Autotaxin is required for the cranial neural tube closure and establishment of the midbrain–hindbrain boundary during mouse development

Autotaxin (ATX) is a lysophospholipid‐generating exoenzyme expressed in embryonic and adult neural tissues. We previously showed that ATX is expressed in the neural organizing centers, anterior head process, and midbrain‐hindbrain boundary (MHB). To elucidate the role of ATX during neural development, here we examined the neural phenotypes of ATX‐deficient mice. Expression analysis of neural marker genes revealed that lateral expansion of the rostral forebrain is reduced and establishment of the MHB is compromised as early as the late headfold stage in ATX mutant embryos. Moreover, ATX mutant embryos fail to complete cranial neural tube closure. These results indicate that ATX is essential for cranial neurulation and MHB establishment. Developmental Dynamics 240:413–421, 2011. © 2011 Wiley‐Liss, Inc.     

Evolutionarily conserved function of Gbx2 in anterior hindbrain development

The amino acid sequence across the DNA‐binding homeodomain of Gbx2 is highly conserved across multiple species. In mice, Gbx2 is essential for establishment of the midbrain–hindbrain boundary (MHB), and in development of anterior hindbrain structures, rhombomeres (r) 1–r3, and the r2/r3‐derived cranial nerve V. In contrast, studies in zebrafish have implicated gbx1 in establishment of the MHB. Therefore, we tested potential roles for gbx2 in anterior hindbrain development in zebrafish. gbx2 knockdown with antisense morpholino results in increased cell death in r2, r3, and r5 and a truncation of the anterior hindbrain, similar to the defect in Gbx2^?/? mice. Moreover, there is abnormal clustering of cranial nerve V cell bodies in r2 and r3 indicative of defects in aspects of anterior hindbrain patterning. These phenotypes can be rescued by expression of the mouse GBX2 protein. These results suggest that gbx2/Gbx2 has an evolutionarily conserved role in anterior hindbrain development. Developmental Dynamics 240:828–838, 2011. © 2011 Wiley‐Liss, Inc.     

Establishing a novel Pig‐a gene mutation assay in L5178YTk+/− mouse lymphoma cells

The X‐linked Pig‐a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig‐a mutant cells fail to synthesize GPI and to express GPI‐anchored protein markers (e.g., CD90) on their surface. Marker deficiency serves as a phenotypic indicator of Pig‐a mutation in various in vivo assays. Here, we describe an in vitro Pig‐a mutation assay in L5178YTk^+/– mouse lymphoma cells, in which mutant‐phenotype cells are measured by flow cytometry using a fluorescent anti‐CD90 antibody. Increased frequencies of CD90‐deficient mutants were detected in cells treated with benzo[a]pyrene (B[a]P), N‐ethyl‐N‐nitrosourea (ENU), ethyl methanesulphonate, and 7,12‐dimethylbenz[a]anthracene, with near maximum mutant frequencies measured eight days after treatment. The CD90 deficiency in mutant cells quantified by flow cytometry was shown to be due to loss of GPI anchors in a limiting‐dilution cloning assay using proaerolysin selection. Individual CD90‐deficient cells from cultures treated with ENU, B[a]P, and vehicle were sorted and clonally expanded for molecular analysis of their Pig‐a gene. Pig‐a mutations with agent‐specific signatures were found in nearly all clones that developed from sorted CD90‐deficient cells. These results indicate that a Pig‐a mutation assay can be successfully conducted in L5178YTk^+/? cells. The assay may be useful for mutagenicity screening of environmental agents as well as for testing hypotheses in vitro before committing to in vivo Pig‐a assays. Environ. Mol. Mutagen. 59:4–17, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.     

