In vivo overexpression of Dad1, the defender against apoptotic death-1, enhances T cell proliferation but does not protect against apoptosis.

The Dad1 protein has been shown to play a role in prevention of apoptosis in certain cell types. Dad1 is also a subunit of the oligosaccharyltransferase enzyme complex that initiates N-linked glycosylation. It is encoded by a gene located adjacent to the TCR alpha and delta genes on mouse chromosome 14. We have investigated the role of Dad1 during T cell development and activation. We observe that endogenous Dad1 levels are modulated during T cell development to reach maximal expression in mature thymocytes. Transgenic mice that overexpress Dad1 in both the thymus and peripheral immune system have been generated. Apoptosis of thymocytes from such mice is largely unaffected, but peripheral T cells display hyperproliferation in response to stimuli. Therefore, the linkage between the TCR and Dad1 genes may have important consequences for T cell function.     

Deletion of Dad1 in mice induces an apoptosis-associated embryonic death

Dad1 is a putative anti-apoptosis gene identified in several distantly related organisms. Expression of Dad1 in transfected cells inhibits apoptosis in vitro. To determine whether Dad1 has a similar function in vivo, we used gene targeting to delete Dad1. Heterozygous adult mice (+/-) show no obvious phenotype or abnormalities, but genotype analysis of over 100 offspring from heterozygous matings detected no weanling, homozygous Dad1 null (-/-) mice. Subsequent analysis of embryos from heterozygous matings detected Dad1 null (-/-) embryos at E3.5 but no later, suggesting Dad1 is required for development beyond the late blastocyst stage. Increased levels of apoptosis were observed in cultured embryos lacking a functional copy of the gene, consistent with an anti-apoptotic role for Dad1.     

Inhibition of N-linked glycosylation causes apoptosis in hamster BHK21 cells

The tsBN7 cell line is one of the temperature-sensitive mutants for cell proliferation derived from hamster BHK21 cell line. It has a mutation in the DAD1 gene and enters apoptosis at the restrictive temperature of 39 degrees C. The defect of Dad1p causes a loss of N-linked glycosylation; therefore, it was thought that an inhibition of N-linked glycosylation induced apoptosis.However, tunicamycin, a potent inhibitor of N-linked glycosylation, had not caused apoptosis in wild-type BHK21 cells. In order to clarify this discrepancy, wild-type BHK21 cells treated with tunicamycin and tsBN7 cells incubated at 39.5 degrees C were examined by the annexin V staining and TUNEL methods. Both methods showed that tunicamycin induces apoptosis in wild-type BHK21 cells, similar to the defect of Dad1p. Thus, we concluded that loss of N-linked glycosylation causes apoptosis.     

PTBP1 is required for embryonic development before gastrulation

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.     

Cse1l is essential for early embryonic growth and development

The CSE1L gene, the human homologue of the yeast chromosome segregation gene CSE1, is a nuclear transport factor that plays a role in proliferation as well as in apoptosis. CSE1 and CSE1L are essential genes in Saccharomyces cerevisiae and mammalian cells, as shown by conditional yeast mutants and mammalian cell culture experiments with antisense-mediated depletion of CSE1L. To analyze whether CSE1L is also essential in vivo and whether its absence can be compensated for by other genes or mechanisms, we have cloned the murine CSE1L gene (Cse1l) and analyzed its tissue- and development-specific expression: Cse1l was detected at embryonic day 7.0 (E7.0), E11.0, E15.0, and E17.0, and in adults, high expression was observed in proliferating tissues. Subsequently, we inactivated the Cse1l gene in embryonic stem cells to generate heterozygous and homozygous knockout mice. Mice heterozygous for Cse1l appear normal and are fertile. However, no homozygous pups were born after interbreeding of heterozygous mice. In 30 heterozygote interbreeding experiments, 50 Cse1l wild-type mice and 100 heterozygotes were born but no animal with both Cse1l alleles deleted was born. Embryo analyses showed that homozygous mutant embryos were already disorganized and degenerated by E5.5. This implicates with high significance (P < 0.0001, Pearson chi-square test) an embryonically lethal phenotype of homozygous murine CSE1 deficiency and suggests that Cse1l plays a critical role in early embryonic development.     

