Podoplanin, PDPN, is a mucin-type transmembrane glycoprotein widely expressed in many tissues, including lung, kidney, lymph nodes, and mineralized tissues. Its function is critical for lymphatic formation, differentiation of type I alveolar epithelial lung cells, and for bone response to biomechanical loading. It has previously been shown that Pdpn null mice die at birth due to respiratory failure emphasizing the importance of Pdpn in alveolar lung development. During the course of generation of Pdpn mutant mice, we found that most Pdpn null mice in the 129S6 and C57BL6/J mixed genetic background die at the perinatal stage, similar to previously published studies with Pdpn null mice, while all Pdpn null mice bred with Swiss outbred mice survived. Surviving mutant mice in the 129S6 and C57BL6/J mixed genetic background showed alterations in the osteocyte lacunocanalicular network, especially reduced osteocyte canaliculi in the tibial cortex with increased tibial trabecular bone. However, adult Pdpn null mice in the Swiss outbred background showed no overt differences in their osteocyte lacunocnalicular network, bone density, and no overt differences when challenged with exercise. Together, these data suggest that genetic variations present in the Swiss outbred mice compensate for the loss of function of PDPN in lung, kidney, and bone.

The study of gene function in normal human physiology and pathophysiology is complicated by countless factors such as genetic diversity (similar to 98 million SNPs identified in the human genome as of June 2015), environmental exposure, epigenetic imprinting, maternal/in utero exposure, diet, exercise, age, sex, socioeconomic factors, and many other variables. Inbred mouse lines have allowed researchers to control for many of the variables that define human diversity but complicate the study of the human genome, gene/protein function, cellular and molecular pathways, and countless other genetic diseases. Furthermore, genetically modified mouse models enable us to generate and study mice whose genomes differ by as little as a single point mutation while controlling for non-genomic variables. This allows researchers to elucidate the quintessential function of a gene, which will further not only our scientific understanding, but provide key insight into human health and disease. Recent advances in CRISPR/Cas9 genome editing have revolutionized scientific protocols for introducing mutations into the mammalian genome. The ensuing chapter provides an overview of CRISPR/Cas9 genome editing in murine embryonic stem cells for the generation of genetically modified mouse models.

Transgenic mouse models can be subdivided into two main categories based on genomic location: (1) targeted genomic manipulation and (2) random integration into the genome. Despite the potential confounding insertional mutagenesis and host locus-dependent expression, random integration transgenics allowed for rapid in vivo assessment of gene/protein function. Since precise genomic manipulation required the time-consuming prerequisite of first generating genetically modified embryonic stem cells, the rapid nature of generating random integration transgenes remained a strong benefit outweighing various disadvantages. The advent of targetable nucleases, such as CRISPR/Cas9, has eliminated the prerequisite of first generating genetically modified embryonic stem cells for some types of targeted genomic mutations. This chapter outlines the generation of mouse models with targeted genomic manipulation using the CRISPR/Cas9 system directly into single cell mouse embryos.

Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factor-β activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment (“drug on”), the impact of drug withdrawal (“drug off”) implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment (“drug on”) and subsequent withdrawal (“drug off”) through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the “drug on” (Cre-mediated inactivation) and “drug off” (Flp-mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766–778

The identity of the gonads is determined by which fate, ovarian granulosa cell or testicular Sertoli cell, the bipotential somatic cell precursors choose to follow. In most vertebrates, the conserved transcription factor FOXL2 contributes to the fate of granulosa cells. To understand FOXL2 functions during gonad differentiation, we performed genome-wide analysis of FOXL2 chromatin occupancy in fetal ovaries and established a genetic mouse model that forces Foxl2 expression in the fetal testis. When FOXL2 was ectopically expressed in the somatic cell precursors in the fetal testis, FOXL2 was sufficient to repress Sertoli cell differentiation, ultimately resulting in partial testis-to-ovary sex-reversal. Combining genome-wide analysis of FOXL2 binding in the fetal ovary with transcriptomic analyses of our Foxl2 gain-of-function and previously published Foxl2 loss-of-function models, we identified potential pathways responsible for the feminizing action of FOXL2. Finally, comparison of FOXL2 genome-wide occupancy in the fetal ovary with testis-determining factor SOX9 genome-wide occupancy in the fetal testis revealed extensive overlaps, implying that antagonistic signals between FOXL2 and SOX9 occur at the chromatin level.

