Dennd1a, a susceptibility gene for polycystic ovary syndrome, is essential for mouse embryogenesis.

BACKGROUND: The DENND1A has been identified as a guanine nucleotide exchange factor for small GTPase Rab35, which functions in endocytic trafficking to mediate the recycling of selective cargos. Genetic alterations within the DENND1A gene have been implicated in human disease such as polycystic ovary syndrome (PCOS). However, the role of DENND1A in developmental and reproductive processes is largely unknown. RESULTS: Using Dennd1a gene knockout mice, we uncovered that homogeneous Dennd1a-/- mutants died around embryonic day (E) 14.5. The brain of Dennd1a-/- embryos exhibited defects, partially attributed to the dysregulation of cell division and survival in the telencephalon. The transcription of Fgf8 mRNA was ectopically elevated in the dorsal midline of telencephalon, concomitant with a decrease of active ß-catenin and Axin2 in the brain of Dennd1a-/- embryos. During liver morphogenesis, the ablation of Dennd1a impaired hepatic cell proliferation, the differentiation of hepatocyte, and hepatic hematopoiesis. In addition, loss of Dennd1a also affected the development of primordial germ cells. CONCLUSIONS: We demonstrate that Dennd1a, a susceptibility gene for PCOS, is essential for embryogenesis, probably through the mediation of endocytic recycling of selective cargos that are involved in cell signaling crucial for the development of multiple embryonic organ systems.

Loss of GRHL3 leads to TARC/CCL17-mediated keratinocyte proliferation in the epidermis.

Identifying soluble factors that influence epidermal integrity is critical for the development of preventative and therapeutic strategies for disorders such as ichthyosis, psoriasis, dermatitis and epidermal cancers. The transcription factor Grainyhead-like 3 (GRHL3) is essential for maintaining barrier integrity and preventing development of cutaneous squamous cell carcinoma (SCC); however, how loss of this factor, which in the skin is expressed exclusively within suprabasal epidermal layers triggers proliferation of basal keratinocytes, had thus far remained elusive. Our present study identifies thymus and activation-regulated chemokine (TARC) as a novel soluble chemokine mediator of keratinocyte proliferation following loss of GRHL3. Knockdown of GRHL3 in human keratinocytes showed that of 42 cytokines examined, TARC was the only significantly upregulated chemokine. Mouse skin lacking Grhl3 presented an inflammatory response with hallmarks of TARC activation, including heightened induction of blood clotting, increased infiltration of mast cells and pro-inflammatory T cells, increased expression of the pro-proliferative/pro-inflammatory markers CD3 and pSTAT3, and significantly elevated basal keratinocyte proliferation. Treatment of skin cultures lacking Grhl3 with the broad spectrum anti-inflammatory 5-aminosalicylic acid (5ASA) partially restored epidermal differentiation, indicating that abnormal keratinocyte proliferation/differentiation balance is a key driver of barrier dysfunction following loss of Grhl3, and providing a promising therapeutic avenue in the treatment of GRHL3-mediated epidermal disorders.

[Generation and characterization of the adult neuron-specific ADAM10 knock-out mice].

A disintegrin and metalloproteinase 10 (ADAM10) is a major sheddase for over 30 different membrane proteins and gets involved in such physiological processes and pathogenesis as embryonic development, cell adhesion, signal transduction, immune reaction, cancer, and Alzheimer's disease. Both ADAM10 knock-out mice and the neural progenitor cell-specific ADAM10 knock-out mice having been reported so far died in the embryonic or perinatal stage, respectively, thus resulting in the failure to investigate ADAM10 function in the adult mouse brain. Through a series of tests, we have succeeded in generating and characterizing the CaMKIIα-Cre/ADAM10(loxP/loxP) mice surviving until adulthood by means of crossing ADAM10(loxP/loxP) mice with newly generated CaMKIIα-Cre transgenic mice. PCR analysis of genomic DNAs from different regions of the ADAM10 cKO mouse brain shows that the deleted ADAM10 alleles are mainly found in the cortex and hippocampus. Real-time RT-PCR findings further confirm that ADAM10 mRNAs decrease in the cortex and hippocampus by 55.7% and 60.8%, respectively. Western-blotting analysis demonstrates 63% and 84.8% loss of mature ADAM10 proteins from the cortex and hippocampus. Immunohistochemical tests show that there is significantly less ADAM10- positive staining in the cortical and hippocampal neurons but not gliocytes of ADAM10 cKO mice compared with control mice. In summary, we established the adult neuron-specific ADAM10 knock-out (cKO) mice for the first time, which prevented ADAM10(-/-) mice from the embryonic and perinatal mortality and laid a firm foundation for the further study of ADAM10 function in the brain of adult mice in vivo.

