Deanne J. Whitworth

Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization

In humans, SOX9 heterozygous mutations cause the severe skeletal dysmorphology syndrome campomelic dysplasia. Except for clinical descriptions, little is known about the pathogenesis of this disease. We have generated heterozygous Sox9 mutant mice that phenocopy most of the skeletal abnormalities of this syndrome. The Sox9+/− mice died perinatally with cleft palate, as well as hypoplasia and bending of many skeletal structures derived from cartilage precursors. In embryonic day (E)14.5 heterozygous embryos, bending of radius, ulna, and tibia cartilages was already prominent. In E12.5 heterozygotes, all skeletal elements visualized by using Alcian blue were smaller. In addition, the overall levels of Col2a1 RNA at E10.5 and E12.5 were lower than in wild-type embryos. We propose that the skeletal abnormalities observed at later embryonic stages were caused by delayed or defective precartilaginous condensations. Furthermore, in E18.5 embryos and in newborn heterozygotes, premature mineralization occurred in many bones, including vertebrae and some craniofacial bones. Because Sox9 is not expressed in the mineralized portion of the growth plate, this premature mineralization is very likely the consequence of allele insufficiency existing in cells of the growth plate that express Sox9. Because the hypertrophic zone of the heterozygous Sox9 mutants was larger than that of wild-type mice, we propose that Sox9 also has a role in regulating the transition to hypertrophic chondrocytes in the growth plate. Despite the severe hypoplasia of cartilages, the overall organization and cellular composition of the growth plate were otherwise normal. Our results suggest the hypothesis that two critical steps of the chondrocyte differentiation pathway are sensitive to Sox9 dosage. First, an early step presumably at the stage of mesenchymal condensation of cartilage primordia, and second, a later step preceding the transition of chondrocytes into hypertrophic chondrocytes.

A transgenic insertion upstream of Sox9 is associated with dominant XX sex reversal in the mouse

In most mammals, male development is triggered by the transient expression of the Y-chromosome gene, Sry, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Several genes, in particular Sox9, have a crucial role in this pathway. Despite this, the direct downstream targets of Sry and the nature of the pathway itself remain to be clearly established. We report here a new dominant insertional mutation, Odsex (Ods), in which XX mice carrying a 150-kb deletion (approximately 1 Mb upstream of Sox9) develop as sterile XX males lacking Sry. During embryogenesis, wild-type XX fetal gonads downregulate Sox9 expression, whereas XY and XX Ods/+ fetal gonads upregulate and maintain its expression. We propose that Ods has removed a long-range, gonad-specific regulatory element that mediates the repression of Sox9 expression in XX fetal gonads. This repression would normally be antagonized by Sry protein in XY embryos. Our data are consistent with Sox9 being a direct downstream target of Sry and provide genetic evidence to support a general repressor model of sex determination in mammals.

High specificity of Müllerian-inhibiting substance signaling in vivo

Female transgenic mice that ectopically express high levels of human Mullerian-inhibiting substance (hMIS) under the control of the mouse metallothionein (MT) promoter lack a uterus, oviducts, and ovaries. The loss of the uterus and oviducts is consistent with the known activities for MIS. However, it is not clear if the loss of the ovaries in these transgenic females is caused by interactions of MIS with its normal receptor signaling pathway or by abnormal interactions with other transforming growth factor-beta (TGF-beta) super family receptor signaling pathways. To address this question, female mice carrying the MT-hMIS transgene that were also homozygous for a targeted deletion of the MIS type II receptor gene were generated. Although these females had high levels of circulating hMIS, they had normal reproductive tracts and ovaries with germ cells. In addition, these females were able to become pregnant and gave birth to pups. These findings demonstrate that all of the abnormalities of the reproductive system that are found in female transgenic mice that ectopically express high levels of hMIS are caused by signaling through the MIS type II receptor. These in vivo data demonstrate a high specificity for MIS and its receptor.

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.

Marsupial Anti-Müllerian Hormone Gene Structure, Regulatory Elements, and Expression

Abstract During male sexual development in reptiles, birds, and mammals, anti-Müllerian hormone (AMH) induces the regression of the Müllerian ducts that normally form the primordia of the female reproductive tract. Whereas Müllerian duct regression occurs during fetal development in eutherian mammals, in marsupial mammals this process occurs after birth. To investigate AMH in a marsupial, we isolated an orthologue from the tammar wallaby (Macropus eugenii) and characterized its expression in the testes and ovaries during development. The wallaby AMH gene is highly conserved with the eutherian orthologues that have been studied, particularly within the encoded C-terminal mature domain. The N-terminus of marsupial AMH is divergent and larger than that of eutherian species. It is located on chromosome 3/4, consistent with its autosomal localization in other species. The wallaby 5′ regulatory region, like eutherian AMH genes, contains binding sites for SF1, SOX9, and GATA factors but also contains a putative SRY-binding site. AMH expression in the developing testis begins at the time of seminiferous cord formation at 2 days post partum, and Müllerian duct regression begins shortly afterward. In the developing testis, AMH is localized in the cytoplasm of the Sertoli cells but is lost by adulthood. In the developing ovary, there is no detectable AMH expression, but in adults it is produced by the granulosa cells of primary and secondary follicles. It is not detectable in atretic follicles. Collectively, these studies suggest that AMH expression has been conserved during mammalian evolution and is intimately linked to upstream sex determination mechanisms.

