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Background Hepatocyte transplantation has been extensively investigated as an alternative to orthotopic liver transplantation. However, its application in routine clinical practice has been restricted because of low initial engraftment and subsequent repopulation. Methods Using mice as a model, we have developed a minimally invasive and nontoxic preconditioning strategy based on preadministration of antibodies against hepsin to increase donor hepatocyte retention and engraftment rate. Results Liver sinusoid diameters decreased significantly with antihepsin pretreatment, and graft cell numbers increased nearly 2-fold in the recipients' liver parenchyma for 20 days after hepatocyte transplantation. Postoperative complications such as hepatic ischemia injury or apparent immune cell accumulation were not observed in recipients. In a hemophilia B mouse model, antihepsin preconditioning enhanced the expression and clotting activity of coagulation factor IX (FIX) to nearly 2-fold that of immunoglobulin G-treated controls and maintained higher plasma FIX clotting activity relative to the prophylactic range for 50 days after hepatocyte transplantation. Antihepsin pretreatment combined with adeno-associated virus-transduced donor hepatocytes expressing human FIX-Triple, a hyperfunctional FIX variant, resulted in plasma FIX levels similar to those associated with mild hemophilia, which protected hemophilia B mice from major bleeding episodes for 50 days after transplantation. Furthermore, antihepsin pretreatment and repeated transplantation resulted in extending the therapeutic period by 30 days relative to the immunoglobulin G control. Conclusions Thus, this antihepsin strategy improved the therapeutic effect of hepatocyte transplantation in mice with tremendous safety and minimal invasion. Taken together, we suggest that preconditioning with antihepsin may have clinical applications for liver cell therapy.

Background and objectivesLiver fibrosis (LF) occurs following chronic liver injuries. Currently, there is no effective therapy for LF. Recently, we identified thioredoxin domain containing 5 (TXNDC5), an ER protein disulfide isomerase (PDI), as a critical mediator of cardiac and lung fibrosis. We aimed to determine if TXNDC5 also contributes to LF and its potential as a therapeutic target for LF.DesignHistological and transcriptome analyses on human cirrhotic livers were performed. Col1a1-GFPTg, Alb-Cre;Rosa26-tdTomato and Tie2-Cre/ERT2;Rosa26-tdTomato mice were used to determine the cell type(s) where TXNDC5 was induced following liver injury. In vitro investigations were conducted in human hepatic stellate cells (HSCs). Col1a2-Cre/ERT2;Txndc5fl/fl (Txndc5cKO) and Alb-Cre;Txndc5fl/fl (Txndc5Hep-cKO) mice were generated to delete TXNDC5 in HSCs and hepatocytes, respectively. Carbon tetrachloride treatment and bile duct ligation surgery were employed to induce liver injury/fibrosis in mice. The extent of LF was quantified using histological, imaging and biochemical analyses.ResultsTXNDC5 was upregulated markedly in human and mouse fibrotic livers, particularly in activated HSC at the fibrotic foci. TXNDC5 was induced by transforming growth factor β1 (TGFβ1) in HSCs and it was both required and sufficient for the activation, proliferation, survival and extracellular matrix production of HSC. Mechanistically, TGFβ1 induces TXNDC5 expression through increased ER stress and ATF6-mediated transcriptional regulation. In addition, TXNDC5 promotes LF by redox-dependent JNK and signal transducer and activator of transcription 3 activation in HSCs through its PDI activity, activating HSCs and making them resistant to apoptosis. HSC-specific deletion of Txndc5 reverted established LF in mice.ConclusionsER protein TXNDC5 promotes LF through redox-dependent HSC activation, proliferation and excessive extracellular matrix production. Targeting TXNDC5, therefore, could be a potential novel therapeutic strategy to ameliorate LF.

Genotoxicity-induced hair loss from chemotherapy and radiotherapy is often encountered in cancer treatment, and there is a lack of effective treatment. In growing hair follicles (HF), quiescent stem cells (SC) are maintained in the bulge region, and hair bulbs at the base contain rapidly dividing, yet genotoxicity-sensitive transit-amplifying cells (TAC) that maintain hair growth. How genotoxicity-induced HF injury is repaired remains unclear. We report here that HFs mobilize ectopic progenitors from distinct TAC compartments for regeneration in adaptation to the severity of dystrophy induced by ionizing radiation (IR). Specifically, after low-dose IR, keratin 5+ basal hair bulb progenitors, rather than bulge SCs, were quickly activated to replenish matrix cells and regenerated all concentric layers of HFs, demonstrating their plasticity. After high-dose IR, when both matrix and hair bulb cells were depleted, the surviving outer root sheath cells rapidly acquired an SC-like state and fueled HF regeneration. Their progeny then homed back to SC niche and supported new cycles of HF growth. We also revealed that IR induced HF dystrophy and hair loss and suppressed WNT signaling in a p53- and dose-dependent manner. Augmenting WNT signaling attenuated the suppressive effect of p53 and enhanced ectopic progenitor proliferation after genotoxic injury, thereby preventing both IR- and cyclophosphamide-induced alopecia. Hence, targeted activation of TAC-derived progenitor cells, rather than quiescent bulge SCs, for anagen HF repair can be a potential approach to prevent hair loss from chemotherapy and radiotherapy. Cancer Res; 77(22); 6083–96. ©2017 AACR.

