BRUCE, a giant E2/E3 ubiquitin ligase and inhibitor of apoptosis protein of the trans-Golgi network, is required for normal placenta development and mouse survival

BRUCE is a highly conserved 528-kDa peripheral membrane protein of the trans-Golgi network. Owing to the presence of an N-terminal single baculovirus inhibitor repeat, BRUCE functions as an inhibitor of apoptosis protein and blocks apoptosis when overexpressed. In addition, due to the presence of a C-terminal ubiquitin-conjugating domain, BRUCE can covalently attach ubiquitin to substrates. Here we report the generation and characterization of BRUCE-deficient mice. Complete inactivation of the BRUCE gene resulted in perinatal lethality and growth retardation discernible after embryonic day 14. The growth defect is linked to impaired placental development and may be caused by insufficient oxygen and nutrient transfer across the placenta. Chorioallantoic placentation initiated normally, but the mutant placenta showed an impaired maturation of the labyrinth layer and a significant reduction of the spongiotrophoblast. No evidence for an elevated apoptosis rate was detectable in embryonic and extraembryonic tissues and in knockout fibroblasts. Thus, although BRUCE is broadly expressed in embryonic, extraembryonic, and adult mouse tissues, this bifunctional protein might play a unique role in normal trophoblast differentiation and embryonic survival.     

Targeted deletion of the tub mouse obesity gene reveals that tubby is a loss-of-function mutation

The mouse tubby phenotype is characterized by maturity-onset obesity accompanied by retinal and cochlear degeneration. A positional cloning effort to find the gene responsible for this phenotype led to the identification of tub, a member of a novel gene family of unknown function. A splice defect mutation in the 3' end of the tub gene, predicted to disrupt the C terminus of the Tub protein, has been implicated in the genesis of the tubby phenotype. It is not clear, however, whether the Tub mutant protein retains any biological activity, or perhaps has some dominant function, nor is it established that the tubby mutation is itself responsible for all of the observed tubby phenotypes. To address these questions, we generated tub-deficient mice and compared their phenotype to that of tubby mice. Our results demonstrate that tubby is a loss-of-function mutation of the tub gene and that loss of the tub gene is sufficient to give rise to the full spectrum of tubby phenotypes. We also demonstrate that loss of photoreceptors in the retina of tubby and tub-deficient mice occurs by apoptosis. In addition, we show that Tub protein expression is not significantly altered in the ob, db, or melanocortin 4 receptor-deficient mouse model of obesity.     

Partial loss of Ascl2 function affects all three layers of the mature placenta and causes intrauterine growth restriction

Several imprinted genes have been implicated in the regulation of placental function and embryonic growth. On distal mouse chromosome 7, two clusters of imprinted genes, each regulated by its own imprinting center (IC), are separated by a poorly characterized region of 280 kb (the IC1–IC2 interval). We previously generated a mouse line in which this IC1–IC2 interval has been deleted (Del^7AI allele) and found that maternal inheritance of this allele results in low birth weights in newborns. Here we report that Del^7AI causes a partial loss of Ascl2, a maternally expressed gene in the IC2 cluster, which when knocked out leads to embryonic lethality at midgestation due to a lack of spongiotrophoblast formation. The hypomorphic Ascl2 allele causes embryonic growth restriction and an associated placental phenotype characterized by a reduction in placental weight, reduced spongiotrophoblast population, absence of glycogen cells, and an expanded trophoblast giant cell layer. We also uncovered severe defects in the labyrinth layer of maternal mutants including increased production of the trilaminar labyrinth trophoblast cell types and a disorganized labyrinthine vasculature. Our results have important implications for our understanding of the role played by the spongiotrophoblast layer during placentation and show that regulation of the dosage of the imprinted gene Ascl2 can affect all three layers of the chorio-allantoic placenta.     