P0-Cre and Wnt1-Cre mouse lines have been widely used in combination with loxP-flanked mice to label and genetically modify neural crest (NC) cells and their derivatives. Wnt1-Cre has been regarded as the gold standard and there have been concerns about the specificity of P0-Cre because it is not clear about the timing and spatial distribution of the P0-Cre transgene in labeling NC cells at early embryonic stages. We re-visited P0-Cre and Wnt1-Cre models in the labeling of NC cells in early mouse embryos with a focus on cranial NC. We found that R26-lacZ Cre reporter responded to Cre activity more reliably than CAAG-lacZ Cre reporter during early embryogenesis. Cre immunosignals in P0-Cre and reporter (lacZ and RFP) activity in P0-Cre/R26-lacZ and P0-Cre/R26-RFP embryos was detected in the cranial NC and notochord regions in E8.0-9.5 (4-19 somites) embryos. P0-Cre transgene expression was observed in migrating NC cells and was more extensive in the forebrain and hindbrain but not apparent in the midbrain. Differences in the Cre distribution patterns of P0-Cre and Wnt1-Cre were profound in the midbrain and hindbrain regions, that is, extensive in the midbrain of Wnt1-Cre and in the hindbrain of P0-Cre embryos. The difference between P0-Cre and Wnt1-Cre in labeling cranial NC may provide a better explanation of the differential distributions of their NC derivatives and of the phenotypes caused by Cre-driven genetic modifications.

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome.

Objective: Our objectives were to determine the expression of EVC2 in craniofacial tissues and investigate the effect of Evc2 deficiency on craniofacial bones using Evc2 knockout (KO) mouse model.Design: Evc2 KO mice were generated by introducing a premature stop codon followed by the Internal Ribosomal Entry Site fused to beta-galactosidase (LacZ). Samples from wild-type (WT), heterozygous (Het) and homozygous Evc2 KO mice were prepared. LacZ staining and immunohistochemistry (IHC) with anti-beta-galactosidase, anti-EVC2 and anti-SOX9 antibodies were performed. The craniofacial bones were stained with alcian blue and alizarin red.Results: The LacZ activity in KO was mainly observed in the anterior parts of viscerocranium. The Evc2-expressing cells were identified in many cartilageous regions by IHC with anti-beta-galactosidase antibody in KO and Het embryos. The endogenous EVC2 protein was observed in these areas in WT embryos. Double labeling with anti-SOX9 antibody showed that these cells were mainly chondrocytes. At adult stages, the expression of EVC2 was found in chondrocytes of nasal bones and spheno-occipital synchondrosis, and osteocytes and endothelial-like cells of the premaxilla and mandible. The skeletal double staining demonstrated that craniofacial bones, where the expression of EVC2 was observed, in KO had the morphological defects as compared to WT.Conclusion: To our knowledge, our study was the first to identify the types of Evc2-expressing cells in craniofacial tissues. Consistent with the expression pattern, abnormal craniofacial bone morphology was found in the Evc2 KO mice, suggesting that EVC2 may be important during craniofacial growth and development. (C) 2016 Elsevier Ltd. All rights reserved.