Background: To avoid complicated procedures requiring in vitro chondrocyte expansion for cartilage repair, the development of a culture-free, 1-stage approach combining platelet-rich fibrin (PRF) and autologous cartilage grafts may be the solution. Purpose: To develop a feasible 1-step procedure to combine PRF and autologous cartilage grafts for articular chondral defects. Study Design: Controlled laboratory study Methods: The chemotactic effects of PRF on chondrocytes harvested from the primary culture of rabbit cartilage were evaluated in vitro and ex vivo. The rabbit chondrocytes were cultured with different concentrations of PRF media and evaluated for their cell proliferation, chondrogenic gene expression, cell viability, and extracellular matrix synthesis abilities. For the in vivo study, the chondral defects were created on established animal models of rabbits. The gross anatomy, histology, and objective scores were evaluated to validate the treatment results. Results: PRF improved the chemotaxis, proliferation, and viability of the cultured chondrocytes. The gene expression of the chondrogenic markers, including type II collagen and aggrecan, revealed that PRF induced the chondrogenic differentiation of cultured chondrocytes. PRF increased the formation and deposition of the cartilaginous matrix produced by cultured chondrocytes. The efficacy of PRF on cell viability was comparable with that of fetal bovine serum. In animal disease models, morphologic, histological, and objectively quantitative evaluation demonstrated that PRF combined with cartilage granules was feasible in facilitating chondral repair. Conclusion: PRF enhances the migration, proliferation, viability, and differentiation of chondrocytes, thus showing an appealing capacity for cartilage repair. The data altogether provide evidence to confirm the feasibility of 1-stage, culture-free method of combining PRF and autologous cartilage graft for repairing articular chondral defects. Clinical Relevance: The single-stage, culture-free method of combining PRF and autologous cartilage is useful for repairing articular chondral defects. These advantages benefit clinical translation by simplifying and potentiating the efficacy of autologous cartilage transplantation.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology facilitates somatic genome editing to reveal cooperative genetic interactions at the cellular level without extensive breeding between different mutant animals. Here we propose a transgenic inducible Cas9 effector-CRISPR mutagen ( ICE CRIM) mouse model in which CRISPR/Cas9-mediated somatic mutagenesis events can occur in response to Cre expression. The well-known tumor suppressor gene, Trp53, and 2 important DNA mismatch repair genes, Mlh1 and Msh2, were selected to be our somatic mutagenesis targets. Amplicon-based sequencing was performed to validate the editing efficiency and to identify the mutant allelic series. Crossed with various Cre lines, the Trp53 ICE CRIM alleles were activated to generate targeted cancer gene somatic or germ line mutant variants. We provide experimental evidence to show that an activated ICE CRIM can mutate both targeted alleles within a cell. Simultaneous disruption of multiple genes was also achieved when there were multiple single-guide RNA expression cassettes embedded within an activated ICE CRIM. Our mouse model can be used to generate mutant pools in vivo, which enables a functional screen to be performed in situ. Our results also provide evidence to support a monoclonal origin of hematopoietic neoplasms and to indicate that DNA mismatch repair deficiency accelerates tumorigenesis in Trp53 mutant genetic background.-Fan, H.-H., Yu, I.-S., Lin, Y.-H., Wang, S.-Y., Liaw, Y.-H., Chen, P.-L., Yang, T.-L., Lin, S.-W., Chen, Y.-T. P53 ICE CRIM mouse: a tool to generate mutant allelic series in somatic cells and germ lines for cancer studies.