Igf2 pathway dependency of the Trp53 developmental and tumour phenotypes.

Insulin-like growth factor 2 (IGF2) and the transformation related protein 53 (Trp53) are potent regulators of cell growth and metabolism in development and cancer. In vitro evidence suggests several mechanistic pathway interactions. Here, we tested whether loss of function of p53 leads to IGF2 ligand pathway dependency in vivo. Developmental lethality occurred in p53 homozygote null mice that lacked the paternal expressed allele of imprinted Igf2. Further lethality due to post-natal lung haemorrhage occurred in female progeny with Igf2 paternal null allele only if derived from double heterozygote null fathers, and was associated with a specific gene expression signature. Conditional deletion of Igf2(fl/fl) attenuated the rapid tumour onset promoted by homozygous deletion of p53(fl/fl) . Accelerated carcinoma and sarcoma tumour formation in p53(+/-) females with bi-allelic Igf2 expression was associated with reductions in p53 loss of heterozygosity and apoptosis. Igf2 genetic dependency of the p53 null phenotype during development and tumour formation suggests that targeting the IGF2 pathway may be useful in the prevention and treatment of human tumours with a disrupted Trp53 pathway.

ΔNp63 transcriptionally regulates brachyury, a gene with diverse roles in limb development, tumorigenesis and metastasis.

The phenotypes of the p63 mutant mice are complex and diverse. The p63-/- mice develop severe defects in morphogenesis of ectodermal appendages, and p63+/- mice are tumor prone. Transcriptional targets of p63 with functions in both of these biological processes likely exist. Here, we identified one such direct transcriptional target of p63, brachyury, a gene with diverse roles in limb development and tumorigenesis. We found that brachyury is not expressed in developing p63-/- mouse embryos, and that in osteosarcomas, ΔNp63 and brachyury are expressed at high levels. Knock down of ΔNp63 in tumor cells resulted in a concomitant diminution of brachyury, cell proliferation, migration and invasion. These data provide evidence that suppression of ΔNp63 in tumors may lead to tumor regression through loss of cell proliferative and metastatic potential.

CDK4 activity in mouse embryos expressing a single D-type cyclin.

D-type cyclins (D1, D2, and D3) are components of the cell cycle machinery. Their association with cyclin-dependent kinase 4 (CDK4) and CDK6 causes activation of these protein kinases and leads to phosphorylation and inactivation of the retinoblastoma protein, pRb. Using embryos expressing single D-type cyclin ('cyclin D1-only', 'cyclin D2-only' and 'cyclin D3-only'), we tested whether each of D-type cyclin plays the same role in CDK activation and phosphorylation of pRb during mouse embryonic development. We found that the level of CDK4 activity was similar in wild-type embryos and those expressing only cyclin D3 or cyclin D2. However, we did not detect CDK4 activity in embryos expressing only cyclin D1, despite the fact that this cyclin was able to form complexes with CDK4 and p27(kip1) in wild-type as well as in mutant embryos. Analysis of the expression pattern of mRNA encoding cyclin D1 revealed that the expression of this RNA is regulated temporally during embryogenesis. These data and results from other laboratories indicate that cyclin D1-dependent CDK4 activity is dispensable for normal development of the mouse embryo.

Regulation of mammalian horizontal gene transfer by apoptotic DNA fragmentation.