CD48‐deficient T‐lymphocytes from DMBA‐treated rats have de novo mutations in the endogenous Pig‐a gene

A major question concerning the scientific and regulatory acceptance of the rodent red blood cell‐based Pig‐a gene mutation assay is the extent to which mutants identified by their phenotype in the assay are caused by mutations in the Pig‐a gene. In this study, we identified T‐lymphocytes deficient for the glycosylphosphatidylinositol‐anchored surface marker, CD48, in control and 7,12‐dimethylbenz[a]anthracene (DMBA)‐treated rats using a flow cytometric assay and determined the spectra of mutations in the endogenous Pig‐a gene in these cells. CD48‐deficient T‐cells were seeded by sorting at one cell per well into 96‐well plates, expanded into clones, and exons of their genomic Pig‐a were sequenced. The majority (78%) of CD48‐deficient T‐cell clones from DMBA‐treated rats had mutations in the Pig‐a gene. The spectrum of DMBA‐induced Pig‐a mutations was dominated by mutations at A:T, with the mutated A being on the nontranscribed strand and A→T transversion being the most frequent change. The spectrum of Pig‐a mutations in DMBA‐treated rats was different from the spectrum of Pig‐a mutations in N‐ethyl‐N‐nitrosourea (ENU)‐treated rats, but similar to the spectrum of DMBA mutations for another endogenous X‐linked gene, Hprt. Only 15% of CD48‐deficient mutants from control animals contained Pig‐a mutations; T‐cell biology may be responsible for a relatively large fraction of false Pig‐a mutant lymphocytes in control animals. Among the verified mutants from control rats, the most common were frameshifts and deletions. The differences in the spectra of spontaneous, DMBA‐, and ENU‐induced Pig‐a mutations suggest that the flow cytometric Pig‐a assay detects de novo mutation in the endogenous Pig‐a gene. Environ. Mol. Mutagen. 56:674–683, 2015. © 2015 Wiley Periodicals, Inc.     

The frequency of Pig‐a mutant red blood cells in rats exposed in utero to N‐ethyl‐N‐nitrosourea

The Pig‐a assay has been developed as a rapid sensitive measure of gene mutation in adult rats; however, no data exist on its ability to detect mutation following in utero exposures or in neonatal animals. Pregnant Sprague‐Dawley rats were treated daily on gestational days 12–18 with oral doses of 0, 6, or 12 mg/kg/day N‐ethyl‐N‐nitrosourea (ENU); following parturition, the offspring and dams were monitored over a period of 5 months for the frequency of CD59‐deficient erythrocytes as a marker of Pig‐a mutation. Significant dose‐related increases in Pig‐a mutant red blood cells (RBCs) were observed in ENU‐treated dams. However, only very weak increases in RBC Pig‐a mutant frequency (MF) were noted in offspring treated in utero with the lower ENU dose. The higher ENU dose produced extremely variable responses in the offspring as a function of age, even among littermates, ranging from a steady low or moderately high Pig‐a MF to a rapidly increasing or decreasing Pig‐a MF. The manifestation kinetics of Pig‐a mutant RBCs in the offspring suggest that the change from predominantly hepatic to predominantly bone marrow erythropoiesis that occurs during early development may have contributed to this variability. Our results indicate that using the RBC Pig‐a model for mutation detection in animals treated in utero may require analysis of multiple offspring from the same litter to account for potential “jack pot” effects, and that detection of the earliest treatment effect (i.e., in neonates using the hepatic RBC fraction) may require optimization of blood processing. Environ. Mol. Mutagen. 2012. Published 2012 Wiley Periodicals, Inc.     

Evaluation of in vivo genotoxicity induced by N‐ethyl‐N‐nitrosourea,benzo[a]pyrene,and 4‐nitroquinoline‐1‐oxide in the Pig‐a and gpt assays

The recently developed Pig‐a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor‐deficient red blood cells caused by a forward mutation in the Pig‐a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig‐a assay could be integrated into repeat‐dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig‐a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N‐ethyl‐N‐nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4‐nitroquinoline‐1‐oxide (4NQO, 50 mg/kg) in the Pig‐a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig‐a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7‐week terminal sacrifice. ENU increased both Pig‐a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig‐a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two‐ or threefold above control) were detected in the bone marrow in both the Pig‐a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig‐a mutation assay in order to use it as an alternative to the TGR mutation assay. Environ. Mol. Mutagen. 54:747–754, 2013. © 2013 Wiley Periodicals, Inc.     

