Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis

Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.     

Molecular cytogenetic analysis in mouse sperm of chemically induced aneuploidy: studies with topoisomerase II inhibitors

The ability of two topoisomerase II (topo II) inhibitors, etoposide (VP-16) and merbarone (MER), to induce meiotic delay and aneuploidy in mouse spermatocytes was investigated. The progression from meiotic divisions to epididymal sperm was determined by injecting male mice with 5-bromo-2′-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. At 20–24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). It was found that VP-16 (50 mg/kg) treatment induced a meiotic delay of about 24 h. A significant reduction of BrdU-labelled sperm was observed at 22 days compared to the controls (VP-16 group: 14.20%; controls: 41.10%, P<0.001). At 23 and 24 days, there were no significant differences between the VP-16 and the control groups. MER (80 mg/kg) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated with 12.5, 25 and 50 mg/kg VP-16 or 15, 30 and 60 mg/kg MER. Sperm were sampled from the Caudae epididymes 24 days after VP-16 treatment or 22 days after MER treatment. Significant increases above the concurrent controls in the frequencies of total hyperhaploid sperm were found after treatment with 25, 50 mg/kg VP-16 (0.074 and 0.122% versus 0.052%) and after treatment with 60 mg/kg MER (0.098% versus 0.044%). Furthermore, significant increases in the frequencies of diploid sperm were found after treatment of mice with all three doses of VP-16 (0.024, 0.032 and 0.056% versus 0.004 and 0.00%, respectively) and with 30 and 60 mg/kg MER (0.022 and 0.05% versus 0.004 and 0.002%, respectively). All dose responses could be expressed by linear equations. The results indicate that cancer patients may stand transient risk for siring chromosomally abnormal offspring after chemotherapy with these topo II inhibitors.     

Transgenerational transmission of radiation- and chemically induced tumors and congenital anomalies in mice: studies of their possible relationship to induced chromosomal and molecular changes

This article provides a broad overview of our earlier studies on the induction of tumors and congenital anomalies in the progeny of X-irradiated or chemically treated mice and our subsequent (published, hitherto unpublished and on-going) investigations aimed at identifying potential relationships between genetic changes induced in germ cells and the adverse effects manifest as tumors and congenital anomalies using cytogenetic and molecular approaches. The earlier studies document the fact that tumors and congenital anomalies can be induced by irradiation or treatment with certain chemicals such as urethane and that these phenotypes are heritable i.e., transmitted to generations beyond the first generation. These findings support the view that transmissible induced genetic changes are involved. The induced rates of congenital abnormalities and tumors are about two orders of magnitude higher than those recorded in the literature from classical mutation studies with specific locus mutations. The cytogenetic studies addressed the question of whether there were any relationships between induced translocations and induced tumors. The available data permit the inference that gross chromosomal changes may not be involved but do not exclude smaller induced genetic changes that are beyond the resolution of the techniques used in these studies. Other work on possible relationship between visible chromosomal anomalies (in bone marrow preparations) and tumors were likewise negative. However, there were indications that some induced cytogenetic changes might underlie induced congenital anomalies, i.e., trisomies, deletions and inversions were observed in induced and transmissible congenital anomalies (such as dwarfs, tail anomalies). Studies that explored possible relationships between induction of minisatellite mutations at the Pc-3 locus and tumors were negative. However, gene expression analysis of tumor (hepatoma)-susceptible offspring of progeny descended from irradiated male mice showed abnormal expression of many genes. Of these, only very few were oncogenes. This lends some support to our hypothesis that cumulative changes in gene expression of many genes, which perform normal cellular functions, may contribute to the occurrence of tumors in the offspring of irradiated or chemically treated mice.     

Intraspecific mating with CzechII/Ei mice rescue lethality associated with loss of function mutations of the imprinted genes, Igf2r and Cdkn1c

Maternal inheritance of targeted loss of function alleles encoding either the cyclin-dependent kinase inhibitor 1C (Cdkn1c) or the insulin-like growth factor 2 receptor (Igf2r) leads to fully penetrant perinatal lethality in C57BL/6J mice due to genomic imprinting. Here, we demonstrate that there is a marked enhancement in postnatal viability of F(1) mice carrying either the ablated Igf2r ( approximately 32%) or Cdkn1c ( approximately 83%) when the paternal genome was derived from the inbred Mus musculus musculus CzechII/Ei strain. Genetic and molecular analyses indicated that the increased viability was not caused by relaxation of imprinted gene expression, but is the consequence of unidentified polygenic modifiers that are not imprinted. In the course of this study, restriction-site polymorphisms between 129S1 and CzechII/Ei in 21 imprinted and 14 biallelically expressed genes were identified. These polymorphisms may prove useful in determining the effects of different mutant backgrounds on genomic imprinting.     