Cul4b-null (Cul4bΔ/Y) mice undergo growth arrest and degeneration during the early embryonic stages and die at E9.5. The pathogenic causes of this lethality remain incompletely characterized. However, it has been hypothesized that the loss of Cul4b function in extraembryonic tissues plays a key role. In this study, we investigated possible causes of death for Cul4b-null embryos, particularly in regard to the role of embryonic Cul4b. First, we show that the loss of embryonic Cul4b affects the growth of the inner cell mass in vitro and delays epiblast development during the gastrulation period at E6.5~E7.5 in vivo, as highlighted by the absence of the epiblastic transcription factor Brachyury from E6.5~E7.5. Additionally, at E7.5, strong and laterally expanded expression of Eomes and Fgf8 signaling was detected. Sectioning of these embryos showed disorganized primitive streak layer cells. Second, we observed that Mash2-expressing cells were present in the extraembryonic tissues of Cul4b-deficient embryos at E6.5 but were absent at E7.5. In addition, the loss of Cul4b resulted in decreased expression of cyclin proteins, which are required for the cell cycle transition from G1 to S. Taken together, these observations suggest that the embryonic expression of Cul4b is important for epiblast growth during E6.5~E7.5, and the loss of Cul4b results in either delayed growth of the epiblast or defective localization of primitive streak layer cells. As a result, the signaling activity mediated by the epiblast for subsequent ectoplacental cone development is affected, with the potential to induce growth retardation and lethality in Cul4bΔ/Y embryos.

WD repeat and HMG-box DNA binding protein 1 (Wdhd1) is the mouse ortholog of budding yeast Chromosome Transmission Fidelity 4 (CTF4), the protein product of which integrates the MCM2-7 helicase and DNA polymerase α/primase complex to initiate DNA replication. Previous work in fruit flies, Xenopus egg extracts, and human cell lines suggest that Wdhd1 is required for efficient DNA synthesis. However, rigorous in vivo functional studies on Wdhd1 in mammals are unavailable. In the present study, we have successfully generated a Wdhd1 null allele in mice through CRISPR/Cas9-mediated genome editing to investigate the role of Wdhd1 in embryogenesis in vivo. We characterized Wdhd1 expression using quantitative reverse-transcription polymerase chain reaction, and assessed embryonic cell proliferation by histology in both pre- and peri-implantation embryos. While Wdhd1 heterozygous mutant mice were grossly normal and fertile, we observed a reduction in cell proliferation by the gastrulation stage in Wdhd1 homozygous null mutant embryos which severely hampered their growth and viability. These results indicate that Wdhd1 plays a major role in cell proliferation during embryogenesis in mice.

Background: Insufficient cell numbers still present a challenge for articular cartilage repair. Converting heterotopic auricular chondrocytes by extracellular matrix may be the solution. Hypothesis: Specific extracellular matrix may convert the phenotype of auricular chondrocytes toward articular cartilage for repair. Study Design: Controlled laboratory study. Methods: For in vitro study, rabbit auricular chondrocytes were cultured in monolayer for several passages until reaching status of dedifferentiation. Later, they were transferred to chondrogenic type II collagen (Col II)–coated plates for further cell conversion. Articular chondrogenic profiles, such as glycosaminoglycan deposition, articular chondrogenic gene, and protein expression, were evaluated after 14-day cultivation. Furthermore, 3-dimensional constructs were fabricated using Col II hydrogel-associated auricular chondrocytes, and their histological and biomechanical properties were analyzed. For in vivo study, focal osteochondral defects were created in the rabbit knee joints, and auricular Col II constructs were implanted for repair. Results: The auricular chondrocytes converted by a 2-step protocol expressed specific profiles of chondrogenic molecules associated with articular chondrocytes. The histological and biomechanical features of converted auricular chondrocytes became similar to those of articular chondrocytes when cultivated with Col II 3-dimensional scaffolds. In an in vivo animal model of osteochondral defects, the treated group (auricular Col II) showed better cartilage repair than did the control groups (sham, auricular cells, and Col II). Histological analyses revealed that cartilage repair was achieved in the treated groups with abundant type II collagen and glycosaminoglycans syntheses rather than elastin expression. Conclusion: The study confirmed the feasibility of applying heterotopic chondrocytes for cartilage repair via extracellular matrix–induced cell conversion. Clinical Relevance: This study proposes a feasible methodology to convert heterotopic auricular chondrocytes for articular cartilage repair, which may serve as potential alternative sources for cartilage repair.