The absence of mitochondrial thioredoxin 2 causes massive apoptosis,exencephaly, and early embryonic lethality in homozygous mice

Thioredoxin 2 (Trx-2) is a small redox protein containing the thioredoxin active site Trp-Cys-Gly-Pro-Cys that is localized to the mitochondria by a mitochondrial leader sequence and encoded by a nuclear gene (Trx-2). Trx-2 plays an important role in cell viability and the regulation of apoptosis in vitro. To investigate the role of Trx-2 in mouse development, we studied the phenotype of mice that have the Trx-2 gene silenced by mutational insertion. Homozygous mutant embryos do not survive to birth and die after implantation at Theiler stage 15/16. The homozygous mutant embryos display an open anterior neural tube and show massively increased apoptosis at 10.5 days postcoitus and are not present by 12.5 days postcoitus. The timing of the embryonic lethality coincides with the maturation of the mitochondria, since they begin oxidative phosphorylation during this stage of embryogenesis. In addition, embryonic fibroblasts cultured from homozygous Trx-2-null embryos were not viable. Heterozygous mice are fertile and have no discernible phenotype visible by external observation, despite having decreased Trx-2 mRNA and protein. These results show that the mitochondrial redox protein Trx-2 is required for normal development of the mouse embryo and for actively respiring cells.     

Missense mutation in the tubulin-specific chaperone E (Tbce) gene in the mouse mutant progressive motor neuronopathy,a model of human motoneuron disease

Progressive motor neuronopathy (pmn) mutant mice have been widely used as a model for human motoneuron disease. Mice that are homozygous for the pmn gene defect appear healthy at birth but develop progressive motoneuron disease, resulting in severe skeletal muscle weakness and respiratory failure by postnatal week 3. The disease starts at the motor endplates, and then leads to axonal loss and finally to apoptosis of the corresponding cell bodies. We localized the genetic defect in pmn mice to a missense mutation in the tubulin-specific chaperone E (Tbce) gene on mouse chromosome 13. The human orthologue maps to chromosome 1q42.3. The Tbce gene encodes a protein (cofactor E) that is essential for the formation of primary alpha-tubulin and beta-tubulin heterodimeric complexes. Isolated motoneurons from pmn mutant mice exhibit shorter axons and axonal swelling with irregularly structured beta-tubulin and tau immunoreactivity. Thus, the pmn gene mutation provides the first genetic evidence that alterations in tubulin assembly lead to retrograde degeneration of motor axons, ultimately resulting in motoneuron cell death.     

Progressive expression of trophoblast-specific genes during formation of mouse trophoblast giant cells in vitro

The expression of a battery of trophoblast-specific mRNAs was studied during trophectoderm development in vivo and in vitro to assess the use of these mRNAs as markers of trophoblast differentiation and to examine lineage relationships between various trophectoderm derivatives. In situ hybridization of sectioned day 6.5–18.5 mouse embryos localized mRNAs for mouse placental lactogens I and II and mouse proliferin (PLF) to trophoblast giant cells and proliferin-related protein mRNA to the spongiotrophoblast and giant cell layers. A fifth marker, cDNA 4311, was found only in spongiotrophoblast. Day 3.5 blastocyst outgrowths and day 7.5 diploid extraembryonic ectoderm (EX) and ectoplacental cone (EPC) were then cultured to produce polyploid giant cells in vitro. Cultures were processed for in situ hybridization after 2, 4, or 6 days. EX and EPC both formed secondary giant cells, which expressed all markers in the same sequence as was observed in vivo, and primary giant cells in blastocyst outgrowths expressed the early giant cell markers PLF and PL-I on days 4 and 6 of culture. EPC progressed through the sequence 2 days ahead of EX, indicating commitment of EPC to giant cell formation. These results suggest that EX, EPC, and primary and secondary giant cells all share in a common pathway of differentiation and that the highly ordered sequence of gene expression characteristic of this pathway occurs similarly in vivo and in vitro. © 1993 Wiley-Liss, Inc.     

Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders

During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders.     

Loss of hairless confers susceptibility to UVB-induced tumorigenesis via disruption of NF-kappaB signaling

In order to model squamous cell carcinoma development in vivo, researchers have long preferred hairless mouse models such as SKH-1 mice that have traditionally been classified as 'wild-type' mice irrespective of the genetic factors underlying their hairless phenotype. The work presented here shows that mutations in the Hairless (Hr) gene not only result in the hairless phenotype of the SKH-1 and Hr(-/-) mouse lines but also cause aberrant activation of NFκB and its downstream effectors. We show that in the epidermis, Hr is an early UVB response gene that regulates NFκB activation and thereby controls cellular responses to irradiation. Therefore, when Hr expression is decreased in Hr mutant animals there is a corresponding increase in NFκB activity that is augmented by UVB irradiation. This constitutive activation of NFκB in the Hr mutant epidermis leads to the stimulation a large variety of downstream effectors including the cell cycle regulators cyclin D1 and cyclin E, the anti-apoptosis protein Bcl-2, and the pro-inflammatory protein Cox-2. Therefore, Hr loss results in a state of uncontrolled epidermal proliferation that promotes tumor development, and Hr mutant mice should no longer be considered merely hairless 'wild-type' mice. Instead, Hr is a crucial UVB response gene and its loss creates a permissive environment that potentiates increased tumorigenesis.     

