metanephroi) xenotransplanted into the omentum of adult recipients continue to develop and display immune protection due to their more nave immune presentation. To date, this has been achieved using rat, pig and human metanephroi, with unilateral nephrectomy (UNX) of recipient rats a requisite of renal development. The aim of this study was to adapt this approach for use in mice and examine the parameters affecting successful onward development in this species. Methods: Metanephroi at embryonic age (E) 13.5 were transplanted either onto the body wall, abdominal fat pads or omentum of recipient isogenic C57/Bl6 mice using either sutures or polyglycolic acid mesh. Having established greatest success with polyglycolic acid mesh on the body wall, E12.5 and 15.5 days metanephroi from C57/Bl6 mice were then transplanted onto the body wall of control (non-pregnant non-UNX), UNX or 12.5 days post-coitum pregnant isogenic recipients. After 7 days, implanted tissue was harvested and examined using histology and immunohistochemistry for markers of renal maturation. The mean number of S-shaped bodies and glomeruli per section were recorded and statistically analysed for significant differences between all recipient groups and untransplanted metanephroi. The degree of development was scored qualitatively. Results: Transplanted E12.5 metanephroi developed S-shaped bodies and glomeruli in all recipient groups, although there were statistically higher numbers of S-shaped bodies in UNX (n = 2) and pregnant recipients (n = 9) than in control recipients (n = 4). Continued development, as indicated by mature vascularized glomeruli, was only observed in those E15.5 metanephroi transplanted into pregnant recipients (n = 11) with a 15.5-fold increase in S-shaped bodies and 4-fold increase in glomeruli compared with control transplants (n = 12). Conclusions: We have successfully established metanephros transplantation in mice and demonstrated enhancement of onward development of E12.5 metanephroi in response to both pregnancy and UNX. Using E15.5 metanephroi, continued development only occurred in pregnant recipients, implying pregnancy provides an environment conducive to continued organogenesis. This murine assay, when coupled with transgenically-tagged strains of mice, will allow the investigation of the relative contribution of donor and recipient cells to this process. Copyright (C) 2005 S. Karger AG, Basel.

Developmental Vitamin D-3 deficiency alters the adult rat brain

There is growing evidence that Vitamin D-3 (1,25-dihydroxyvitamin D-3) is involved in brain development. We have recently shown that the brains of newborn rats from Vitamin D-3 deficient dams were larger than controls, had increased cell proliferation, larger lateral ventricles, and reduced cortical thickness. Brains from these animals also had reduced expression of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor. The aim of the current study was to examine if there were any permanent outcomes into adulthood when the offspring of Vitamin D-3 deficient dams were restored to a normal diet. The brains of adult rats were examined at 10 weeks of age after Vitamin D-3 deficiency until birth or weaning. Compared to controls animals that were exposed to transient early Vitamin D-3 deficiency had larger lateral ventricles, reduced NGF protein content, and reduced expression of a number genes involved in neuronal structure, i.e. neurofilament or MAP-2 or neurotransmission, i.e. GABA-(alpha 4). We conclude that transient early life hypovitaminosis D-3 not only disrupts brain development but leads to persistent changes in the adult brain. In light of the high incidence of hypovitammosis D-3 in women of child-bearing age, the public health implications of these findings warrant attention. (c) 2005 Elsevier Inc. All rights reserved.

Neonatal calyceal dilation and renal fibrosis resulting from loss of Adamts-1 in mouse kidney is due to a developmental dysgenesis

Background. A disintegrin and metalloproteinase with thrombospondin motifs 1, Adamts-1, is important for the development and function of the kidney. Mice lacking this protein present with renal lesions comprising enlarged calyces, and reduced cortex and medulla layers. Our current findings are consistent with the defect occurring due to a developmental dysgenesis. Methods. We generated Adamts-1 null mice, and further investigated their kidney phenotype in a time course study ranging from E18.5 to 12 months of age. Immunohistochemistry was used to assess the localization of type IV collagen, TGF-beta and F4/80-positive macrophages in the kidneys of Adcants-1 null mice compared to wild-type control animals. The expression of Adamts-1 mRNA was determined in metanephric kidney explants by in situ hybridization. Results. Adamts-1 null mice have a gross kidney defect. At day 18.5 of gestation, the Adcants-1 null kidney has a normal appearance but at birth when the kidney begins to function, the defect becomes evident. During development of the kidney Adamts-1 expression was specifically detected in the developing loops of Henle, as well as in the proximal and distal convoluted tubules. Expression was not detected in the ureter, ureteric bud or its derivatives as had been previously suggested. At 6 months and I year of age, the Adamts-1 null mice displayed interstitial fibrosis in the cortical and medullary regions of the kidney. At I year of age, the Adamts-1 null mice displayed mild interstitial matrix expansion associated with increased collagen type IV expression, without apparent tubular dilatation, compared to wild-type animals. Immunohistochemical analysis demonstrated TGF-beta protein localized to infiltrating macrophages and glomeruli of Adamts-1 null mice. Conclusions. Adamts-1 is required for the normal development of the kidney. The defect observed in its absence results from a dysgenic malformation affecting the medulla that becomes apparent at birth, once the kidneys start to function.

Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y-DOM sex reversal

The phenomenon of B6-Y-DOM sex reversal arises when certain variants of the Mus domesticus Y chromosome are crossed onto the genetic background of the C57BL/6J (136) inbred mouse strain, which normally carries a Mus musculus-derived Y chromosome. While the sex reversal has been assumed to involve strain-specific variations in structure or expression of Sry, the actual cause has not been identified. Here we used in situ hybridization to study expression of Sry, and the critical downstream gene Sox9, in strains containing different chromosome combinations to investigate the cause of B6-Y-DOM sex reversal. Our findings establish that a delay of expression of Sry(DOM) relative to Sry(B6) underlies B6-Y-DOM sex reversal and provide the first molecular confirmation that Sry must act during a critical time window to appropriately activate Sox9 and effect male testis determination before the onset of the ovarian-determining pathway. (C) 2004 Elsevier Inc. All rights reserved.

Developmental vitamin D (DVD) deficiency in the rat alters adult behaviour independently of HPA function

Developmental vitamin D deficiency (DVD) has been shown to alter the orderly pattern of brain development. Even though the period of vitamin D deficiency is restricted to gestation this is sufficient to induce behavioural abnormalities in the adult offspring consistent with those seen in many animal models of schizophrenia. Given that some of these behavioural alterations could also be an indirect result of either impaired maternal hypothalamic pituitary axis (HPA) function (which in turn could influence maternal care) or the result of a permanent alteration in HPA function in the adult offspring we have examined HPA status in both maternal animals and adult offspring. In this study we have established that HPA function is normal in the maternally vitamin D deficient rat. We replicate the behavioural phenotype of hyperlocomotion whilst establishing that HPA function is also unchanged in the adult mate offspring. We conclude that the behavioural alterations induced by DVD deficiency are due to some adverse event in brain development rather than via an alteration in stress response. (c) 2006 Elsevier Ltd. All rights reserved.

Expression analysis of the Epha1 receptor tyrosine kinase and its high-affinity ligands Efna1 and Efna3 during early mouse development

Interaction of Eph receptor tyrosine kinases with their membrane bound ephrin ligands initiates bidirectional signaling events that regulate cell migratory and adhesive behavior. Whole-mount in situ hybridization revealed overlapping expression of the Epha1 receptor and its high-affinity ligands ephrin A1 (Efna1) and ephrin A3 (Efna3) in the primitive streak and the posterior paraxial mesoderm during early mouse development. These results show complex and dynamic expression for all three genes with expression domains that are successively complementary, overlapping, and divergent. (c) 2006 Elsevier B.V. All rights reserved.

Differential gene expression in the developing mouse ureter

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPAR gamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Co15a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations. (c) 2005 Elsevier B.V. All rights reserved.

Glucose affects monocarboxylate cotransporter (MCT) 1 expression during mouse preimplantation development

Cleavage-stage embryos have an absolute requirement for pyruvate and lactate, but as the morula compacts, it switches to glucose as the preferred energy source to fuel glycolysis. Substrates such as glucose, amino acids, and lactate are moved into and out of cells by facilitated diffusion. in the case of lactate and pyruvate, this occurs via H+-monocarboxylate cotransporter (MCT) proteins. To clarify the role of MCT in development, transport characteristics for DL-lactate were examined, as were mRNA expression and protein localisation for MCT1 and MCT3, using confocal laser scanning immunofluorescence in freshly collected and cultured embryos. Blastocysts demonstrated significantly higher affinity for DL-lactate than zygotes (K-m 20 +/- 10 vs 87 +/- 35 mmol lactate/l; P = 0.03 by linear regression) but was similar for all stages. For embryos derived in vivo and those cultured with glucose, MCT1 mRNA was present throughout preimplantation development, protein immunoreactivity appearing diffuse throughout the cytoplasm with brightest intensity in the outer cortical region of blastomeres. in expanding blastocysts, MCT1 became more prominent in the cytoplasmic cortex of blastomeres, with brightest intensity in the polar trophectoderm. Without glucose, MCT1 mRNA was not expressed, and immunoreactivity dramatically reduced in intensity as morulae died. MCT3 mRNA and immunoreactivity were not detected in early embryos. The differential expression of MCT1 in the presence or absence of glucose demonstrates that it is important in the critical regulation of pH and monocarboxylate transport during preimplantation development, and implies a role for glucose in the control of MCT1, but not MCT3, expression.