XX germ cells: the difference between an ovary and a testis.

In mammals, gonadal sex is determined by the action of the testis-determining gene, SRY. In the absence of SRY, the indifferent gonad follows an alternative pathway and develops as an ovary. Both mitotic and meiotic germ cells appear to play an essential role in ensuring ovarian development. Ovaries depleted of germ cells before or after ovarian differentiation has commenced can develop seminiferous cords, suggesting that XX germ cells may inhibit testicular differentiation in the ovary.

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.

Generation and characterization of leukemia inhibitory factor-dependent equine induced pluripotent stem cells from adult dermal fibroblasts

In this study we have reprogrammed dermal fibroblasts from an adult female horse into equine induced pluripotent stem cells (equiPSCs). These equiPSCs are dependent only on leukemia inhibitory factor (LIF), placing them in striking contrast to previously derived equiPSCs that have been shown to be co-dependent on both LIF and basic fibroblast growth factor (bFGF). These equiPSCs have a normal karyotype and have been maintained beyond 60 passages. They possess alkaline phosphatase activity and express eqNANOG, eqOCT4, and eqTERT mRNA. Immunocytochemistry confirmed that they produce NANOG, REX1, SSEA4, TRA1-60, and TRA1-81. While our equiPSCs are LIF dependent, bFGF co-stimulates their proliferation via the PI3K/AKT pathway. EquiPSCs lack expression of eqXIST and immunostaining for H3K27me3, suggesting that during reprogramming the inactive X chromosome has likely been reactivated to generate cells that have two active X chromosomes. EquiPSCs form embryoid bodies and in vitro teratomas that contain derivatives of all three germ layers. These LIF-dependent equiPSCs likely reflect a more naive state of pluripotency than equiPSCs that are co-dependent on both LIF and bFGF and so provide a novel resource for understanding pluripotency in the horse.

Stem cell therapies for treating osteoarthritis: prescient or premature?

There has been unprecedented interest in recent years in the use of stem cells as therapy for an array of diseases in companion animals. Stem cells have already been deployed therapeutically in a number of clinical settings, in particular the use of mesenchymal stem cells to treat osteoarthritis in horses and dogs. However, an assessment of the scientific literature highlights a marked disparity between the purported benefits of stem cell therapies and their proven abilities as defined by rigorously controlled scientific studies. Although preliminary data generated from clinical trials in human patients are encouraging, therapies currently available to treat animals are supported by very limited clinical evidence, and the commercialisation of these treatments may be premature. This review introduces the three main types of stem cells relevant to veterinary applications, namely, embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells, and draws together research findings from in vitro and in vivo studies to give an overview of current stem cell therapies for the treatment of osteoarthritis in animals. Recent advances in tissue engineering, which is proposed as the future direction of stem cell-based therapy for osteoarthritis, are also discussed.

Overtraining syndrome in horses

Overtraining is a significant cause of poor athletic performance in both human and equine athletes. In humans, overtraining syndrome has been defined as an imbalance between training and recovery manifesting as a syndrome of chronic fatigue and poor performance that may be accompanied by physiological and psychological changes. A similar syndrome has been described in horses using both cross-sectional observations and longitudinal studies with progressively increasing training loads until signs of overtraining were observed. The original crosssectional studies of overtraining in horses linked the syndrome to increased red cell volume. However, more recent longitudinal studies revealed that overtraining is not always associated with increased red cell volume. Once other causes of poor performance have been ruled out, overtraining syndrome should be suspected in horses with evidence of sustained decreased performance in association with one or more physiological or psychological (behavioural) signs. While no single physiological marker is able to identify the syndrome, accompanying physiological signs in horses may include a decrease in body weight, elevated heart rates during exercise, lower plasma cortisol response to exercise, or elevated muscle enzymes or gamma glutamyl transferase concentrations. Behavioural signs were consistent and an early marker of overtraining syndrome in longitudinal studies in horses, and more research on developing behavioural scores to assist in early detection of overtraining syndrome in horses -as has been achieved for humans- is warranted. Two successful models of overtraining syndrome in horses have been developed, both of which appear to meet the criteria of overtraining syndrome rather than overreaching. Overtraining syndrome in horses is real, reproducible and future research should ensure the criteria for diagnosis of overtraining syndrome are met.