玉米种子发育过程中Dad1基因的表达(英文)

Dad1是一个高度保守的细胞程序性死亡抑制基因。以拟南芥Dad1 (AtDad1) cDNA为模板,利用体外RT-PCR对其进行地高辛标记。以产物反义RNA作探针 (顺义RNA为对照),对Dad1在玉米种子发育过程中的时空表达图谱进行了研究。结果表明Dad1在一些衰老组织如子房壁、珠被(Fig.1, B&C)、成熟的胚乳 (Fig.1, E) 以及脱落的根冠细胞 (Fig.2 A)中表达量均较高,同时在新陈代谢活跃的组织如根尖分生组织细胞 (Fig.2, A&B)、发育的子叶(Fig.2, C&D)、雌性生殖细胞 (Fig.1 C)以及发育的胚乳细胞(Fig.1 C)中表达量也较高。结论:同其它植物中的Dad1同源基因一样,玉米中的Dad1基因在胚胎发育及胚乳PCD等多种发育过程中也可能起重要作用。     

PINCH1 plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes

PINCH1, an adaptor protein composed of five LIM domains, mediates protein-protein interactions and functions as a component of the integrin-integrin-linked kinase (ILK) complex. The integrin-ILK signaling complex plays a pivotal role in cell motility, proliferation, and survival during embryonic development of many animal species. To elucidate the physiological function of PINCH1 in mouse embryonic development, we have deleted the mouse PINCH1 gene by homologous recombination. Mice heterozygous for PINCH1 are viable and indistinguishable from wild-type littermates. However, no viable homozygous offspring were observed from PINCH1+/- intercrosses. Histological analysis of homozygous mutant embryos revealed that they had a disorganized egg cylinder by E5.5, which degenerated by E6.5. Furthermore, E5.5 PINCH1-/- embryos exhibited decreased cell proliferation and excessive cell death. We have also generated and analyzed mice in which PINCH1 has been specifically deleted in ventricular cardiomyocytes. These mice exhibit no basal phenotype, with respect to mouse survival, cardiac histology, or cardiac function as measured by echocardiography. Altogether, these data indicate that PINCH1 plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes.     

Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development.

Complex asparagine (N)-linked oligosaccharides appear late in phylogeny and are highly regulated in vertebrates. Variations in these structures are found on the majority of cell-surface and secreted proteins. Complex N-linked oligosaccharide biosynthesis is initiated in the Golgi apparatus by the action of Mgat-1-encoded UDP-N-acetylglucosamine:alpha-3-D- mannoside beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI). To determine if these structures govern ontogenic processes in mammals, mouse embryos were generated that lacked a functional Mgat-1 gene. Inactivation of both Mgat-1 alleles produced deficiencies in GlcNAc-TI activity and complex N-linked oligosaccharides. Embryonic lethality occurred by day 10.5, thus establishing that complex N-linked oligosaccharides are required during post-implantation development. Remarkably, embryonic development proceeded into day 9 with the differentiation of multiple cell types. Complex N-linked oligosaccharides are important for morphogenic processes as neural tube formation, vascularization and the determination of left-right body plan asymmetry were impaired in the absence of a functional Mgat-1 gene.     

The Decreased apical dominance1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE8 gene affects branch production and plays a role in leaf senescence, root growth, and flower development

Carotenoids and carotenoid cleavage products play an important and integral role in plant development. The Decreased apical dominance1 (Dad1)/PhCCD8 gene of petunia (Petunia hybrida) encodes a hypothetical carotenoid cleavage dioxygenase (CCD) and ortholog of the MORE AXILLARY GROWTH4 (MAX4)/AtCCD8 gene. The dad1-1 mutant allele was inactivated by insertion of an unusual transposon (Dad-one transposon), and the dad1-3 allele is a revertant allele of dad1-1. Consistent with its role in producing a graft-transmissible compound that can alter branching, the Dad1/PhCCD8 gene is expressed in root and shoot tissue. This expression is upregulated in the stems of the dad1-1, dad2, and dad3 increased branching mutants, indicating feedback regulation of the gene in this tissue. However, this feedback regulation does not affect the root expression of Dad1/PhCCD8. Overexpression of Dad1/PhCCD8 in the dad1-1 mutant complemented the mutant phenotype, and RNA interference in the wild type resulted in an increased branching phenotype. Other differences in phenotype associated with the loss of Dad1/PhCCD8 function included altered timing of axillary meristem development, delayed leaf senescence, smaller flowers, reduced internode length, and reduced root growth. These data indicate that the substrate(s) and/or product(s) of the Dad1/PhCCD8 enzyme are mobile signal molecules with diverse roles in plant development.     