PAX2 activates WNT4 expression during mammalian kidney development

The transcription factor PAX2 is expressed during normal kidney development and is thought to influence outgrowth and branching of the ureteric bud. Mice with homozygous null Pax2 mutations have developmental defects of the midbrain-hindbrain region, optic nerve, and ear and are anephric. During nephrogenesis, PAX2 is also expressed by mesenchymal cells as they cluster and reorganize to form proximal elements of each nephron, but the function of PAX2 in these cells is unknown. In this study we hypothesized that PAX2 activates expression of WNT4, a secreted glycoprotein known to be critical for successful nephrogenesis. PAX2 protein was identified in distal portions of the S-shaped body, and the protein persists in the emerging proximal tubules of murine fetal kidney. PAX2 activated WNT4 promoter activity 5-fold in co-transfection assays with JTC12 cells derived from the proximal tubule. Inspection of the 5'-flanking sequence of the human WNT4 gene identified three novel PAX2 recognition motifs; each exhibited specific PAX2 protein binding in electromobility shift assays. Two motifs were contained within a completely duplicated 0.66-kb cassette. Transfection of JTC12 cells with a PAX2 expression vector was associated with a 7-fold increase in endogenous WNT4 mRNA. In contrast, Wnt4 mRNA was decreased by 60% in mesenchymal cell condensates of fetal kidney from mice with a heterozygous Pax2 mutation. We speculated that a key function of PAX2 is to activate WNT4 gene expression in metanephric mesenchymal cells as they differentiate to form elements of the renal tubules.

The effect of larval age on morphology and gene expression during ascidian metamorphosis

Metamorphosis is both an ecological and a developmental genetic transition that an organism undergoes as a normal part of ontogeny. Many organisms have the ability to delay metamorphosis when conditions are unsuitable. This strategy carries obvious benefits, but may also result in severe consequences for older larvae that run low on energy. In the marine environment, some lecithotrophic larvae that have prolonged periods in the plankton may begin forming postlarval and juvenile structures that normally do not appear until after settlement and the initiation of metamorphosis. This precocious activation of the postlarval developmental program may reflect an adaptation to increase the survival of older, energy-depleted larvae by allowing them to metamorphose more quickly. In the present study, we investigate morphological and genetic consequences of delay of metamorphosis in larvae of Herdmania momus (a solitary stolidobranch ascidian). We observe significant morphological and genetic changes during prolonged larval life, with older larvae displaying significant changes in RNA levels, precocious migration of mesenchyme cells, and changes in larval shape including shortening of the tail. While these observations suggest that the older H. momus larvae are functionally different from younger larvae and possibly becoming more predisposed to undergo metamorphosis, we did not find any significant differences in gene expression levels between postlarvae arising from larvae that metamorphosed as soon as they were competent and postlarvae developing from larvae that postponed metamorphosis. This recalibration, or convergence, of transcript levels in the early postlarva suggests that changes that occur during prolonged larval life of H. momus are not necessarily associated with early activation of adult organ differentiation. Instead, it suggests that an autonomous developmental program is activated in H. momus upon the induction of metamorphosis regardless of the history of the larva.

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.

Differentiating human NT2/D1 neurospheres as a versatile in vitro 3D model system for developmental neurotoxicity testing

Developmental neurotoxicity is a major issue in human health and may have lasting neurological implications. In this preliminary study we exposed differentiating Ntera2/clone D1 (NT2/D1) cell neurospheres to known human teratogens classed as non-embryotoxic (acrylamide), weakly embryotoxic (lithium, valproic acid) and strongly embryotoxic (hydroxyurea) as listed by European Centre for the Validation of Alternative Methods (ECVAM) and examined endpoints of cell viability and neuronal protein marker expression specific to the central nervous system, to identify developmental neurotoxins. Following induction of neuronal differentiation, valproic acid had the most significant effect on neurogenesis, in terms of reduced viability and decreased neuronal markers. Lithium had least effect on viability and did not significantly alter the expression of neuronal markers. Hydroxyurea significantly reduced cell viability but did not affect neuronal protein marker expression. Acrylamide reduced neurosphere viability but did not affect neuronal protein marker expression. Overall, this NT2/D1 -based neurosphere model of neurogenesis, may provide the basis for a model of developmental neurotoxicity in vitro.