Rationale: Reducing cardiomyocyte death and enhancing their proliferation after myocardial infarction is perhaps the single largest challenge for cardiac tissue regeneration. Survivin (SVV) is the smallest member of the inhibitor of apoptosis (IAP) family but plays two important roles; inhibiting caspase-9 activation in the intrinsic apoptosis pathway, and regulating microtubule dynamics and chromosome segregation during cell division. Genetic depletion of cardiac SVV leads to incomplete cardiomyocyte division and abnormal heart development. However, the function of SVV in adult hearts after myocardial infarction remains unclear. Methods: A homozygous inducible cardiomyocyte-specific SVV knockout transgenic mouse model was established through crossbreeding SVVflox/flox and αMHC-MCM transgenic mice. Adult mice received consecutive intraperitoneal injection of tamoxifen to induce genetic removal of SVV in cardiomyocytes. A SVV overexpressing model was established via local delivery of SVV in wild-type mouse hearts. Results: We found that 30.82% of cardiomyocytes in the peri-infarct region of SVV knockout mice were apoptotic, significantly higher than the 22.18% in control mice. In addition, ejection fraction was 29.00±0.40% in knockout mice compared to 38.04±0.50% in control mice 21 days after myocardial infarction. On the contrary, locally overexpressing SVV in the heart improved cardiac functions. Unexpectedly, we found that altering the subcellular localization of SVV overexpression produced different outcomes. Overexpression of SVV in the cytoplasm decreased cardiomyocyte apoptosis, whereas overexpression of SVV in the nucleus enhanced cardiac regeneration. The ejection fraction of mice overexpressing SVV was 36.58±0.91%, significantly higher than 28.18±1.70% in the GFP control group. Apoptotic cardiomyocytes were only 4.63% in mouse overexpressing cytosolic SVV, compared to 9.31% in the GFP group, and activation of caspase-3 was also reduced. Moreover, mice overexpressing NLS-SVV exhibited a better ejection fraction (36.19±1.02%,) than GFP controls (26.69±0.75%). NLS-SVV enhanced H3P-positive cardiomyocytes in the border zone to 0.28%, compared to only 0.08% in GFP group, through interacting with Aurora B. Conclusions: We demonstrate the importance of SVV subcellular localization in regulating post-MI cardiac repair and regeneration. We hope that this will open new translational approaches through targeted delivery of SVV.

The salivary gland is characterized by ramified epithelial branches, a specific tissue structure responsible for saliva production and regulation. To regenerate the salivary gland function, it is important to establish the tissue structure. Chitosan is a deacetylated derivative of chitin with wide biomedical applications. Because of its deacetylated nature, chitosan has different properties when prepared with different degrees of deacetylation (DDA). However, the impact of chitosan DDA on the effect of regulating tissue structure formation remains unexplored. In this study, the embryonic murine submandibular gland (SMG) was used as a model to investigate the role of chitosan DDA in regulating tissue structure formation of the salivary gland. When chitin substrates with different DDA were used, the branching numbers of cultured SMG explants changed. Similar effects were observed in the culture with chitosan prepared using different degrees of acetylation. The mRNA expressions of type I and type III collagen were elevated in SMG explants with enhanced branching morphogenesis, as was the protein level. In addition to the amounts of collagen, type I and type III collagen fibers were spatially present in the epithelial-mesenchymal junction of developing branches in the culture with chitosan of a specific range of DDA. The branch-promoting effect of chitosan DDA was abolished when SMG explants were treated with collagenase, both early in the stage of branch initiation and with the establishment of the branching structure. The branch-promoting effect of chitosan DDA disappeared when antisense oligonucleotides were applied to specifically block type III collagen. This study demonstrates for the first time that DDA of chitosan affects tissue structure formation. The different proportions of side-chain components of chitin derivatives regulate structural formation of cultured SMG, indicating that DDA is an important parameter using chitosan as a biomaterial for tissue structure formation of the salivary glands.

The basic helix–loop–helix transcription factor Twist1 is a key regulator of craniofacial development. Twist1-null mouse embryos exhibit failure of cephalic neural tube closure and abnormal head development and die at E11.0. To dissect the function of Twist1 in the cranial mesoderm beyond mid-gestation, we used Mesp1-Cre to delete Twist1 in the anterior mesoderm, which includes the progenitors of the cranial mesoderm. Deletion of Twist1 in mesoderm cells resulted in loss and malformations of the cranial mesoderm-derived skeleton. Loss of Twist1 in the mesoderm also resulted in a failure to fully segregate the mesoderm and the neural crest cells, and the malformation of some cranial neural crest-derived tissues. The development of extraocular muscles was compromised whereas the differentiation of branchial arch muscles was not affected, indicating a differential requirement for Twist1 in these two types of craniofacial muscle. A striking effect of the loss of Twist1 was the inability of the mesodermal cells to maintain their mesenchymal characteristics, and the acquisition of an epithelial-like morphology. Our findings point to a role of Twist1 in maintaining the mesenchyme architecture and the progenitor state of the mesoderm, as well as mediating mesoderm–neural crest interactions in craniofacial development.