Requirement for the Xrcc1 DNA base excision repair gene during early mouse development

Surveillance and repair of DNA damage are essential for maintaining the integrity of the genetic information that is needed for normal development. Several multienzyme pathways, including the excision repair of damaged or missing bases, carry out DNA repair in mammals. We determined the developmental role of the X-ray cross-complementing (Xrcc)-1 gene, which is central to base excision repair, by generating a targeted mutation in mice. Heterozygous matings produced Xrcc1-/- embryos at early developmental stages, but not Xrcc1-/- late-stage fetuses or pups. Histology showed that mutant (Xrcc1-/-) embryos arrested at embryonic day (E) 6.5 and by E7.5 were morphologically abnormal. The most severe abnormalities observed in mutant embryos were in embryonic tissues, which showed increased cell death in the epiblast and an altered morphology in the visceral embryonic endoderm. Extraembryonic tissues appeared relatively normal at E6.5-7.5. Even without exposure to DNA-damaging agents, mutant embryos showed increased levels of unrepaired DNA strand breaks in the egg cylinder compared with normal embryos. Xrcc1-/- cell lines derived from mutant embryos were hypersensitive to mutagen-induced DNA damage. Xrcc1 mutant embryos that were also made homozygous for a null mutation in Trp53 underwent developmental arrest after only slightly further development, thus revealing a Trp53-independent mechanism of embryo lethality. These results show that an intact base excision repair pathway is essential for normal early postimplantation mouse development and implicate an endogenous source of DNA damage in the lethal phenotype of embryos lacking this repair capacity.     

Targeted disruption of the mouse Asna1 gene results in embryonic lethality

The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Homologues of the bacterial ArsA ATPase are widespread in nature. We had earlier identified the mouse homologue (Asna1) that exhibits 27% identity to the bacterial ArsA ATPase. To identify the physiological role of the protein, heterozygous Asna1 knockout mice (Asna1+/-) were generated by homologous recombination. The Asna1+/- mice displayed similar phenotype as the wild-type mice. However, early embryonic lethality was observed in homozygous Asna1 knockout embryos, between E3.5 (E=embryonic day) and E8.5 stage. These findings indicate that Asna1 plays a crucial role during early embryonic development.     

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is required for branching morphogenesis in the chorioallantoic placenta

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-associated Kunitz-type serine proteinase inhibitor that was initially identified as a potent inhibitor of hepatocyte growth factor activator. HAI-1 is also a cognate inhibitor of matriptase, a membrane-associated serine proteinase. HAI-1 is expressed predominantly in epithelial cells in the human body. Its mRNA is also abundant in human placenta, with HAI-1 specifically expressed by villous cytotrophoblasts. In order to address the precise roles of HAI-1 in vivo, we generated HAI-1 mutant mice by homozygous recombination. Heterozygous HAI-1+/- mice underwent normal organ development. However, homozygous HAI-1-/- mice experienced embryonic lethality which became evident at embryonic day 10.5 postcoitum (E10.5). As early as E9.5, HAI-1-/- embryos showed growth retardation that did not reflect impaired cell proliferation but resulted instead from failed placental development and function. Histological analysis revealed severely impaired formation of the labyrinth layer, in contrast all other placental layers, such as the spongiotrophoblast layer and giant cell layer, which were formed. Our results indicate that mouse HAI-1 is essential for branching morphogenesis in the chorioallantoic placenta and lack of HAI-1 function may result in placental failure.     

Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation

The Notch signaling pathway is an evolutionarily conserved signaling mechanism and mutations in its components disrupt cell fate specification and embryonic development in many organisms. To analyze the in vivo role of the Notch3 gene in mice, we created a deletion allele by gene targeting. Embryos homozygous for this mutation developed normally and homozygous mutant adults were viable and fertile. We also examined whether we could detect genetic interactions during early embryogenesis between the Notch3 mutation and a targeted mutation of the Notch1 gene. Double homozygous mutant embryos exhibited defects normally observed in Notch1-deficient embryos, but we detected no obvious synergistic effects in the double mutants. These data demonstrate that the Notch3 gene is not essential for embryonic development or fertility in mice, and does not have a redundant function with the Notch1 gene during early embryogenesis.     

Disruption of muREC2/RAD51L1 in mice results in early embryonic lethality which can Be partially rescued in a p53(-/-) background

muREC2/RAD51L1 is a radiation-inducible gene that regulates cell cycle progression. To elucidate the biological function of muREC2/RAD51L1, the gene was disrupted in embryonic stem cells by homologous recombination. Mice heterozygous for muREC2/RAD51L1 appear normal and fertile; however, no homozygous pups were born after interbreeding of heterozygous mice. Timed pregnancy studies showed that homozygous mutant embryos were severely retarded in growth as early as ca. 5 days gestation (E5.5) and were completely resorbed by E8.5. Mutant blastocyst outgrowth was also severely impaired in a double-knockout embryo, but embryonic development did progress further in a p53-null background. These results suggest that muREC2/RAD51L1 plays a role in cell proliferation and early embryonic development, perhaps through interaction with p53.     