Genetic disruption of CYP26B1 severely affects development of neural crest derived head structures,but does not compromise hindbrain patterning

Cyp26b1 encodes a cytochrome‐P450 enzyme that catabolizes retinoic acid (RA), a vitamin A derived signaling molecule. We have examined Cyp26b1^?/? mice and report that mutants exhibit numerous abnormalities in cranial neural crest cell derived tissues. At embryonic day (E) 18.5 Cyp26b1^?/? animals exhibit a truncated mandible, abnormal tooth buds, reduced ossification of calvaria, and are missing structures of the maxilla and nasal process. Some of these abnormalities may be due to defects in formation of Meckel's cartilage, which is truncated with an unfused distal region at E14.5 in mutant animals. Despite the severe malformations, we did not detect any abnormalities in rhombomere segmentation, or in patterning and migration of anterior hindbrain derived neural crest cells. Abnormal migration of neural crest cells toward the posterior branchial arches was observed, which may underlie defects in larynx and hyoid development. These data suggest different periods of sensitivity of anterior and posterior hindbrain neural crest derivatives to elevated levels of RA in the absence of CYP26B1. Developmental Dynamics 238:732–745, 2009. © 2009 Wiley‐Liss, Inc.     

Presynaptic secretion of mind‐the‐gap organizes the synaptic extracellular matrix‐integrin interface and postsynaptic environments

Mind‐the‐Gap (MTG) is required during synaptogenesis of the Drosophila glutamatergic neuromuscular junction (NMJ) to organize the postsynaptic domain. Here, we generate MTG::GFP transgenic animals to demonstrate MTG is synaptically targeted, secreted, and localized to punctate domains in the synaptic extracellular matrix (ECM). Drosophila NMJs form specialized ECM carbohydrate domains, with carbohydrate moieties and integrin ECM receptors occupying overlapping territories. Presynaptically secreted MTG recruits and reorganizes secreted carbohydrates, and acts to recruit synaptic integrins and ECM glycans. Transgenic MTG::GFP expression rescues hatching, movement, and synaptogenic defects in embryonic‐lethal mtg null mutants. Targeted neuronal MTG expression rescues mutant synaptogenesis defects, and increases rescue of adult viability, supporting an essential neuronal function. These results indicate that presynaptically secreted MTG regulates the ECM‐integrin interface, and drives an inductive mechanism for the functional differentiation of the postsynaptic domain of glutamatergic synapses. We suggest that MTG pioneers a novel protein family involved in ECM‐dependent synaptic differentiation. Developmental Dynamics 238:554–571, 2009. © 2009 Wiley‐Liss, Inc.     