Telomerase is highly expressed in cancer and embryonic stem cells (ESCs) and implicated in controlling genome integrity, cancer formation and stemness. Previous studies identified that Krüppel-like transcription factor 4 (KLF4) activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in ESCs. However, little is known about how KLF4 regulates TERT expression. Here, we discover poly(ADP-ribose) polymerase 1 (PARP1) as a novel KLF4-interacting partner. Knockdown of PARP1 reduces TERT expression and telomerase activity not only in cancer cells, but also in human and mouse ESCs. Recruitment of KLF4 to TERT promoter is reduced in PARP1-suppressed cells. The poly(ADP-ribose) polymerase activity is dispensable, while the oligo(ADP-ribose) polymerase activity is required for the PARP1- and KLF4-mediated TERT activation. Repression of Parp1 in mouse ESCs decreases expression of pluripotent markers and induces differentiation. These results suggest that PARP1 recruits KLF4 to activate telomerase expression and stem cell pluripotency, indicating a positive regulatory role of the PARP1–KLF4 complex in telomerase expression in cancer and stem cells.

Development of the mouse forelimb bud depends on normal Twist1 activity. Global loss of Twist1 function before limb bud formation stops limb development and loss of Twist1 throughout the mesenchyme after limb bud initiation leads to polydactyly, the ulnarization or loss of the radius and malformations and reductions of the shoulder girdle. Here we show that conditional deletion of Twist1 by Mesp1-Cre in the mesoderm that migrates into the anterior-proximal part of the forelimb bud results in the development of supernumerary digits and carpals, the acquisition of ulna-like characteristics by the radius and malformations of the humerus and scapula. The mirror-like duplications and posteriorization of pre-axial tissues are preceded by disruptions to anterior-posterior Shh, Bmp and Fgf signaling gradients and dysregulation of transcription factors that regulate anterior-posterior limb patterning.

Preaxial polydactyly (PPD) is a common limb-associated birth defect characterized by extra digit(s) in the anterior autopod. It often results from ectopic sonic hedgehog (Shh) expression in the anterior limb bud. Although several transcription factors are known to restrict Shh expression to the posterior limb bud, how they function together remains unclear. Here we provide evidence from mouse conditional knockout limb buds that the bHLH family transcription factor gene Twist1 is required to inhibit Shh expression in the anterior limb bud mesenchyme. More importantly, we uncovered genetic synergism between Twist1 and the ETS family transcription factor genes Etv4 and Etv5 (collectively Etv), which also inhibit Shh expression. Biochemical data suggest that this genetic interaction is a result of direct association between TWIST1 and ETV proteins. Previous studies have shown that TWIST1 functions by forming homodimers or heterodimers with other bHLH factors including HAND2, a key positive regulator of Shh expression. We found that the PPD phenotype observed in Etv mutants is suppressed by a mutation in Hand2, indicative of genetic antagonism. Furthermore, overexpression of ETV proteins influences the dimerization of these bHLH factors. Together, our data suggest that through biochemical interactions, the Shh expression regulators ETV, TWIST1 and HAND2 attain a precise balance to establish anterior-posterior patterning of the limb.

Using a Cre-mediated conditional deletion approach, we have dissected the function of Twist1 in the morphogenesis of the craniofacial skeleton. Loss of Twist1 in neural crest cells and their derivatives impairs skeletogenic differentiation and leads to the loss of bones of the snout, upper face and skull vault. While no anatomically recognizable maxilla is formed, a malformed mandible is present. Since Twist1 is expressed in the tissues of the maxillary eminence and the mandibular arch, this finding suggests that the requirement for Twist1 is not the same in all neural crest derivatives. The effect of the loss of Twist1 function is not restricted to neural crest-derived bones, since the predominantly mesoderm-derived parietal and interparietal bones are also affected, presumably as a consequence of lost interactions with neural crest-derived tissues. In contrast, the formation of other mesodermal skeletal derivatives such as the occipital bones and most of the chondrocranium are not affected by the loss of Twist1 in the neural crest cells.