Ellis-van Creveld (EvC) syndrome is a genetic disorder with mutations in either EVC or EVC2 gene. Previous case studies reported that EvC patients underwent orthodontic treatment, suggesting the presence of craniofacial bone phenotypes. To investigate whether a mutation in EVC2 gene causes a craniofacial bone phenotype, Evc2 knockout (KO) mice were generated and cephalometric analysis was performed. The heads of wild type (WT), heterozygous (Het) and homozygous Evc2 KO mice (1-, 3-, and 6-week-old) were prepared and cephalometric analysis based on the selected reference points on lateral X-ray radiographs was performed. The linear and angular bone measurements were then calculated, compared between WT, Het and KO and statistically analyzed at each time point. Our data showed that length of craniofacial bones in KO was significantly lowered by ∼20% to that of WT and Het, the growth of certain bones, including nasal bone, palatal length, and premaxilla was more affected in KO, and the reduction in these bone length was more significantly enhanced at later postnatal time points (3 and 6 weeks) than early time point (1 week). Furthermore, bone-to-bone relationship to cranial base and cranial vault in KO was remarkably changed, i.e. cranial vault and nasal bone were depressed and premaxilla and mandible were developed in a more ventral direction. Our study was the first to show the cause-effect relationship between Evc2 deficiency and craniofacial defects in EvC syndrome, demonstrating that Evc2 is required for craniofacial bone development and its deficiency leads to specific facial bone growth defect. Anat Rec, 299:1110-1120, 2016. © 2016 Wiley Periodicals, Inc.

Ellis-van Creveld (EvC) syndrome (OMIM 225500) is an autosomal recessive disease characterized with chondrodysplastic dwarfism in association with abnormalities in oral cavity. Ciliary proteins EVC and EVC2 have been identified as causative genes and they play an important role on Hedgehog signal transduction. We have also identified a causative gene LIMBIN for bovine chondrodysplastic dwarfism (bcd) that is later identified as the bovine ortholog of EVC2. Here, we report generation of conventional and conditional mutant Evc2/Limbin alleles that mimics mutations found in EvC patients and bcd cattle. Resulted homozygous mice showed no ciliary localization of EVC2 and EVC and displayed reduced Hedgehog signaling activity in association with skeletal and oral defects similar to the EvC patients. Cartilage-specific disruption of Evc2/Limbin resulted in similar but milder skeletal defects, whereas osteoblast-specific disruption did not cause overt changes in skeletal system. Neural crest-specific disruption of Evc2/Limbin resulted in defective incisor growth similar to that seen in conventional knockouts; however, differentiation of amelobolasts was relatively normal in the conditional knockouts. These results showcased functions of EVC2/LIMBIN during formation of mineralized tissues. Availability of the conditional allele for this gene should facilitate further detailed analyses of the role of EVC2/LIMBIN in pathogenesis of EvC syndrome. genesis 53:612-626, 2015. © 2015 Wiley Periodicals, Inc.

We generated a new Bmp2 conditional knock-out allele without a neo cassette and removed Bmp2 gene in osteoblasts (Bmp2-cKOob) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKOob mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in reduced bone formation rate, and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKOob osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSC), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Several marker genes of MSC (α-SMA, CD146 and Angiopoietin-1), in vitro CFU assays and deletion of the Bmp2 gene in vitro in α-SMA+ BMSC support our conclusions. Critical roles of the Bmp2 gene in osteoblasts and MSC are a vital link between bone formation, vascularization and mesenchymal stem cells.

Dentin matrix protein1 (DMP1), highly conserved in humans and mice, is highly expressed in teeth, the skeleton, and to a lesser extent in nonskeletal tissues such as brain, kidney, and salivary gland. Pathologically, DMP1 is associated with several forms of cancers and with tumor‐induced osteomalacia. Conventional disruption of the murine Dmp1 gene results in defects in dentin in teeth and in the skeleton, including hypophosphatemic rickets, and abnormalities in phosphate homeostasis. Human DMP1 mutations are responsible for the condition known as autosomal recessive hypophosphatemic rickets. For better understanding of the roles of DMP1 in different tissues at different stages of development and in pathological conditions, we generated Dmp1 floxed mice in which loxP sites flank exon 6 that encodes for over 80% of DMP1 protein. We demonstrate that Cre‐mediated recombination using Sox2‐Cre, a Cre line expressed in epiblast during early embryogenesis, results in early deletion of the gene and protein. These homozygous Cre‐recombined null mice display an identical phenotype to conventional null mice. This animal model will be useful to reveal distinct roles of DMP1 in different tissues at different ages. genesis 46:87–91, 2008. © 2008 Wiley‐Liss, Inc.