In Vivo Pig‐a gene mutation assay: Guidance for 3Rs‐friendly implementation

The rodent Pig‐a assay is an in vivo method for the detection of gene mutation, where lack of glycosylphosphatidylinositol‐anchored proteins on the surface of circulating red blood cells (RBCs) serves as a reporter for Pig‐a gene mutation. In the case of rats, the frequency of mutant phenotype RBCs is measured via fluorescent anti‐CD59 antibodies and flow cytometry. The Pig‐a assay meets the growing expectations for novel approaches in animal experimentation not only focusing on the scientific value of the assay but also on animal welfare aspects (3Rs principles), for example, amenable to integration into pivotal rodent 28‐day general toxicology studies. However, as recommended in the Organisation for Economic Co‐operation and Development Test Guidelines for genotoxicity testing, laboratories are expected to demonstrate their proficiency. While this has historically involved the extensive use of animals, here we describe an alternative approach based on a series of blood dilutions covering a range of mutant frequencies. The experiments described herein utilized either non‐fluorescent anti‐CD59 antibodies to provide elevated numbers of mutant‐like cells, or a low volume blood sample from a single N‐ethyl‐N‐nitrosourea treated animal. Results from these so‐called reconstruction experiments from four independent laboratories showed good overall precision (correlation coefficients: 0.9979–0.9999) and accuracy (estimated slope: 0.71–1.09) of mutant cell scoring, which was further confirmed by Bland–Altman analysis. These data strongly support the use of reconstruction experiments for training purposes and demonstrating laboratory proficiency with very few animals, an ideal situation given the typically conflicting goals of demonstrating laboratory proficiency and reducing the use of animals. Environ. Mol. Mutagen. 57:678–686, 2016. © 2016 Wiley Periodicals, Inc.     

The in vivo Pig‐a gene mutation assay is useful for evaluating the genotoxicity of ionizing radiation in mice

The in vivo Pig‐a mutation assay has been adapted for measuring mutation in rats, mice, monkeys, and humans. To date, the assay has been used mainly to assess the mutagenicity of chemicals that are known to be powerful point mutagens. The assay has not been used to measure the biological effects associated with ionizing radiation. In this study, we modified the Pig‐a gene mutation assay (Kimoto et al. [2011b]: Mutat Res 723:36‐42) and used 3‐color staining with fluorescently labeled anti‐CD24, anti‐TER‐119, and anti‐CD71 to detect the Pig‐a mutant frequencies in total red blood cells (RBCs) and in reticulocytes (RETs) from X‐irradiated mice. Single exposures to X‐irradiation resulted in dose‐ and time‐dependent increases in Pig‐a mutant frequencies, and these subsequently declined over time returning to background frequencies. The same total amount of radiation, delivered either as a single dose or as four repeat doses at weekly intervals, increased Pig‐a mutant frequencies to comparable levels, reaching maxima 2–3 weeks after the single dose or 2–3 weeks after the last of the repeat doses. These increased frequencies subsequently returned to background levels. Our results indicated that the 3‐color Pig‐a assay was useful for evaluating the in vivo genotoxicity of radiation. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

Involvement of the centrosomal protein 55 (cep55) gene in zebrafish head formation

Mammalian CEP55 (centrosomal protein 55 kDa) is a coiled‐coil protein localized to the centrosome in interphase cells and is required for cytokinesis. A homozygous non‐sense mutation in human CEP55 has been recently identified in perinatal lethal MARCH (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly) syndrome. We have isolated zebrafish cep55 mutants defective in head morphology. The zebrafish cep55 gene was expressed in the head including the retina and the pectoral fin at 1 day post‐fertilization (dpf), and extensive cell death was widely observed in the head and tail of the cep55 mutant. In the cep55 mutant, the anterior–posterior distance of the ventral pharyngeal arches was short, and retinal lamination was disorganized. Neural cells, such as islet1‐positive cells and pax2‐positive cells, and fli1b‐positive vascular cells were reduced in the head of the cep55 mutant. Thus, we propose that the zebrafish cep55 mutant is a model organism for human MARCH syndrome.     

Novel markers of early ovarian pre‐granulosa cells are expressed in an Sry‐like pattern

Mammalian gonad differentiation involves sexually dimorphic cell‐fate decisions within the bipotential gonadal primordia. Testis differentiation is initiated by a center‐to‐poles wave of Sry expression that induces supporting cell precursors (SCPs) to become Sertoli rather than granulosa cells. The initiation of ovary differentiation is less well understood. We identified two novel SCP markers, 1700106J16Rik and Sprr2d, whose expression is ovary‐biased during early gonad development, and altered in Wnt4, Sf1, Wt1, and Fog2 mutant gonads. In XX and XY gonads, both genes were up‐regulated at ～E11 in a center‐to‐poles wave, and then rapidly down‐regulated in XY gonads in a center‐to‐poles wave, which is reminiscent of Sry expression in XY gonads. Our data suggest that 1700106J16Rik and Sprr2d may have important roles in early gonad development, and are consistent with the hypothesis that ovarian SCP differentiation occurs in a center‐to‐poles wave with similar timing to that of testicular SCP differentiation. Developmental Dynamics 238:812–825, 2009. © 2009 Wiley‐Liss, Inc.     