Twist1 is the mouse ortholog of TWIST1, the human gene mutated in Saethre-Chotzen syndrome. Previously, a Twist1 null allele was generated by gene targeting in mouse embryonic stem cells. Twist1 heterozygous mice develop polydactyly and a craniofacial phenotype similar to Saethre-Chotzen patients. Mice homozygous for the Twist1 null allele die around embryonic day 11.5 (E11.5) with cranial neural tube closure and vascular defects, hindering in vivo studies of Twist1 function at later stages of development. Here, we report the generation of a Twist1 conditional null allele in mice that functions like a wild-type allele but can be converted to a null allele upon Cre-mediated recombination. genesis 45:588–592, 2007. © 2007 Wiley-Liss, Inc.

The mesenchyme of the developing vertebrate limb responds to inductive signals, giving rise to skeletal elements that define limb shape and size. Several signals emanate from the limb ectoderm and in particular from the specialized epithelium of the apical ectodermal ridge (AER), including three members of the bone morphogenetic protein (BMP) family of signaling molecules, BMP2, BMP4 and BMP7. Using the Cre/loxP system in mice, we rendered limb bud mesenchyme insensitive to BMP signals through the type I receptor, BMPR-IA. Conditional mutants had shortened limbs and almost complete agenesis of the autopod because of reduced cell proliferation. Reduced expression of downstream BMP signaling targets, Msx1, Msx2 and gremlin in the distal mesenchyme (progress zone) correlated with decreased levels of cyclin D1 and Wnt5a. Ectopic anterior activation of sonic hedgehog (SHH) signaling and Hox expression revealed alterations in anterior–posterior (AP) patterning. Abnormal localization of Lmx1b-expressing cells in the ventral mesenchyme, along with histological alterations and an abnormal melanization pattern of the limb, indicate altered dorsal–ventral (DV) boundaries. These findings suggest that signaling through BMPR-IA in limb mesenchyme is essential for distal outgrowth and also influences AP and DV patterning.

Dravet syndrome (DS) is characterized by severe infant-onset myoclonic epilepsy along with delayed psychomotor development and heightened premature mortality. A primary monogenic cause is mutation of the SCN1A gene, which encodes the voltage-gated sodium channel subunit Na v 1.1. The nature and timing of changes caused by SCN1A mutation in the hippocampal dentate gyrus (DG) network, a core area for gating major excitatory input to hippocampus and a classic epileptogenic zone, are not well known. In particularly, it is still not clear whether the developmental deficit of this epileptogenic neural network temporally matches with the progress of seizure development. Here, we investigated the emerging functional and structural deficits of the DG network in a novel mouse model ( Scn1a E1099X/+ ) that mimics the genetic deficit of human DS. Scn1a E1099X/+ (Het) mice, similarly to human DS patients, exhibited early spontaneous seizures and were more susceptible to hyperthermia-induced seizures starting at postnatal week (PW) 3, with seizures peaking at PW4. During the same period, the Het DG exhibited a greater reduction of Na v 1.1-expressing GABAergic neurons compared to other hippocampal areas. Het DG GABAergic neurons showed altered action potential kinetics, reduced excitability, and generated fewer spontaneous inhibitory inputs into DG granule cells. The effect of reduced inhibitory input to DG granule cells was exacerbated by heightened spontaneous excitatory transmission and elevated excitatory release probability in these cells. In addition to electrophysiological deficit, we observed emerging morphological abnormalities of DG granule cells. Het granule cells exhibited progressively reduced dendritic arborization and excessive spines, which coincided with imbalanced network activity and the developmental onset of spontaneous seizures. Taken together, our results establish the existence of significant structural and functional developmental deficits of the DG network and the temporal correlation between emergence of these deficits and the onset of seizures in Het animals. Most importantly, our results uncover the developmental deficits of neural connectivity in Het mice. Such structural abnormalities likely further exacerbate network instability and compromise higher-order cognitive processing later in development, and thus highlight the multifaceted impacts of Scn1a deficiency on neural development.

PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.

Mutation in CUL4B, which encodes a scaffold protein of the E3 ubiquitin ligase complex, has been found in patients with X-linked mental retardation (XLMR). However, early deletion of Cul4b in mice causes prenatal lethality, which has frustrated attempts to characterize the phenotypes in vivo. In this report, we successfully rescued Cul4b mutant mice by crossing female mice in which exons 4-5 of Cul4b were flanked by loxP sequences with Sox2-Cre male mice. In Cul4b-deficient (Cul4b(Δ)/Y) mice, no CUL4B protein was detected in any of the major organs, including the brain. In the hippocampus, the levels of CUL4A, CUL4B substrates (TOP1, β-catenin, cyclin E and WDR5) and neuronal markers (MAP2, tau-1, GAP-43, PSD95 and syn-1) were not sensitive to Cul4b deletion, whereas the number of parvalbumin (PV)-positive GABAergic interneurons was decreased in Cul4b(Δ)/Y mice, especially in the dentate gyrus (DG). Some dendritic features, including the complexity, diameter and spine density in the CA1 and DG hippocampal neurons, were also affected by Cul4b deletion. Together, the decrease in the number of PV-positive neurons and altered dendritic properties in Cul4b(Δ)/Y mice imply a reduction in inhibitory regulation and dendritic integration in the hippocampal neural circuit, which lead to increased epileptic susceptibility and spatial learning deficits. Our results identify Cul4b(Δ)/Y mice as a potential model for the non-syndromic model of XLMR that replicates the CUL4B-associated MR and is valuable for the development of a therapeutic strategy for treating MR.