Inactivation of a testis-specific Lis1 transcript in mice prevents spermatid differentiation and causes male infertility

Lis1 protein is the non-catalytic component of platelet-activating factor acetylhydrolase 1b (PAF-AH 1B) and associated with microtubular structures. Hemizygous mutations of the LIS1 gene cause type I lissencephaly, a brain abnormality with developmental defects of neuronal migration. Lis1 is also expressed in testis, but its function there has not been determined. We have generated a mouse mutant (LIS1GT/GT) by gene trap integration leading to selective disruption of a Lis1 splicing variant in testis. Homozygous mutant males are infertile with no other apparent phenotype. We demonstrate that Lis1 is predominantly expressed in spermatids, and spermiogenesis is blocked when Lis1 is absent. Mutant spermatids fail to form correct acrosomes and nuclei appear distorted in size and shape. The tissue architecture in mutant testis appears severely disturbed displaying collapsed seminiferous tubules, mislocated germ cells, and increased apoptosis. These results provide evidence for an essential and hitherto uncharacterized role of the Lis1 protein in spermatogenesis, particularly in the differentiation of spermatids into spermatozoa.     

The role of p53 and pRB in apoptosis and cancer.

Loss of function of both the p53 pathway and the retinoblastoma protein (pRB) pathway plays a significant role in the development of most human cancers. Loss of pRB results in deregulated cell proliferation and apoptosis, whereas loss of p53 desensitizes cells to checkpoint signals, including apoptosis. In the past two years, mouse genetics and gene expression profiling have led to major advances in our understanding of how the pRB and p53 pathways regulate apoptosis and thus the development of tumours.     

X-Linked inhibitor of apoptosis protein is involved in mutant SOD1-mediated neuronal degeneration

Mutations in the superoxide dismutase 1 (SOD1) gene cause the degeneration of motor neurons in familial amyotrophic lateral sclerosis (FALS). An apoptotic process including caspase-1 and -3 has been shown to participate in the pathogenesis of FALS transgenic (Tg) mouse model. Here we report that IAP proteins, potent inhibitors of apoptosis, are involved in the FALS Tg mouse pathologic process. The levels of X-linked inhibitor of apoptosis protein (XIAP) mRNA and protein were significantly decreased in the spinal cord of symptomatic G93A-SOD1 Tg mice compared with littermates. In contrast, the levels of cIAP-1 mRNA and protein were increased in symptomatic G93A-SOD1 Tg mice, whereas the levels of cIAP-2 mRNA and protein were unchanged. In situ hybridization showed that the expression of XIAP was remarkably reduced in the motor neurons of Tg mice, and the expression of cIAP-1 was strongly increased in the reactive astrocytes of Tg mice. Overexpression of XIAP markedly inhibited the cell death and caspase-3 activity in the neuro2a cells expressing mutant SOD1. Deletional mutant analysis revealed that the N-terminal domain of XIAP, the BIR1-2 domains, was essential for this inhibitory activity. These results suggest that XIAP plays a role in the apoptotic mechanism in the progression of disease in mutant SOD1 Tg mice and holds therapeutic possibilities for FALS.     

Dysfunction of the ubiquitin ligase ube3a may be associated with synaptic pathophysiology in a mouse model of huntington disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by progressive cognitive, psychiatric, and motor symptoms. The disease is caused by abnormal expansion of CAG repeats in the gene encoding huntingtin, but how mutant huntingtin leads to early cognitive deficits in HD is poorly understood. Here, we demonstrate that the ubiquitin ligase Ube3a, which is implicated in synaptic plasticity and involved in the clearance of misfolded polyglutamine protein, is strongly recruited to the mutant huntingtin nuclear aggregates, resulting in significant loss of its functional pool in different regions of HD mouse brain. Interestingly, Arc, one of the substrates of Ube3a linked with synaptic plasticity, is also associated with nuclear aggregates, although its synaptic level is increased in the hippocampus and cortex of HD mouse brain. Different regions of HD mouse brain also exhibit decreased levels of AMPA receptors and various pre- and postsynaptic proteins, which could be due to the partial loss of function of Ube3a. Transient expression of mutant huntingtin in mouse primary cortical neurons further demonstrates recruitment of Ube3a into mutant huntingtin aggregates, increased accumulation of Arc, and decreased numbers of GluR1 puncta in the neuronal processes. Altogether, our results suggest that the loss of function of Ube3a might be associated with the synaptic abnormalities observed in HD.     