Previously it was shown that horizontal DNA transfer between mammalian cells can occur through the uptake of apoptotic bodies, where genes from the apoptotic cells were transferred to neighbouring cells phagocytosing the apoptotic bodies. The regulation of this process is poorly understood. It was shown that the ability of cells as recipient of horizontally transferred DNA was enhanced by deficiency of p53 or p21. However, little is known with regard to the regulation of DNA from donor apoptotic cells. Here we report that the DNA fragmentation factor/caspase-activated DNase (DFF/CAD), which is the endonuclease responsible for DNA fragmentation during apoptosis, plays a significant role in regulation of horizontal DNA transfer. Cells with inhibited DFF/CAD function are poor donors for horizontal gene transfer (HGT) while their ability of being recipients of HGT is not affected.

The chemokine stromal cell-derived factor-1 regulates the migration of sensory neuron progenitors.

Chemokines and their receptors are essential for the development and organization of the hematopoietic/lymphopoietic system and have now been shown to be expressed by different types of cells in the nervous system. In mouse embryos, we observed expression of the chemokine (CXC motif) receptor 4 (CXCR4) by neural crest cells migrating from the dorsal neural tube and in the dorsal root ganglia (DRGs). Stromal cell-derived factor-1 (SDF-1), the unique agonist for CXCR4, was expressed along the path taken by crest cells to the DRGs, suggesting that SDF-1/CXCR4 signaling is needed for their migration. CXCR4 null mice exhibited small and malformed DRGs. Delayed migration to the DRGs was suggested by ectopic cells expressing tyrosine receptor kinase A (TrkA) and TrkC, neurotrophin receptors required by DRG sensory neuron development. In vitro, the CXCR4 chemokine receptor was upregulated by migratory progenitor cells just as they exited mouse neural tube explants, and SDF-1 acted as a chemoattractant for these cells. Most CXCR4-expressing progenitors differentiated to form sensory neurons with the properties of polymodal nociceptors. Furthermore, DRGs contained a population of progenitor cells that expressed CXCR4 receptors in vitro and differentiated into neurons with a similar phenotype. Our findings indicate an important role for SDF-1/CXCR4 signaling in directing the migration of sensory neuron progenitors to the DRG and potentially in other aspects of development once the DRGs have coalesced.

Identification of signaling pathways in early mammary gland development by mouse genetics.

The mammary gland develops as an appendage of the ectoderm. The prenatal stage of mammary development is hormone independent and is regulated by sequential and reciprocal signaling between the epithelium and the mesenchyme. A number of recent studies using human and mouse genetics, in particular targeted gene deletion and transgenic expression, have identified some of the signals that control specific steps in development. This process involves cell specification and proliferation, reciprocal tissue interactions and cell migration. Since some of these events are recapitulated during tumorigenesis, an understanding of these signaling pathways may contribute to the development of targeted therapies and novel drugs.

Human T-cell lymphotropic virus type 1 tax induction of biologically Active NF-kappaB requires IkappaB kinase-1-mediated phosphorylation of RelA/p65.

Activation of the NF-kappaB/Rel family of transcription factors proceeds through a catalytic complex containing IkappaB kinase (IKK)-1 and IKK2. Targeted disruption of each of the IKK genes suggests that these two kinases may mediate distinct functions in the activation pathway. In our studies of the human T-cell lymphotropic virus type 1 (HTLV-1) Tax oncoprotein, we have uncovered a new function of IKK1 required for complete activation of the NF-kappaB transcriptional program. In IKK1(-/-) murine embryonic fibroblasts (MEFs), Tax normally induced early NF-kappaB activation events. However, NF-kappaB induced by Tax in these IKK1(-/-) cells was functionally impaired. In IKK1(-/-) (but not wild-type) MEFs, Tax failed to activate several different kappaB reporter constructs or to induce the endogenous IkappaBalpha gene. In contrast, Tax normally activated the cAMP-responsive element-binding protein/activating transcription factor pathway, leading to full stimulation of an HTLV-1 long terminal repeat reporter construct in IKK1(-/-) cells. Furthermore, reconstitution of IKK1(-/-) cells with kinase-proficient (but not kinase-deficient) forms of IKK1 restored the Tax induction of full NF-kappaB transactivation. We further found that the defect in NF-kappaB action in IKK1(-/-) cells correlated with a failure of Tax to induce phosphorylation of the RelA/p65 subunit of NF-kappaB at Ser(529) and Ser(536). Such phosphorylation of RelA/p65 was readily detected in wild-type MEFs. Phosphorylation of Ser(536) was required for a complete response to Tax expression, whereas phosphorylation of Ser(529) appeared to be less critical. Together, these findings highlight distinct roles for the IKK1 and IKK2 kinases in the activation of NF-kappaB in response to HTLV-1 Tax. IKK2 plays a dominant role in signaling for IkappaBalpha degradation, whereas IKK1 appears to play an important role in enhancing the transcriptional activity of NF-kappaB by promoting RelA/p65 phosphorylation.

