Dmitry A. Ovchinnikov

Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice.

Type II collagen is one of the principal markers of chondrocyte differentiation (Mayne, 1990) and is encoded by the proa1(II) collagen gene (Col2a1) (Cheah et al., 1985). In the mouse, Col2a1 transcripts are first detected at 9.5 days post-coitum (dpc) in the sclerotome of the differentiating somites and the cranial mesenchyme destined to give rise to the cartilage. Expression is also observed in the notochord and later in some neural tissues, including regions of the rhombencephalic basal plate and the ventricular layer of the hindbrain (Cheah et al., 1991). The regulatory elements that direct chondrocyte-specific expression in tissue culture cells and transgenic mice are located within the first intron of the Col2a1 gene (Mukhopadhyay et al., 1995; Zhou et al., 1995). To direct the expression of Cre recombinase to developing chondrocytes, we generated transgenic mice expressing a Col2a1-Cre gene construct (Fig. 1). The gene construct consisted of 3 kb of the Col2a1 promoter region, the first exon with a mutated initiation codon, and a 3.02 kb fragment of intron 1 ligated to a splice acceptor sequence (Zhou et al., 1995) followed by an internal ribosome-entry site (IRES), Cre recombinase coding region, and the SV40 large T antigen polyadenylation signal. The 8.4 kb gene construct was purified from vector sequences and microinjected into the pronuclei of fertilized C57BL/6 3 SJL F2 hybrid eggs to generate transgenic mice (Brinster et al., 1985). To analyze the expression pattern of Cre recombinase in these Col2-Cre transgenic mice, we utilized the ROSA26 Cre reporter mouse strain, R26R (Soriano, 1999). Col2-Cre transgenic males from line A were bred with R26R/1 females to establish timed matings. Embryos were stained with X-gal to detect b-galactosidase (b-gal) activity (Hogan et al., 1994). Reciprocal crosses, using Col2-Cre females, yielded identical patterns. Another Col2-Cre transgenic mouse line (B) demonstrated essentially identical patterns of Cre activity. b-gal activity was first detected between 8.75 and 9.0 dpc in the notochord and cranial mesenchyme of Col2Cre, R26R compound heterozygotes (Fig. 2A). In somites, b-gal activity was first detected at 9.5 dpc in the sclerotomes (Fig. 2b). b-gal activity was strongest in the posterior portion of the sclerotome. Additionally, strong b-gal activity was observed in the otic vesicle region. By 11.5–12.0 dpc, intense b-gal activity was observed in the notochord and the surrounding sclerotomal cells of the vertebral anlagen undergoing chondrocytic differentiation (Fig. 2C,D). In the limb buds, b-gal activity was observed in the developing cartilaginous anlagen of the long bones (Fig. 2C,D). At 15 dpc, b-gal activity was detected in virtually all of the existing cartilaginous primordia of the bones of the axial and appendicular skeleton, temporal and basioccipital bones, and the other elements of the base of the skull developing by endochondral bone formation. b-gal activity was also observed in the submandibular glands (Fig. 2E). Osteoblasts are also derived from the sclerotome of somites (Aubin, 1998). Therefore, Col2a1-directed expression of Cre may also lead to the activation of the R26R locus in the osteoblast lineage. To address this question, we analyzed the long bones of Col2-Cre mice for Cre activity. Longitudinal sections of the X-gal stained hindlimbs of neonatal Col2-Cre/R26R compound heterozygotes show specific staining in the chondrocytes of the epiphysis of the bone but not in the osteoblasts or perichondrial fibroblasts (Fig. 2F,G). Some mosaicism in b-gal activity was observed in the cartilage, with approximately 5% of chondrocytes being b-gal negative. The Col2-Cre transgenic mice described here should be a useful resource for analysis of gene function, employing conditional genetics approaches in differentiating chondrocytes, notochord, and submandibular glands.