Compound genetic ablation of nidogen 1 and 2 causes basement membrane defects and perinatal lethality in mice

Nidogen 1 and 2 are basement membrane glycoproteins, and previous biochemical and functional studies indicate that they may play a crucial role in basement membrane assembly. While they show a divergent expression pattern in certain adult tissues, both have a similar distribution during development. Gene knockout studies in mice demonstrated that the loss of either isoform has no effect on basement membrane formation and organ development, suggesting complementary functions. Here, we show that this is indeed the case. Deficiency of both nidogens in mice resulted in perinatal lethality. Nidogen 1 and 2 do not appear to be crucial in establishing tissue architecture during organ development; instead, they are essential for late stages of lung development and for maintenance and/or integrity of cardiac tissue. These organ defects are not compatible with postnatal survival. Ultrastructural analysis suggests that the phenotypes directly result from basement membrane changes. However, despite the ubiquitous presence of nidogens in basement membranes, defects do not occur in all tissues or in all basement membranes, suggesting a varying spectrum of roles for nidogens in the basement membrane.     

Molecular lesions associated with white gene mutations induced by I-R hybrid dysgenesis in Drosophila melanogaster

We have identified molecular lesions associated with six mutations, w^IR2 and w^IR4-8, of the white gene of Drosophila melanogaster. These mutations arose in flies subject to I-R hybrid dysgenesis. Four of the mutations give rise to coloured eyes and are associated with insertions of 5.4-kb elements indistinguishable from the I factor controlling I-R dysgenesis. The insertion associated with w^IR4 is at a site which, within the resolution of these experiments, is identical to that of two previously studied I factors. This appears to be a hot-spot for I factor insertion. We have compared the sites of these insertions with sequences complementary to white gene mRNA identified by Pirrotta and Bröckl. The hot-spot is in the fourth intron. The insertion carried by w^IR5 is either within, or just beyond, the last exon. The insertion carried by w^IR6 is near the junction of the first exon and first intron. The w^IR2 mutation is a derivative of w¹. It contains an insertion of I factor DNA within, or immediately adjacent to, the F-like element associated with w¹, and results in restoration of some eye colour. This insertion is just upstream of the start of the white mRNA. Mutations w^IR7 and w^IR8 are deletions removing mRNA coding sequences. Both determine a bleached white phenotype.     

Molecular mapping of albino deletions associated with early embryonic lethality in the mouse

The albino-deletion complex consists of more than 37 deletions that remove an area of mouse chromosome 7 including the albino coat-color locus. Previous genetic and embryological studies with five of these deletions (C11DSD, c5FR60Hg, c4FR60Hd, c2YPSj, c6H) defined at least two genes required for normal development of the embryonic and extraembryonic ectoderm of early postimplantation embryos. A molecular genetic analysis of this region has been initiated using palb18, a genomic clone that defines the D7TM18 locus that maps to a region of chromosome 7 removed by the c11DSD deletion but not by the c5FR60Hg, c4FR60Hd, c2YPSj, or c6H deletions. palb18 was obtained by chromosomal microdissection and microcloning of the wild-type albino region. A genomic clone isolated with palb18 contains a repeat sequence localized primarily to the proximal region of the five deletions. The repeat sequence hybridizes differentially to the five deletion DNAs. The patterns of hybridization associated with these DNAs were used to define the order of the proximal breakpoints as centromere-c11DSD-c2YPSj-(c5FR60Hg-c4FR60Hd)- c6H. This order was confirmed by isolation of additional single-copy sequences. The molecular probes described here should allow for identification and isolation of the deletion breakpoints and thus provide immediate access to the distal side of the deletions where the genes affecting the development of the embryonic and extraembryonic ectoderm are located.     