A regenerative erythropoietic response does not increase the frequency of Pig‐a mutant reticulocytes and erythrocytes in Sprague‐Dawley rats

The in vivo rodent Pig‐a mutation assay is a sensitive test to identify exposure to mutagenic substances, and has been proposed as an assay for the identification of impurities for pharmaceuticals. Red blood cells (RBCs) and reticulocytes (RETs) are analyzed by flow cytometry after exposure to potentially mutagenic chemicals for cells deficient in the cell surface anchored protein CD59, representing mutation in the X‐linked Pig‐a gene. The full potential of the assay as well as its limitations are currently being explored. The current study investigated the effects of regenerative erythropoietic bone marrow responses on the frequency of Pig‐a mutated reticulocytes (RET^CD59‐) and erythrocytes (RBC^CD59‐). We hypothesized that a robust regenerative erythropoietic response would not increase the basal frequency of RET^CD59‐ or RBC^CD59‐ cells. Two groups of six male Sprague‐Dawley rats either had 2 mL of blood sampled each day via an indwelling catheter over a period of 5 days or were minimally sampled for hematology and used as controls. Blood was also then collected and evaluated 5, 18, and 49 days after the initial bleed period for the number of Pig‐a mutant cells in either the RET or RBC population. Despite the expected decrease in hematocrit and the correlative increase in reticulocytes after bleeding, no increase in the number of Pig‐a mutant cells was observed in male Sprague‐Dawley rats that were bled for five consecutive days. These results indicate that changes in erythropoiesis and hematology parameters in rats appear to have no effect on the background levels of Pig‐a mutated RETs and RBCs. Environ. Mol. Mutagen. 59:91–95, 2018. © 2017 Wiley Periodicals, Inc.     

Hereditary pancreatitis caused by mutation‐induced misfolding of human cationic trypsinogen: A novel disease mechanism

We investigated the biochemical properties and cellular expression of the c.346C>T (p.R116C) human cationic trypsinogen (PRSS1) mutant, which we identified in a German family with autosomal dominant hereditary pancreatitis. This mutation leads to an unpaired Cys residue with the potential to interfere with protein folding via incorrect disulfide bond formation. Recombinantly expressed p.R116C trypsinogen exhibited a tendency for misfolding in vitro. Biochemical analysis of the correctly folded, purified p.R116C mutant revealed unchanged activation and degradation characteristics compared to wild type trypsinogen. Secretion of mutant p.R116C from transfected 293T cells was reduced to ～20% of wild type. A similar secretion defect was observed with another rare PRSS1 variant, p.C139S, whereas mutants p.A16V, p.N29I, p.N29T, p.E79K, p.R122C, and p.R122H were secreted normally. All mutants were detected in cell extracts at comparable levels but a large portion of mutant p.R116C was present in an insoluble, protease‐sensitive form. Consistent with intracellular retention of misfolded trypsinogen, the endoplasmic reticulum (ER) stress markers immunoglobulin‐binding protein (BiP) and the spliced form of the X‐box binding protein‐1 (XBP1s) were elevated in cells expressing mutant p.R116C. The results indicate that mutation‐induced misfolding and intracellular retention of human cationic trypsinogen causes hereditary pancreatitis in carriers of the p.R116C mutation. ER stress triggered by trypsinogen misfolding represents a new potential disease mechanism for chronic pancreatitis. Hum Mutat 0, 1–8, 2009. © 2009 Wiley‐Liss, Inc.     