Amniotic membrane (AM) has been widely used in the reconstruction of oral epithelial defects. However, whether it is also effective in facilitating tissue formation of salivary gland, an appendix of oral epithelia, has never been explored. To investigate the effects and the underlying mechanism of AM on salivary gland morphogenesis, murine fetal submandibular gland (SMG) explants were cultured on different preparations of AM scaffolds. It was found that, on AM stromal scaffold, SMG demonstrated well-developed branching morphogenesis. Nonetheless, on AM epithelial scaffold, SMG epithelial cell converted to a spindle-shape, lost intercellular connection, changed cytoskeletal organization, and exhibited scattering behaviors. Meanwhile, the integrity of SMG basement membrane was dismantled as well. However, when acellular AM epithelial scaffold was used, cultured SMG demonstrated organized morphology, indicating that AM epithelial component provided specific surface features for SMG morphogenesis. To further investigate AM scaffold morphogenetic effect, it was found hepatocyte growth factor (HGF), an epithelial scattering factor, was expressed abundantly in cultivated AM epithelia. After blocking HGF function of AM, cultured SMG regained branching activity, reorganized cell adhesion and subcellular organization, and reproduced basement membranes. Therefore, AM-derived bioactive factor profoundly influences cell behaviors and structure formation of SMG. Together, this study showed that compositional topography of AM scaffold is important in affecting SMG morphogenesis. By understanding the effects of AM scaffold on SMG morphogenesis, it provides important information for rationally designing and fabricating AM scaffold for salivary gland regeneration.

The mammalian constitutive photomorphogenesis 9 (COP9) signalosome (CSN), a protein complex involved in embryonic development, is implicated in cell cycle regulation and the DNA damage response. Its role in tumor development, however, remains unclear. Here, we have shown that the COP9 subunit 6 (CSN6) gene is amplified in human breast cancer specimens, and the CSN6 protein is upregulated in human breast and thyroid tumors. CSN6 expression positively correlated with expression of murine double minute 2 (MDM2), a potent negative regulator of the p53 tumor suppressor. Expression of CSN6 appeared to prevent MDM2 autoubiquitination at lysine 364, resulting in stabilization of MDM2 and degradation of p53. Mice in which Csn6 was deleted died early in embryogenesis (E7.5). Embryos lacking both Csn6 and p53 survived to later in embryonic development (E10.5), which suggests that loss of p53 could partially rescue the effect of loss of Csn6. Mice heterozygous for Csn6 were sensitized to γ-irradiation-induced, p53-dependent apoptosis in both the thymus and the developing CNS. These mice were also less susceptible than wild-type mice to γ-irradiation-induced tumorigenesis. These results suggest that loss of CSN6 enhances p53-mediated tumor suppression in vivo and that CSN6 plays an important role in regulating DNA damage-associated apoptosis and tumorigenesis through control of the MDM2-p53 signaling pathway.

The basic helix-loop-helix transcription factor Twist1 is essential for normal limb development. Twist1−/− embryos die at midgestation. However, studies on early limb buds found that Twist1−/− mutant limb mesenchyme has an impaired response to FGF signaling from the apical ectodermal ridge, which disrupts the feedback loop between the mesenchyme and AER, and reduces and shifts anteriorly Shh expression in the zone of polarizing activity. We have combined Twist1 null, hypomorph and conditional alleles to generate a Twist1 allelic series that survives to birth. As Twist1 activity is reduced, limb skeletal defects progress from preaxial polydactyly to girdle reduction combined with hypoplasia, aplasia or mirror symmetry of all limb segments. With reduced Twist1 activity there is striking and progressive upregulation of ectopic Shh expression in the anterior of the limb, combined with an anterior shift in the posterior Shh domain, which is expressed at normal intensity, and loss of the posterior AER. Consequently limb outgrowth is initially impaired, before an ectopic anterior Shh domain expands the AER, promoting additional growth and repatterning. Reducing the dosage of FGF targets of the Etv gene family, which are known repressors of Shh expression in anterior limb mesenchyme, strongly enhances the anterior skeletal phenotype. Conversely this and other phenotypes are suppressed by reducing the dosage of the Twist1 antagonist Hand2. Our data support a model whereby multiple Twist1 activity thresholds contribute to early limb bud patterning, and suggest how particular combinations of skeletal defects result from differing amounts of Twist1 activity.