Macrophage Activation and Differentiation Signals Regulate Schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

Background The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity. Methodology/Principal Findings Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1−/− BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens. Conclusions Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in vivo perturbs myelopoiesis. We hypothesise that the down-regulation of Slfn4 gene expression during macrophage differentiation is a necessary step in development of this lineage.

Integration‐Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology

Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild‐type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress‐induced apoptosis, and this could be prevented by the antioxidant N‐acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform. STEM CELLS2013;31:467–478

Macrophages in the embryo and beyond: Much more than just giant phagocytes

Originally recognized as an essential part of the innate and acquired immune systems, macrophages emerged as omnipresent and influential regulators of embryo‐ and organo‐genesis, as well as of tissue and tumor growth. Macrophages are present essentially in all tissues, beginning with embryonic development and, in addition to their role in host defense and in the clearance of apoptotic cells, are being increasingly recognized for their trophic function and role in regeneration. Some tissue macrophages are also found to posses a substantial potential for autonomous self‐renewal. Macrophages are associated with a significant proportion of malignant tumors and are widely recognized for their angiogenesis‐promoting and trophic roles, making them one of the new promising targets for cancer therapies. Recent expression profiling of embryonic macrophages from different tissues revealed remarkable consistency of their gene expression profiles, independent of their tissue of origin, as well as their similarities with tumor‐associated macrophages. Macrophages are also capable of fusion with other cells in tissue repair and metastasizing tumors, as well as with each other in the immune response and osteoclastogenesis. genesis 46:447–462, 2008.

Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice

We generated double‐transgenic mice carrying cointegrated tissue‐specific Gal4 and Gal4 reporter transgenes to direct transgene overexpression in the mononuclear phagocyte system (MPS). A modified promoter of the Csf1r (c‐fms) gene, containing a deletion of the trophoblast‐specific promoter, was used to drive the expression of Gal4VP16 transcriptional activator specifically in macrophages. This module was cointegrated with a fluorescent reporter, enhanced cyan fluorescent protein (ECFP), driven by a Gal4‐dependent promoter. ECFP fluorescence was first detected in forming blood islands of the yolk sac at 8 dpc, then in macrophages in the yolk sac and the embryo proper. In adult mice ECFP was detected primarily in monocytes, tissue macrophages, microglia, and dendritic cells, including Langerhans cells of the skin. Crossing of these mice to transgenics containing tagged protein under control of a Gal4‐dependent promoter directed expression of that protein in mononuclear phagocytes of double‐transgenic animals. The new mouse line provides a useful tool for overexpression of transgenes in cells of the myeloid lineage, while simultaneously labeling them by ECFP expression.

Multiple calvarial defects in lmx1b mutant mice

The vertebrate cranial vault, or calvaria, forms during embryonic development from cranial mesenchyme of multiple embryonic origins. Inductive interactions are thought to specify the number and location of the calvarial bones, including interactions between the neuroepithelium and cranial mesenchyme. An important feature of calvarial development is the local inhibition of osteogenic potential which occurs between specific bones and results in the formation of the cranial sutures. These sutures allow for postnatal growth of the skull to accommodate postnatal increase in brain size. The molecular genetic mechanisms responsible for the patterning of individual calvarial bones and for the specification of the number and location of sutures are poorly understood at the molecular genetic level. Here we report on the function and expression pattern of the LIM-homeodomain gene, lmx1b, during calvarial development. Lmx1b is expressed in the neuroepithelium underlying portions of the developing skull and in cranial mesenchyme which contributes to portions of the cranial vault. Lmx1b is essential for proper patterning and morphogenesis of the calvaria since the supraoccipital and interparietal bones of lmx1b mutant mice are either missing or severely reduced. Moreover, lmx1b mutant mice have severely abnormal sutures between the frontal, parietal, and interparietal bones. Our results indicate that lmx1b is required for multiple events in calvarial development and suggest possible genetic interaction with other genes known to regulate skull development and suture formation.