In Vitro studies of non poly alanine PHOX2B mutations argue against a loss‐of‐function mechanism for congenital central hypoventilation

A wide range of autonomic dysfunctions, i.e. Central Hypoventilation Syndromes, Hirschsprung disease and Tumours of the Sympathetic Nervous System have been ascribed to heterozygous PHOX2B mutations in man. The PHOX2B mutations reported include polyalanine expansions in a 20 alanines tract, missense, frameshift mutations and nonsense mutation. Some genotype/phenotype correlations have been drawn, but the molecular mechanism(s) underlying them remain(s) unclear. So far, loss‐of‐function, gain‐of‐function and dominant negative effects have been proposed as disease‐causing mechanisms for polyalanine expansions. Indeed, mutant with an expanded polyalanine tract result in decreased transactivation of known target genes and protein misfolding leading to oligomerisation in vitro for all expansions and to cytoplasmic protein aggregation for longer expansions. We extended the molecular studies to other non‐polyalanine expansion mutations and show that most PHOX2B protein mutants oligomerize even in the absence of the normal 20 alanines tract. Conversely, a premature stop codon mutation in a CHS patient leads to the production of an N‐terminally truncated protein by re‐initiation of translation that does not form oligomers. Therefore, PHOX2B misfolding is not the only mechanism leading to dysfunction of the ventilatory autonomic system. © 2008 Wiley‐Liss, Inc.     

A rapid and efficient method of genotyping zebrafish mutants

In order to facilitate high throughput genotyping of zebrafish, we have developed a novel technique that uses High Resolution Melting Analysis (HRMA) to distinguish wild‐type, heterozygous mutants and homogyzous mutants. This one hour technique removes the need for restriction enzymes and agarose gels. The generated melting curve profiles are sensitive enough to detect non‐specific PCR products. We have been able to reliably genotype three classes of mutations in zebrafish, including point mutants, apc^hu745 (apc^mcr), and p53^zy7 (p53^I166T), a small deletion mutant (bap28^y75) and a retroviral insertion mutant (wdr43^hi821a). This technique can genotype individual zebrafish embryos and adults (by tail‐clip) and is applicable to other model organisms. Developmental Dynamics 238:3168–3174, 2009. © 2009 Wiley‐Liss, Inc.     

Geranylgeranyltransferase Cwg2‐Rho4/Rho5 module is implicated in the Pmk1 MAP kinase‐mediated cell wall integrity pathway in fission yeast

Pmk1, a fission yeast homologue of mammalian ERK MAPK, regulates cell wall integrity, cytokinesis, RNA granule formation and ion homeostasis. Our screen for vic (viable in the presence of immunosuppressant and chloride ion) mutants identified regulators of the Pmk1 MAPK signaling, including Cpp1 and Rho2, based on the genetic interaction between calcineurin and Pmk1 MAPK. Here, we identified the vic2‐1 mutants carrying a mis‐sense mutation in the cwg2⁺ gene encoding a beta subunit of geranylgeranyltransferase I (GGTase I), which participates in the post‐translational C‐terminal modification of several small GTPases, allowing their targeting to the membrane. Analysis of the vic2‐1/cwg2‐v2 mutant strain showed that the localization of Rho1, Rho4, Rho5 and Cdc42, both at the plasma and vacuolar membranes, was impaired in the vic2‐1/cwg2‐v2 mutant cells. In addition, Rho4 and Rho5 deletion cells exhibited the vic phenotype and cell wall integrity defects, shared phenotypes among the components of the Pmk1 MAPK pathway. Consistently, the phosphorylation of Pmk1 MAPK on heat shock was decreased in the cwg2‐v2 mutants, and rho4‐ and rho5‐null cells. Moreover, Rho4 and Rho5 associate with Pck1/Pck2. Possible roles of Cwg2, Rho4 and Rho5 in the Pmk1 signaling will be discussed.