Permanent and reversible genetic modifications are important approaches to study gene function in different cell types. They are also important for stem cell researchers to explore and test the therapeutic potential of stem cells. The piggyBac transposon from insects is a rising nonviral system that efficiently mutagenizes and mediates gene transfer into the mammalian genome. It is also characterized by its precise excision, leaving no trace sequence behind so that the genomic integrity of the mutated cell can be restored. Here, we use an optimized piggyBac transposon system to mediate gene transfer and expression of a bifunctional fluorescent reporter in human embryonic stem (ES) cells. We provide molecular evidence for transposase-mediated piggyBac integration events and functional evidence for successful expression of a transferred fluorescent protein genes in human ES cells and their in vitro differentiated derivatives. We also demonstrate that the integrated piggyBac transposon can be removed and an undisrupted insertion site can be restored, which implies potential applications for its use in gene therapy and genetics studies.

Gene trapping in mouse embryonic stem cells is an important genetic approach that allows simultaneous mutation of genes and generation of corresponding mutant mice. We designed a selection scheme with drug selection markers and Cre/loxP technology which allows screening of gene trap events that responded to a signaling molecule in a 96-well format. Nine hundred twenty gene trap clones were assayed, and 258 were classified as gene traps induced by in vitro differentiation. Sixty-five of the in vitro differentiation-inducible gene traps were also responsive to retinoic acid treatment. In vivo analysis revealed that 85% of the retinoic acid-inducible gene traps trapped developmentally regulated genes, consistent with the observation that genes induced by retinoic acid treatment are likely to be developmentally regulated. Our results demonstrate that the inducible gene trapping system described here can be used to enrich in vitro for traps in genes of interest. Furthermore, we demonstrate that the cre reporter is extremely sensitive and can be used to explore chromosomal regions that are not detectable with neo as a selection cassette.

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.

Genetic ablation experiments are used to resolve problems regarding cell lineages and the in vivo function of certain groups of cells. We describe a two-component conditional ablation technology using a mouse carrying an X-linked puDeltatk transgene, which is only activated in cells expressing Cre. Ablation of the Cre-expressing cells can be temporally regulated by the time of ganciclovir (GCV) administration. This strategy was demonstrated using a Col2Cre transgenic line. Differentiating chondrocytes in bigenic animals could be ablated at different developmental stages resulting in disorganized growth plates and dwarfism. Macrocephaly, macroglossia and umbilical hernia were also observed in ablated 18.5 dpc embryos. Crosses between the puDeltatk selector transgenic line and existing cre lines will facilitate numerous temporally regulated tissue-specific ablation experiments.

Background Lim1 is a homeobox gene that is essential for nephrogenesis. During metanephric kidney development, Lim1 is expressed in the nephric duct, ureteric buds, and the induced metanephric mesenchyme. Conditional ablation of Lim1 in the metanephric mesenchyme blocks the formation of nephrons at the nephric vesicle stage, leading to the production of small, non-functional kidneys that lack nephrons. Methods In the present study, we used Affymetrix probe arrays to screen for nephron-specific genes by comparing the expression profiles of control and Lim1 conditional mutant kidneys. Kidneys from two developmental stages, embryonic day 14.5 (E14.5) and 18.5 (E18.5), were examined. Results Comparison of E18.5 kidney expression profiles generated a list of 465 nephron-specific gene candidates that showed a more than 2-fold increase in their expression level in control kidney versus the Lim1 conditional mutant kidney. Computational analysis confirmed that this screen enriched for kidney-specific genes. Furthermore, at least twenty-eight of the top fifty (56%) candidates (or their vertebrate orthologs) were previously reported to have a nephron-specific expression pattern. Our analysis of E14.5 expression data yielded 41 candidate genes that are up-regulated in the control kidneys compared to the conditional mutants. Three of them are related to the Notch signaling pathway that is known to be important in cell fate determination and nephron patterning. Conclusion Therefore, we demonstrate that Lim1 conditional mutant kidneys serve as a novel tissue source for comprehensive expression studies and provide a means to identify nephron-specific genes.