Cullin-3 dependent deregulation of ACTN1 represents a new pathogenic mechanism in nemaline myopathy.

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.

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.

Loss of Extended Synaptotagmins ESyt2 and ESyt3 does not affect mouse development or viability, but in vitro cell migration and survival under stress are affected.

The Extended Synaptotagmins (Esyts) are a family of multi-C2 domain membrane proteins with orthologs in organisms from yeast to human. Three Esyt genes exist in mouse and human and these have most recently been implicated in the formation of junctions between endoplasmic reticulum and plasma membrane, as well as the Ca(2+) dependent replenishment of membrane phospholipids. The data are consistent with a function in extracellular signal transduction and cell adhesion, and indeed Esyt2 was previously implicated in both these functions in Xenopus. Despite this, little is known of the function of the Esyts in vivo. We have generated mouse lines carrying homozygous deletions in one or both of the genes encoding the highly homologous Esyt2 and Esyt3 proteins. Surprisingly, esyt2(-/-)/esyt3(-/-) mice develop normally and are both viable and fertile. In contrast, esyt2(-/-)/esyt3(-/-) mouse embryonic fibroblasts display a reduced ability to migrate in standard in vitro assays, and are less resistant to stringent culture conditions and to oxidative stress than equivalent wild type fibroblasts.

Fkbp1a controls ventricular myocardium trabeculation and compaction by regulating endocardial Notch1 activity.

Trabeculation and compaction of the embryonic myocardium are morphogenetic events crucial for the formation and function of the ventricular walls. Fkbp1a (FKBP12) is a ubiquitously expressed cis-trans peptidyl-prolyl isomerase. Fkbp1a-deficient mice develop ventricular hypertrabeculation and noncompaction. To determine the physiological function of Fkbp1a in regulating the intercellular and intracellular signaling pathways involved in ventricular trabeculation and compaction, we generated a series of Fkbp1a conditional knockouts. Surprisingly, cardiomyocyte-restricted ablation of Fkbp1a did not give rise to the ventricular developmental defect, whereas endothelial cell-restricted ablation of Fkbp1a recapitulated the ventricular hypertrabeculation and noncompaction observed in Fkbp1a systemically deficient mice, suggesting an important contribution of Fkbp1a within the developing endocardia in regulating the morphogenesis of ventricular trabeculation and compaction. Further analysis demonstrated that Fkbp1a is a novel negative modulator of activated Notch1. Activated Notch1 (N1ICD) was significantly upregulated in Fkbp1a-ablated endothelial cells in vivo and in vitro. Overexpression of Fkbp1a significantly reduced the stability of N1ICD and direct inhibition of Notch signaling significantly reduced hypertrabeculation in Fkbp1a-deficient mice. Our findings suggest that Fkbp1a-mediated regulation of Notch1 plays an important role in intercellular communication between endocardium and myocardium, which is crucial in controlling the formation of the ventricular walls.

Pax1(EGFP): new wildtype and mutant EGFP mouse lines for molecular and fate mapping studies.

The Paired box gene 1 (Pax1) transcription factor plays essential roles in the development of axial skeleton, scapula, pelvic girdle, and thymus. Delineating its pleiotropic and molecular roles in the various tissues requires the ability to track and isolate the Pax1-expressing cells for downstream high-throughput experiments such as microarray and RNA-sequencing. With these applications in mind, we have generated two new mouse lines-a Pax1 wildtype (WT) mouse line that co-expresses enhanced green fluorescent protein (EGFP) with functional Pax1, and a Pax1 knockout mouse line which expresses EGFP under the control of Pax1 promoter, using the internal ribosome entry site (IRES) and 2A-peptide multi-cistron concatenating strategies. These mouse lines facilitate the isolation and enrichment of Pax1-specific cells from Pax1-positive and Pax1-null embryos using fluorescence activated cell sorting (FACS). They can be also be used in parallel to investigate the stage- and tissue-specific molecular functions of Pax1.

[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.

Generation of mice with a conditional allele for the p75(NTR) neurotrophin receptor gene.

The p75(NTR) neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75(NTR) , many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (Exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75(NTR) (p75(NTR-FX) ) allele made by flanking Exons 4-6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75(NTR) /Wnt1-Cre mutants and conventional p75(NTR) null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75(NTR) in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75(NTR) mutants. This novel conditional p75(NTR) allele will offer new opportunities to investigate the role of p75(NTR) in specific tissues and cells.

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.

Cautionary insights on knockout mouse studies: the gene or not the gene?

Gene modification technologies play a vital role in the study of biological systems and pathways. Although there is widespread and beneficial use of genetic mouse models, potential shortcomings of gene targeting technology exist, and are not always taken into consideration. Oversights associated with the technology can lead to misinterpretation of results; for example, ablation of a gene of interest can appear to cause an observed phenotype when, in fact, residual embryonic stem cell-derived genetic material in the genetic background or in the area immediately surrounding the ablated gene is actually responsible. The purpose of this review is to remind researchers, regardless of scientific discipline, that the background genetics of a knockout strain can have a profound influence on any observed phenotype. It is important that this issue be appropriately addressed during data collection and interpretation.

Generation of C57BL/6 knockout mice using C3H x BALB/c blastocysts.