Hydrops fetalis, cardiovascular defects, and embryonic lethality in mice lacking the calcitonin receptor-like receptor gene

Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. To date, numerous in vitro studies have suggested that AM can mediate its biological effects through at least three different receptors. To determine the in vivo importance of the most likely candidate receptor, calcitonin receptor-like receptor, a gene-targeted knockout model of the gene was generated. Mice heterozygous for the targeted Calcrl allele appear normal, survive to adulthood, and reproduce. However, heterozygote matings fail to produce viable Calcrl-/- pups, demonstrating that Calcrl is essential for survival. Timed matings confirmed that Calcrl-/- embryos die between embryonic day 13.5 (E13.5) and E14.5 of gestation. The Calcrl-/- embryos exhibit extreme hydrops fetalis and cardiovascular defects, including thin vascular smooth muscle walls and small, disorganized hearts remarkably similar to the previously characterized AM-/- phenotype. In vivo assays of cellular proliferation and apoptosis in the hearts and vasculature of Calcrl-/- and AM-/- embryos support the concept that AM signaling is a crucial mediator of cardiovascular development. The Calcrl gene targeted mice provide the first in vivo genetic evidence that CLR functions as an AM receptor during embryonic development.     

Molecular and morphological characterization of neural tube defects in embryos of diabetic Swiss Albino mice

BACKGROUND AND RESULTS: Embryos from diabetic mice exhibit several forms of neural tube defects, including non-closure of the neural tube. In the present study, embryos collected at embryonic day 11.5 from diabetic pregnancies displayed open neural tube with architectural disruption of the surrounding tissues. The percentage of proliferating cells was found to be increased in the dorsal and ventral domains of the spinal neural tube of embryos from diabetic mice, indicating a defect in the proliferation index. We have analyzed the development of various cell types, including motoneurons, interneurons, oligodendrocytes and migrating neurons, as well as radial glial cells in the open neural tube using specific molecular markers. Immunofluorescence results revealed a significantly reduced number of Pax2+ interneurons and increased number of Isl-1+ motoneurons, as well as Olig2+ oligodendrocytes in the neural tube of embryos from diabetic mice as compared to controls. In addition, these embryos exhibited a decreased number of doublecortin positive migrating neurons and Glast/Blbp positive radial glial cells with shortened processes in the neural tube. Expression levels of several developmental control genes involved in the generation of different neuronal cell types (such as Shh, Ngn, Ngn2, Ascl1) were also found to be altered in the neural tube of embryos from diabetic mice. CONCLUSIONS: Overall, the open neural tube in embryos of diabetic mice exhibits defects in the specification of different cell types, including motoneurons and interneurons, as well as glial cells along the dorsoventral axis of the developing spinal cord. Although these defects are associated with altered expression of several development control genes, the exact mechanisms by which maternal diabetes contributes to these changes remain to be investigated.     

Alterations in the testis of hormone sensitive lipase-deficient mice is associated with decreased sperm counts, sperm motility, and fertility

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) functions as a triglyceride and cholesteryl esterase, supplying fatty acids, and cholesterol to cells. Gene-targeted HSL-deficient (HSL(-/-)) mice reveal abnormal spermatids and are infertile at 24 weeks after birth. The purpose of this study was to follow the evolution of spermatid abnormalities as HSL(-/-) mice age, characterize sperm motility in older HSL(-/-) mice, and determine if mice expressing a human testicular HSL transgene (HSL(-/-)ttg) produce normal motile sperm. In situ hybridization indicated that HSL is expressed exclusively in steps 5-16 spermatids, but not in Sertoli cells. In HSL(-/-) mice, abnormalities were evident in step 16 spermatids at 5 weeks after birth, with defects progressively increasing in spermatids with age. The defects included multinucleation of spermatids, abnormal shapes and a reduction of elongating spermatids. In older HSL(-/-) mice, sperm counts appeared reduced by 42%, but this value was lower because samples were compromised by the presence of small degenerating germ cells in addition to sperm, both of which appeared of similar size and density. Sperm motility was dramatically reduced with only 11% classified as motile in HSL(-/-) mice compared to 76-78% of sperm in wild-type and HSL(-/-)ttg mice. Sperm morphology, counts, and motility were normal in HSL(-/-)ttg mice, as was their fertility. Collectively, the data indicate that HSL deficiency results in abnormal spermatid development with defects arising at 5 weeks of age and progressively increasing at later ages. HSL(-/-) mice also show a dramatic reduction in sperm counts and motility and are infertile.     