Alcian Blue/Alizarin Red Staining of Cartilage and Bone in Mouse

The vertebrate skeleton forms by endochondral and intramembranous bone formation. During endochondral bone formation, mesenchyme condensations give rise to cartilages that are eventually replaced by bone. However, there are some permanent cartilages that do not ossify, such as the cartilage of the trachea and articular cartilage of the joints, and intramembranous bone formation occurs directly without a cartilage template. This article describes a method for simultaneously visualizing cartilage and mineralized tissues (notably bone) in the developing mouse. It makes use of alcian blue and alizarin red stains, and it is best used for later fetal, newborn, and early post-natal stages of development. Neonatal skeletons are especially well suited for this technique.

Generation and Characterization of LIF-dependent Canine Induced Pluripotent Stem Cells from Adult Dermal Fibroblasts

Dogs provide a more clinically relevant model of human disease than rodents, particularly with respect to hereditary diseases. Thus, the availability of canine stem cells will greatly facilitate the use of the dog in the development of stem cell-based gene therapies and regenerative medicine. In this study we describe the production of canine induced pluripotent stem cells (ciPSCs) from adult dermal fibroblasts. These cells have a morphology resembling previously described canine embryonic stem cells, a normal karyotype, and express pluripotency markers including alkaline phosphatase, Nanog, Oct4, Telomerase, SSEA1, SSEA4, TRA1-60, TRA1-81, and Rex1. Furthermore, the inactive X chromosome is reactivated indicating a ground-state pluripotency. In culture they readily form embryoid bodies, which in turn give rise to cell types from all 3 embryonic germ layers, as indicated by expression of the definitive endoderm markers Cxcr4 and α-fetoprotein, mesoderm markers Collagen IIA and Gata2, and ectoderm markers βIII-tubulin, Enolase, and Nestin. Of particular significance is the observation that these ciPSCs are dependent only on leukemia inhibitory factor (LIF), making them similar to mouse and canine embryonic stem cells, but strikingly unlike the ciPSCs recently described in two other studies, which were dependent on both basic fibroblast growth factor and LIF in order to maintain their pluripotency. Thus, our ciPSCs closely resemble mouse ESCs derived from the inner cell mass of preimplantation embryos, while the previously described ciPSCs appear to be more representative of cells from the epiblast of mouse postimplantation embryos.

Derivation of Mesenchymal Stromal Cells from Canine Induced Pluripotent Stem Cells by Inhibition of the TGFβ/Activin Signaling Pathway

In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFβ)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRβ and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro- and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases.

Variability of Gene Expression Identifies Transcriptional Regulators of Early Human Embryonic Development

An analysis of gene expression variability can provide an insightful window into how regulatory control is distributed across the transcriptome. In a single cell analysis, the inter-cellular variability of gene expression measures the consistency of transcript copy numbers observed between cells in the same population. Application of these ideas to the study of early human embryonic development may reveal important insights into the transcriptional programs controlling this process, based on which components are most tightly regulated. Using a published single cell RNA-seq data set of human embryos collected at four-cell, eight-cell, morula and blastocyst stages, we identified genes with the most stable, invariant expression across all four developmental stages. Stably-expressed genes were found to be enriched for those sharing indispensable features, including essentiality, haploinsufficiency, and ubiquitous expression. The stable genes were less likely to be associated with loss-of-function variant genes or human recessive disease genes affected by a DNA copy number variant deletion, suggesting that stable genes have a functional impact on the regulation of some of the basic cellular processes. Genes with low expression variability at early stages of development are involved in regulation of DNA methylation, responses to hypoxia and telomerase activity, whereas by the blastocyst stage, low-variability genes are enriched for metabolic processes as well as telomerase signaling. Based on changes in expression variability, we identified a putative set of gene expression markers of morulae and blastocyst stages. Experimental validation of a blastocyst-expressed variability marker demonstrated that HDDC2 plays a role in the maintenance of pluripotency in human ES and iPS cells. Collectively our analyses identified new regulators involved in human embryonic development that would have otherwise been missed using methods that focus on assessment of the average expression levels; in doing so, we highlight the value of studying expression variability for single cell RNA-seq data.