Sox8 encodes a high-mobility group transcription factor that is widely expressed during development. Sox8, -9 and -10 form group E of the Sox gene family which has been implicated in several human developmental disorders. In contrast to other SoxE genes, the role of Sox8 is unclear and Sox8 mouse mutants reportedly showed only idiopathic weight loss and reduced bone density. The careful analysis of our Sox8 null mice, however, revealed a progressive male infertility phenotype. Sox8 null males only sporadically produced litters of reduced size at young ages. We have shown that SOX8 protein is a product of adult Sertoli cells and its elimination results in an age-dependent deregulation of spermatogenesis, characterized by sloughing of spermatocytes and round spermatids, spermiation failure and a progressive disorganization of the spermatogenic cycle, which resulted in the inappropriate placement and juxtaposition of germ cell types within the epithelium. Those sperm that did enter the epididymides displayed abnormal motility. These data show that SOX8 is a critical regulator of adult Sertoli cell function and is required for both its cytoarchitectural and paracrine interactions with germ cells.

TAK1 binding protein 1 (TAB1) binds and induces autophosphorylation of TGF-beta activating kinase (TAK1). TAK1, a mitogen-activated kinase kinase kinase, is involved in several distinct signaling pathways including non-Smad pathways for TGF-beta superfamily members and inflammatory responses caused by cytokines. Conventional disruption of the murine Tab1 gene results in late gestational lethality showing intraventricular septum defects and underdeveloped lung alveoli. To gain a better understanding of the roles of TAB1 in different tissues, at different stages of development, and in pathological conditions, we generated Tab1 floxed mice in which the loxP sites flank Exons 9 and 10 to remove the C-terminal region of TAB1 protein necessary for activation of TAK1. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in the epiblast during early embryogenesis, results in deletion of the gene and protein. These homozygous Cre-recombined null embryos display an identical phenotype to conventional null embryos. This animal model will be useful in revealing distinct roles of TAB1 in different tissues at different stages.

TAK1 kinase is an indispensable intermediate in several cytokine signaling pathways including tumor necrosis factor, interleukin-1, and transforming growth factor-beta signaling pathways. TAK1 also participates in stress-activated intracellular signaling pathways such as osmotic stress signaling pathway. TAK1-binding protein 1 (TAB1) is constitutively associated with TAK1 through its C-terminal region. Although TAB1 is known to augment TAK1 catalytic activity when it is overexpressed, the role of TAB1 under physiological conditions has not yet been identified. In this study, we determined the role of TAB1 in TAK1 signaling by analyzing TAB1-deficient mouse embryonic fibroblasts (MEFs). Tumor necrosis factor- and interleukin-1-induced activation of TAK1 was entirely normal in Tab1-deficient MEFs and could activate both mitogen-activated protein kinases and NF-kappaB. In contrast, we found that osmotic stress-induced activation of TAK1 was largely impaired in Tab1-deficient MEFs. Furthermore, we showed that the C-terminal 68 amino acids of TAB1 were sufficient to mediate osmotic stress-induced TAK1 activation. Finally, we attempted to determine the mechanism by which TAB1 activates TAK1. We found that TAK1 is spontaneously activated when the concentration is increased and that it is totally dependent on TAB1. Cell shrinkage under the osmotic stress condition increases the concentration of TAB1-TAK1 and may oligomerize and activate TAK1 in a TAB1-dependent manner. These results demonstrate that TAB1 mediates TAK1 activation only in a subset of TAK1 pathways that are mediated through spontaneous oligomerization of TAB1-TAK1.

Fertility-linked demethylase The histone H3K9 demethylase JHDM2A was known to play a role in transcriptional activation mediated by the androgen receptor. Now targeted disruption of the Jhdm2a gene in mice shows that the enzyme is involved in spermatogenesis and regulation of transition nuclear protein and protamine genes. This work points to a role for this histone demethylase in the late stages of sperm production and maturation, and Jhdm2a becomes a possible candidate gene for infertility syndromes that have not yet been fully characterized.