Overexpression of Smurf1 negatively regulates mouse embryonic lung branching morphogenesis by specifically reducing Smad1 and Smad5 proteins

Early embryonic lung branching morphogenesis is regulated by many growth factor-mediated pathways. Bone morphogenetic protein 4 (BMP4) is one of the morphogens that stimulate epithelial branching in mouse embryonic lung explant culture. To further understand the molecular mechanisms of BMP4-regulated lung development, we studied the biological role of Smad-ubiquitin regulatory factor 1 (Smurf1), an ubiquitin ligase specific for BMP receptor-regulated Smads, during mouse lung development. The temporo-spatial expression pattern of Smurf1 in mouse embryonic lung was first determined by quantitative real-time PCR and immunohistochemistry. Overexpression of Smurf1 in airway epithelial cells by intratracheal introduction of recombinant adenoviral vector dramatically inhibited embryonic day (E) 11.5 lung explant growth in vitro. This inhibition of lung epithelial branching was restored by coexpression of Smad1 or by addition of soluble BMP4 ligand into the culture medium. Studies at the cellular level show that overexpression of Smurf1 reduced epithelial cell proliferation and differentiation, as documented by reduced PCNA-positive cell index and by reduced mRNA levels for surfactant protein C and Clara cell protein 10 expression. Further studies found that overexpression of Smurf1 reduced BMP-specific Smad1 and Smad5, but not Smad8, protein levels. Thus overexpression of Smurf1 specifically promotes Smad1 and Smad5 ubiquitination and degradation in embryonic lung epithelium, thereby modulating the effects of BMP4 on embryonic lung growth.     

The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain

The Wnt-1 (int-1) proto-oncogene, which encodes a putative signaling molecule, is expressed exclusively in the developing central nervous system and adult testes. To examine the role of Wnt-1, we generated six independent embryonic stem cell lines in which insertion of a neoR gene by homologous recombination inactivated a Wnt-1 allele. Germline chimeras were generated from two lines, and progeny from matings between heterozygous parents were examined. In all day 9.5 fetuses homozygous for mutated Wnt-1 alleles, most of the midbrain and some rostral metencephalon were absent. The remainder of the neural tube and all other tissues were normal. In late-gestation homozygotes, there was virtually no midbrain and no cerebellum, while the rest of the fetus was normal. Homozygotes are born, but die within 24 hr. Thus the normal role of Wnt-1 is in determination or subsequent development of a specific region of the central nervous system.     

Expression and regulation of α1β1 integrin in Schwann cells

The interaction of cells with the extracellular matrix plays a critical role in morphogenesis and cell differentiation. To define how Schwann cells might interact with the extracellular matrix, we chose to study the expression of the laminin/collagen receptor α1β1 integrin during nerve development in the rat from embryonic day 14 to maturity. We found that this integrin is expressed predominantly on mature non-myelin-forming cells and only at very low levels on myelin-forming cells. Significant levels of this integrin were not detected on Schwann cell precursors or embryonic Schwann cells in vivo. Experiments using transected and crushed sciatic nerve showed that α1β1 integrin expression is regulated at least in part by axonal contact. Furthermore, Schwann cell culture experiments showed that α1β1 integrin levels are strongly upregulated by transforming growth factor-βs and phorbol esters. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 914–928, 1997     

Regulation of programmed cell death during neural induction in the chick embryo

To study early responses to neural inducing signals from the organizer (Hensen's node), a differential screen was performed in primitive streak stage chick embryos, comparing cells that had or had not been exposed to a node graft for 5 hours. Three of the genes isolated have been implicated in Programmed Cell Death (PCD): Defender Against Cell Death (Dad1), Polyubiquitin II (UbII) and Ferritin Heavy chain (fth1). We therefore explored the potential involvement of PCD in neural induction. Dad1, UbII and fth1 are expressed in partly overlapping domains during early neural plate development, along with the pro-apoptotic gene Cas9 and the death effector Cas3. Dad1 and UbII are induced by a node graft within 3 hours. TUNEL staining revealed that PCD is initially random, but both during normal development and following neural induction by a grafted node, it becomes concentrated at the border of the forming neural plate and anterior non-neural ectoderm and downregulated from the neural plate itself. PCD was observed in regions of Caspase expression that are free from Dad1, consistent with the known anti-apoptotic role of Dad1. However, gain- and loss-of-function of any of these genes had no detectable effect on cell identity or on neural plate development. This study reveals that early development of the neural plate is accompanied by induction of putative pro- and anti-apoptotic genes in distinct domains. We suggest that the neural plate is protected against apoptosis, confining cell death to its border and adjacent non-neural ectoderm.