Loss of CCAAT/enhancer binding protein delta promotes chromosomal instability.

The transcription factor CCAAT/enhancer binding protein delta (Cebpd, also known as C/EBPdelta, CRP3, CELF, NF-IL6beta) is implicated in diverse cellular functions such as the acute phase response, adipocyte differentiation, learning and memory, and mammary epithelial cell growth control. Here, we report that lack of Cebpd causes genomic instability and centrosome amplifications in primary embryonic fibroblasts derived from 129S1 mice. Upon spontaneous immortalization, Cebpd-deficient fibroblasts acquire transformed features such as impaired contact inhibition and reduced serum dependence. These data identify a novel role for Cebpd in the maintenance of chromosomal stability and suggest a potential tumor suppressor function in vivo.

Atm and c-Abl cooperate in the response to genotoxic stress during nervous system development.

The c-Abl proto-oncogene is a target of the ATM kinase after DNA double strand breaks, although the physiological significance of these signaling events is not clear. Therefore, to delineate the roles of c-Abl and Atm during mouse development we generated mice with combinations of c-Abl and Atm mutant alleles. We found that dual inactivation of Atm and c-Abl usually resulted in midgestational lethality. However, mice with three mutant alleles, c-Abl(-/-)Atm(+/-) or c-Abl(+/-)Atm(-/-), were viable but predisposed to neuro-developmental abnormalities after genotoxic insult. Thus, these genetic data link Atm and c-Abl signaling and underscore a significant interrelationship between the two during neural development.

Physiological levels of tumstatin, a fragment of collagen IV alpha3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via alphaV beta3 integrin.

We demonstrate a physiological role for tumstatin, a cleavage fragment of the alpha3 chain of type IV collagen (Col IValpha3), which is present in the circulation. Mice with a genetic deletion of Col IValpha3 show accelerated tumor growth associated with enhanced pathological angiogenesis, while angiogenesis associated with development and tissue repair are unaffected. Supplementing Col IValpha3-deficient mice with recombinant tumstatin to a normal physiological concentration abolishes the increased rate of tumor growth. The suppressive effects of tumstatin require alphaVbeta3 integrin expressed on pathological, but not on physiological, angiogenic blood vessels. Mice deficient in matrix metalloproteinase-9, which cleaves tumstatin efficiently from Col IValpha3, have decreased circulating tumstatin and accelerated growth of tumor. These results indicate that MMP-generated fragments of basement membrane collagen can have endogenous function as integrin-mediated suppressors of pathologic angiogenesis and tumor growth.

Knockout mice as model systems for studying nm23/NDP kinase gene functions. Application to the nm23-M1 gene.

Mice carrying a homozygous germ-line mutation in the nm23-M1 gene that eliminates its protein expression and drives expression of beta-galactosidase by nm23-M1 promoter have been generated. nm23-M1 gene inactivation is not teratogenic and the pups can grow to adult age without apparent health problems. However, they undergo a growth retardation and knocked out females cannot feed their pups. Both effects are background dependent. Beta-galactosidase mapping of nm23-M1 promoter activation during embryogenesis shows that the nm23-M1 gene is principally expressed in epithelial layer of tissues which require inductive epithelial-mesenchymal interactions for their formation. In conclusion, invalidated mice could be interesting models to analyze the role of nm23-M1 on signal transduction pathway regulation, or cancer induction and proliferation.