Abnormal intranuclear and cytoplasmic formations associated with a chemically induced,transplantable chicken sarcoma

Thirty GRCH/15 tumors (a 1, 2, 5, 6-dibenzanthracene-induced chicken sarcoma) were examined in the light and the electron microscope. Associated with the sarcoma were two types of abnormal intranuclear lesions, one in the form of a vacuole, the other as an aggregate containing glycogen. In the electron microscope, one type of lesion observed showed an organized microfibrillar structure. Abnormal cytoplasmic formations occurred as massed clusters of thread-like or tubular material, which gave rise to small bodies with concentric shell structure; similar bodies were found associated with vacuoles.     

Whole genome profiling of spontaneous and chemically induced mutations in Toxoplasma gondii

Background

Next generation sequencing is helping to overcome limitations in organisms less accessible to classical or reverse genetic methods by facilitating whole genome mutational analysis studies. One traditionally intractable group, the Apicomplexa, contains several important pathogenic protozoan parasites, including the Plasmodium species that cause malaria.Here we apply whole genome analysis methods to the relatively accessible model apicomplexan, Toxoplasma gondii, to optimize forward genetic methods for chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) and ethylmethane sulfonate (EMS) at varying dosages.

Results

By comparing three different lab-strains we show that spontaneously generated mutations reflect genome composition, without nucleotide bias. However, the single nucleotide variations (SNVs) are not distributed randomly over the genome; most of these mutations reside either in non-coding sequence or are silent with respect to protein coding. This is in contrast to the random genomic distribution of mutations induced by chemical mutagenesis. Additionally, we report a genome wide transition vs transversion ratio (ti/tv) of 0.91 for spontaneous mutations in Toxoplasma, with a slightly higher rate of 1.20 and 1.06 for variants induced by ENU and EMS respectively. We also show that in the Toxoplasma system, surprisingly, both ENU and EMS have a proclivity for inducing mutations at A/T base pairs (78.6% and 69.6%, respectively).

Conclusions

The number of SNVs between related laboratory strains is relatively low and managed by purifying selection away from changes to amino acid sequence. From an experimental mutagenesis point of view, both ENU (24.7%) and EMS (29.1%) are more likely to generate variation within exons than would naturally accumulate over time in culture (19.1%), demonstrating the utility of these approaches for yielding proportionally greater changes to the amino acid sequence. These results will not only direct the methods of future chemical mutagenesis in Toxoplasma, but also aid in designing forward genetic approaches in less accessible pathogenic protozoa as well.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-354) contains supplementary material, which is available to authorized users.     

Molecular characterization of tumor associated antigen in mice exposed to a hepatocarcinogen

The present investigation is aimed to identify and characterize tumor-associated antigen (TAA) in animals exposed to hepatocarcinogen. Swiss albino mice showed an enhanced expression of an approximately 58 kDa glycoprotein in liver cells upon exposure to a potential hepatocarcinogen N-nitrosodibutylamine (DBN). Carcinogenesis induction in mice was monitored by assays of -glutamyl transpeptidase (GGT), acetylcholine esterase (AChE), glutathione-S-transferase (GST) activities and the level of glutathione (GSH) in liver. DBN treated animals showed cell distortion and extensive necrosis as observed by histological examination. The over-expressed TAA was purified by ion-exchange chromatography and further characterized by SDS-PAGE. The carbohydrate contents and glycan linkage to the polypeptide backbone was analyzed by using the DIG glycan differentiation and de-glycosylation kits. The glycoprotein has glycan chains that are N-linked via asparagines to the polypeptide backbone. It was also observed that the molecule is rich in sialic acid residues with a significantly high carbohydrate to protein ratio (2:1). The over-expressed high molecular weight glycoprotein TAA was found to be highly immunogenic and could eventually be used to induce immune response in order to counter tumor regression. (Mol Cell Biochem 271: 177–188, 2005)     

Disruption of the phosphatidylserine decarboxylase gene in mice causes embryonic lethality and mitochondrial defects