The International Mouse Knockout Consortium aims to generate a knockout mouse for every single gene on a C57BL/6 background. Our ability to generate such mice is hampered by the poor economics of producing blastocysts to achieve germline transmission of C57BL/6 embryonic stem (ES) cells. We demonstrate superior utility of (C3H x BALB/c)F1 blastocysts compared with BALB/c blastocysts, with blastocyst numbers and germline transmission from subsequent chimeras at a rate 2- to 3-fold higher than that produced with BALB/c blastocysts.

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.

The TGF-beta pseudoreceptor gene Bambi is dispensable for mouse embryonic development and postnatal survival.

The Bambi (Bmp and activin membrane-bound inhibitor) gene encodes a transmembrane protein highly similar in amino acid sequence to transforming growth factor-beta (TGF-beta receptors, however, the Bambi intracellular domain is short and lacks a serine/threonine-kinase domain that is essential for transducing TGF beta signaling. Previous biochemical assays showed that Bambi interacts directly with BMP receptors and antagonizes BMP signaling. Interestingly, the expression of Bambi largely overlaps, both temporally and spatially, with that of Bmp4 during early embryonic development in Xenopus, zebrafish, and mice, which led to the hypothesis that Bambi may function to regulate BMP signaling during embryogenesis. To directly analyze the roles of Bambi during embryonic development, we generated mice carrying a conditional allele of Bambi, Bambi(flox), with loxP sequences flanking the first exon that encodes the N-terminus and signal peptide region of the Bambi protein. Mice homozygous for this targeted conditional allele appear normal and fertile. We crossed the Bambi(flox)/+ mice to the EIIa-Cre transgenic mice and generated mice carrying deletion of the first exon of the Bambi gene. Surprisingly, mice homozygous for the deleted allele were viable, fertile and did not exhibit any discernible developmental defect. Our data exclude an essential role for Bambi in mouse embryonic development and postnatal survival.

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.

Mammalian motoneuron axon targeting requires receptor protein tyrosine phosphatases sigma and delta.

The leukocyte common antigen-related (LAR) subfamily of receptor protein tyrosine phosphatases (RPTPs), LAR, RPTP-sigma, and RPTP-delta, regulate neuroendocrine development, axonal regeneration, and hippocampal long-term potentiation in mammals. In Drosophila, RPTPs are required for appropriate axon targeting during embryonic development. In contrast, deletion of any one of the three LAR-RPTP family members in mammals does not result in gross axon targeting defects. Both RPTP-sigma and RPTP-delta are highly expressed in the developing mammalian nervous system, suggesting they might be functionally redundant. To test this hypothesis, we generated RPTP-sigma and RPTP-delta (RPTP-sigma/delta) double-mutant mice. Although embryonic day 18.5 RPTP-sigma and RPTP-delta single-mutant embryos were viable, RPTP-sigma/delta double mutants were paralyzed, were never observed to draw a breath, and died shortly after cesarean section. RPTP-sigma/delta double mutants exhibit severe muscle dysgenesis and severe loss of motoneurons in the spinal cord. Detailed analysis of the projections of phrenic nerves in RPTP-sigma/delta double mutants indicated that these motoneuron axons emerge normally from the cervical spinal cord, but stall on reaching the diaphragm. Our results demonstrate that RPTP-sigma and RPTP-delta complement each other functionally during mammalian development, and reveal an essential contribution of RPTP-sigma and RPTP-delta to appropriate motoneuron axon targeting during mammalian axonogenesis.

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.

Gene trap and gene inversion methods for conditional gene inactivation in the mouse.

Conditional inactivation of individual genes in mice using site-specific recombinases is an extremely powerful method for determining the complex roles of mammalian genes in developmental and tissue-specific contexts, a major goal of post-genomic research. However, the process of generating mice with recombinase recognition sequences placed at specific locations within a gene, while maintaining a functional allele, is time consuming, expensive and technically challenging. We describe a system that combines gene trap and site-specific DNA inversion to generate mouse embryonic stem (ES) cell clones for the rapid production of conditional knockout mice, and the use of this system in an initial gene trap screen. Gene trapping should allow the selection of thousands of ES cell clones with defined insertions that can be used to generate conditional knockout mice, thereby providing extensive parallelism that eliminates the time-consuming steps of targeting vector construction and homologous recombination for each gene.

AP endonuclease and poly(ADP-ribose) polymerase-1 interact with the same base excision repair intermediate.

Base excision repair (BER) is a defense system that protects cells from deleterious effects secondary to modified or missing DNA bases. BER is known to involve apurinic/apyrimidinic endonuclease (APE) and DNA polymerase ss (ss-pol) among other enzymes, and recent studies have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) also plays a role by virtue of its binding to BER intermediates. The main role of APE is cleavage of the DNA backbone at abasic sites, and the enzyme also can catalyze 3'- to 5'-exonuclease activity at the cleaved abasic site. Photocross-linking studies with mouse embryonic fibroblast (MEF) cell extracts described here indicated that APE and PARP-1 interact with the same APE-cleaved abasic site BER intermediate. The model BER intermediate used includes a synthetic abasic site sugar, i.e. tetrahydrofuran (THF), in place of the natural deoxyribose. APE cross-linked efficiently with this intermediate, but not with a molecule lacking the 5'-THF phosphate group, and the same property was demonstrated for PARP-1. The addition of purified APE to the MEF extract reduced the amount of PARP-1 cross-linked to the BER intermediate, suggesting that APE can compete with PARP-1. APE and PARP-1 were antagonists of each other in in vitro BER related reactions on this model BER intermediate. These results suggest that PARP-1 and APE can interact with the same BER intermediate and that competition between these two proteins may influence their respective BER related functions.