Molecular cloning of a human cDNA encoding a novel protein, DAD1, whose defect causes apoptotic cell death in hamster BHK21 cells.

The tsBN7 cell line, one of the mutant lines temperature sensitive for growth which have been isolated from the BHK21 cell line, was found to die by apoptosis following a shift to the nonpermissive temperature. The induced apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, but not by the bcl-2-encoded protein. By DNA-mediated gene transfer, we cloned a cDNA that complements the tsBN7 mutation. It encodes a novel hydrophobic protein, designated DAD1, which is well conserved (100% identical amino acids between humans and hamsters). By comparing the base sequences of the parental BHK21 and tsBN7 DAD1 cDNAs, we found that the DAD1-encoding gene is mutated in tsBN7 cells. The DAD1 protein disappeared in tsBN7 cells following a shift to the nonpermissive temperature, suggesting that loss of the DAD1 protein triggers apoptosis.     

Functional mutation of SMAC/DIABLO, encoding a mitochondrial proapoptotic protein, causes human progressive hearing loss DFNA64

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLO(S71L) mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLO(S71L) might cause mitochondrial dysfunction.     

Cytokeratins 8 and 19 in the mouse placental development

To investigate the expression and biological roles of cytokeratin 19 (K19) in development and in adult tissues, we inactivated the mouse K19 gene (Krt1-19) by inserting a bacterial beta-galactosidase gene (lacZ) by homologous recombination in embryonic stem cells, and established germ line mutant mice. Both heterozygous and homozygous mutant mice were viable, fertile, and appeared normal. By 7.5-8.0 days post coitum (dpc), heterozygous mutant embryos expressed lacZ in the notochordal plate and hindgut diverticulum, reflecting the fact that the notochord and the gut endoderm are derived from the axial mesoderm-originated cells. In the adult mutant, lacZ was expressed mainly in epithelial tissues. To investigate the possible functional cooperation and synergy between K19 and K8, we then constructed compound homozygous mutants, whose embryos died approximately 10 dpc. The lethality resulted from defects in the placenta where both K19 and K8 are normally expressed. As early as 9. 5 dpc, the compound mutant placenta had an excessive number of giant trophoblasts, but lacked proper labyrinthine trophoblast or spongiotrophoblast development, which apparently caused flooding of the maternal blood into the embryonic placenta. These results indicate that K19 and K8 cooperate in ensuring the normal development of placental tissues.     

Connexin31-deficiency in mice causes transient placental dysmorphogenesis but does not impair hearing and skin differentiation

Mutations in the human GJB3 gene that codes for Connexin31 (Cx31), a protein subunit of gap junction channels, have recently been reported to cause deafness and the skin disorder erythrokeratodermia variabilis. To study the function of this gene in mice, we generated animals with targeted replacement of the Cx31 gene (Gjb3) by a lacZ reporter gene. Although homozygous Cx31-deficient adult mice (Gjb3(-/-)) were found among the offspring of heterozygous Cx31-deficient parents (Gjb3(+/-)), 60% of the animals expected according to Mendelian inheritance were lost between ED 10.5 and 13.5. Placentas of Gjb3(-/-) embryos at ED 9.5 were smaller than controls as a result of severely reduced labyrinth and spongiotrophoblast size. From ED 10.5 onward, placentas of surviving Gjb3(-/-) embryos recovered progressively and reached normal size and morphology by ED 18.5. This corresponds to a time period in which another connexin isoform, Connexin43, is upregulated in spongiotrophoblast cells of Cx31-deficient and control placentas. No morphological or functional defects of skin or inner ear were observed in surviving adult Gjb3(-/-) mice. We conclude that Cx31 is essential for early placentation but can be compensated for by other connexins in the embryo proper and adult mouse.