Effects of lithium and valproic acid on gene expression and phenotypic markers in an NT2 neurosphere model of neural development

Mood stabilising drugs such as lithium (LiCl) and valproic acid (VPA) are the first line agents for treating conditions such as Bipolar disorder and Epilepsy. However, these drugs have potential developmental effects that are not fully understood. This study explores the use of a simple human neurosphere-based in vitro model to characterise the pharmacological and toxicological effects of LiCl and VPA using gene expression changes linked to phenotypic alterations in cells. Treatment with VPA and LiCl resulted in the differential expression of 331 and 164 genes respectively. In the subset of VPA targeted genes, 114 were downregulated whilst 217 genes were upregulated. In the subset of LiCl targeted genes, 73 were downregulated and 91 were upregulated. Gene ontology (GO) term enrichment analysis was used to highlight the most relevant GO terms associated with a given gene list following toxin exposure. In addition, in order to phenotypically anchor the gene expression data, changes in the heterogeneity of cell subtype populations and cell cycle phase were monitored using flow cytometry. Whilst LiCl exposure did not significantly alter the proportion of cells expressing markers for stem cells/undifferentiated cells (Oct4, SSEA4), neurons (Neurofilament M), astrocytes (GFAP) or cell cycle phase, the drug caused a 1.4-fold increase in total cell number. In contrast, exposure to VPA resulted in significant upregulation of Oct4, SSEA, Neurofilament M and GFAP with significant decreases in both G2/M phase cells and cell number. This neurosphere model might provide the basis of a human-based cellular approach for the regulatory exploration of developmental impact of potential toxic chemicals.

Prenatal environmental exposure and neurodevelopmentally important gene expression in malformed brain tissue from pediatric intractable epilepsy patients

The primary objective of this proposal was to determine whether mitochondrial oxidative stress and variation in a particular mtDNA lineage contribute to the risk of developing cortical dysplasia and are potential contributing factors in epileptogenesis in children. The occurrence of epilepsy in children is highly associated with malformations of cortical development (MCD). It appears that MCD might arise from developmental errors due to environmental exposures in combination with inherited variation in response to environmental exposures and mitochondrial function. Therefore, it is postulated that variation in a particular mtDNA lineage of children contributes to the effects of mitochondrial DNA damage on MCD phenotype. Quantitative PCR and dot blot were used to examine mitochondrial oxidative damage and single nucleotide polymorphism (SNP) in the mitochondrial genome in brain tissue from 48 pediatric intractable epilepsy patients from Miami Children’s Hospital and 11 control samples from NICHD Brain and Tissue Bank for Developmental Disorders. Epilepsy patients showed higher mtDNA copy number compared to normal health subjects (controls). Oxidative mtDNA damage was lower in non-neoplastic but higher in neoplastic epilepsy patients compared to controls. There was a trend of lower mtDNA oxidative damage in the non-neoplastic (MCD) patients compared to controls, yet, the reverse was observed in neoplastic (MCD and Non-MCD) epilepsy patients. The presence of mtDNA SNP and haplogroups did not show any statistically significant relationships with epilepsy phenotypes. However, SNPs G9804A and G9952A were found in higher frequencies in epilepsy samples. Logistic regression analysis showed no relationship between mtDNA oxidative stress, mtDNA copy number, mitochondrial haplogroups and SNP variations with epilepsy in pediatric patients. The levels of mtDNA copy number and oxidative mtDNA damage and the SNPs G9952A and T10010C predicted neoplastic epilepsy, however, this was not significant due to a small sample size of pediatric subjects. Findings of this study indicate that an increase in mtDNA content may be compensatory mechanisms for defective mitochondria in intractable epilepsy and brain tumor. Further validation of these findings related to mitochondrial genotypes and mitochondrial dysfunction in pediatric epilepsy and MCD may lay the ground for the development of new therapies and prevention strategies during embryogenesis.