Most of the phosphatidylethanolamine (PE) in mammalian cells is synthesized by two pathways, the CDP-ethanolamine pathway and the phosphatidylserine (PS) decarboxylation pathway, the final steps of which operate at spatially distinct sites, the endoplasmic reticulum and mitochondria, respectively. We investigated the importance of the mitochondrial pathway for PE synthesis in mice by generating mice lacking PS decarboxylase activity. Disruption of Pisd in mice resulted in lethality between days 8 and 10 of embryonic development. Electron microscopy of Pisd-/- embryos revealed large numbers of aberrantly shaped mitochondria. In addition, fluorescence confocal microscopy of Pisd-/- embryonic fibroblasts showed fragmented mitochondria. PS decarboxylase activity and mRNA levels in Pisd+/- tissues were approximately one-half of those in wild-type mice. However, heterozygous mice appeared normal, exhibited normal vitality, and the phospholipid composition of livers, testes, brains, and of mitochondria isolated from livers, was the same as in wild-type littermates. The amount and activity of a key enzyme of the CDP-ethanolamine pathway for PE synthesis, CTP:phosphoethanolamine cytidylyltransferase, were increased by 35-40 and 100%, respectively, in tissues of Pisd+/- mice, as judged by immunoblotting; PE synthesis from [3H]ethanolamine was correspondingly increased in hepatocytes. We conclude that the CDP-ethanolamine pathway in mice cannot substitute for a lack of PS decarboxylase during development. Moreover, elimination of PE production in mitochondria causes fragmented, misshapen mitochondria, an abnormality that likely contributes to the embryonic lethality.     

Expression of myc, fos and Ha-ras associated with chemically induced cell proliferation in the rat liver

Abstract. Events secondary to induced cell proliferation may play a role in the carcinogenic process. These studies investigated the expression of genes associated with growth control in response to two types of cell proliferation stimuli in the livers of male F344 rats. Regenerative hepatocyte proliferation after partial hepatectomy or a single dose of carbon tetrachloride, and mitogenic liver hyperplasia induced by a single dose of pheno-barbital or WY-14,643 were assessed by thymidine incorporation and quantitative autoradiography. The expression of myc, fos , and Ha- ras was evaluated by Northern blot analysis of liver derived poly(A)⁺ mRNA from these same animals. After each treatment, the level of hepatocyte proliferation (labelling index 4–32%) was observed to peak between 24 and 48 h and return to control values by 8 days. In every case, a peak in myc expression was seen between 0.5 and 18 h depending on the proliferative stimulus treatment. A large peak in fos expression was seen at 0.5–2 h but only with the cytotoxic and regenerative proliferative treatments partial hepatectomy or carbon tetrachloride. A broad peak in Ha -ras expression was observed 12 to 36 h after each treatment. These data demonstrate transient expression of these genes following the synchronous induction of hepatocyte proliferation. The increased expression of fos upon treatment with cytotoxicants, but not mitogens, suggests different modes of growth regulation that may be important in understanding the induction of cell proliferation by these two types of agents.     

Molecular cloning and characterization of Pals, proteins associated with mLin-7

In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologues of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins that can bind to the amino terminus of mLin-7 and cloned the genes encoding these proteins using bacterial expression cloning. We call these proteins Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, we have identified the conserved residues among these proteins crucial for mLin-7 binding. Two of these proteins, Pals1 and Pals2, are newly described. Pals1 consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7.     

Compensatory defects associated with mutations in Hoxa1 restore normal palatogenesis to Hoxa2 mutants.

The rhombencephalic neural crest play several roles in craniofacial development. They give rise to the cranial sensory ganglia and much of the craniofacial skeleton, and are vital for patterning of the craniofacial muscles. The loss of Hoxa1 or Hoxa2 function affects the development of multiple neural crest-derived structures. To understand how these two genes function together in craniofacial development, an allele was generated that disrupts both of these linked genes. Some of the craniofacial defects observed in the double mutants were additive combinations of those that exist in each of the single mutants, indicating that each gene functions independently in the formation of these structures. Other defects were found only in the double mutants demonstrating overlapping or synergistic functions. We also uncovered multiple defects in the attachments and trajectories of the extrinsic tongue and hyoid muscles in Hoxa2 mutants. Interestingly, the abnormal trajectory of two of these muscles, the styloglossus and the stylohyoideus, blocked the attachment of the hyoglossus to the greater horn of the hyoid, which in turn correlated exactly with the presence of cleft palate in Hoxa2 mutants. We suggest that the hyoglossus, whose function is to depress the lateral edges of the tongue, when unable to make its proper attachment to the greater horn of the hyoid, forces the tongue to adopt an abnormal posture which blocks closure of the palatal shelves. Unexpectedly, in Hoxa1/Hoxa2 double mutants, the penetrance of cleft palate is dramatically reduced. We show that two compensatory defects, associated with the loss of Hoxa1 function, restore normal attachment of the hyoglossus to the greater horn thereby allowing the palatal shelves to lift and fuse above the flattened tongue.