Activity-dependent neuroprotective protein: a novel gene essential for brain formation.

We have recently cloned the novel homeobox-containing activity-dependent neuroprotective protein (ADNP). In the current study, mouse ADNP was shown to be expressed at the time of neural tube closure, detected at E7.5 and increased on E9.5. Expression was augmented in the brain (E12.5), sustained throughout embryogenesis and regulated by VIP. To assess the function of ADNP, knockout mice were established. Detailed analysis revealed cranial neural tube closure failure and death on E8.5-9.0 of the ADNP-knockout embryos. The expression of Oct4, a gene associated with germ-line maintenance was markedly augmented in the knockout embryos. In contrast, the expression of Pax6, a gene crucial for cerebral cortex formation, was abolished in the brain primordial tissue of the knockout embryos. Thus, Pax6 and Oct4 constitute a part of the mechanism of action of ADNP on brain formation, inhibiting germ-line division while activating morphogenesis. In conclusion, ADNP is identified here as a new key gene essential for organogenesis in the developing embryo and may be implicated as a clinical target associated with proper neurodevelopment.

Mouse proteasomal ATPases Psmc3 and Psmc4: genomic organization and gene targeting.

PSMC3 and PSMC4, components of the 19S complex of the 26S proteasome, show a significant degree of amino acid similarity, especially in the conserved ATPase domain (CAD). In this study, we characterized the mouse Psmc3 and Psmc4 genes. The genomic structures of both genes showed a significant degree of similarity. The Psmc3 gene was composed of 12 coding exons, whereas the Psmc4 gene had 11 exons. Exons encoding the leucine zipper domain and CAD were identical in number between the Psmc3 and Psmc4 genes. The Psmc3 gene mapped to mouse chromosome 2, whereas Psmc4 mapped to chromosome 7. We further addressed the biological roles of Psmc3 and Psmc4 through the generation of gene targeted mice. Both Psmc3- and Psmc4-deficient mice died before implantation, displaying defective blastocyst development. These findings indicate that Psmc3 and Psmc4 have similar and essential roles in early embryogenesis and further that both ATPases have noncompensatory functions in vivo.

Mouse models in the study of the Ets family of transcription factors.

The Ets family of transcription factors is one of a growing number of master regulators of development. This family was originally defined by the presence of a conserved DNA binding domain, the Ets domain. To date, nearly 30 members of this family have been identified and implicated in a wide range of physiological and pathological processes. Despite the likely importance of Ets-family members, each of their precise roles has not been delineated. Herein, we describe the elucidation of essential functions of a few of these family members in vivo using knockout mouse models. Of the knockouts generated to date, the majority shows important functions in hematopoiesis, ranging from PU.1, a principle regulator of myelo-lymphopoiesis, to Spi-B which regulates the proper function of terminally differentiated cells. Ets1 was shown to be of intermediate importance as a regulator of pan-lymphoid development. Other Ets family members such as Fli1 and TEL1 display distinct and/or overlapping functions in vasculo/angiogenesis, hemostasis and hematopoiesis. The remaining knockouts generated, Ets2 and Er81, show non-hematopoietic defects related to extraembryonic development and neurogenesis, respectively. The pioneering group of knockout models described reveals only the most distinct functions of each of these Ets family members. A better understanding of the roles and hierarchies of Ets family members in cellular differentiation will come with the generation of new null alleles in previously untargeted family members, more mutant alleles in members already disrupted, double knockouts, ES cell differentiation and chimera rescue experiments, and tissue-specific inducible knockouts.

Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally.

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.

Erythroid maturation and globin gene expression in mice with combined deficiency of NF-E2 and nrf-2.

NF-E2 binding sites, located in distant regulatory sequences, may be important for high level alpha- and beta-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2-related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2-deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of alpha- and beta-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2-deficient mice is not the result of compensation by Nrf-2.