Disruption of the Selenocysteine Lyase-Mediated Selenium Recycling Pathway Leads to Metabolic Syndrome in Mice

Selenium (Se) is an essential trace element used for biosynthesis of selenoproteins and is acquired either through diet or cellular recycling mechanisms. Selenocysteine lyase (Scly) is the enzyme that supplies Se for selenoprotein biosynthesis via decomposition of the amino acid selenocysteine (Sec). Knockout (KO) of Scly in a mouse affected hepatic glucose and lipid homeostasis. Mice lacking Scly and raised on an Se-adequate diet exhibit hyperinsulinemia, hyperleptinemia, glucose intolerance, and hepatic steatosis, with increased hepatic oxidative stress, but maintain selenoprotein levels and circulating Se status. Insulin challenge of Scly KO mice results in attenuated Akt phosphorylation but does not decrease phosphorylation levels of AMP kinase alpha (AMPKα). Upon dietary Se restriction, Scly KO animals develop several characteristics of metabolic syndrome, such as obesity, fatty liver, and hypercholesterolemia, with aggravated hyperleptinemia, hyperinsulinemia, and glucose intolerance. Hepatic glutathione peroxidase 1 (GPx1) and selenoprotein S (SelS) production and circulating selenoprotein P (Sepp1) levels are significantly diminished. Scly disruption increases the levels of insulin-signaling inhibitor PTP1B. Our results suggest a dependence of glucose and lipid homeostasis on Scly activity. These findings connect Se and energy metabolism and demonstrate for the first time a unique physiological role of Scly in an animal model.     

Mice Long-Term High-Fat Diet Feeding Recapitulates Human Cardiovascular Alterations: An Animal Model to Study the Early Phases of Diabetic Cardiomyopathy

Background/Aim

Hypercaloric diet ingestion and sedentary lifestyle result in obesity. Metabolic syndrome is a cluster of clinical features secondary to obesity, considered as a pre-diabetic condition and recognized as an independent risk factor for cardiovascular diseases. To better understand the relationship between obesity, metabolic syndrome and cardiovascular disease as well as for the development of novel therapeutic strategies, animal models that reproduce the etiology, course and outcomes of these pathologies are required. The aim of this work was to characterize the long-term effects of high-fat diet-induced obesity on the mice cardiovascular system, in order to make available a new animal model for diabetic cardiomyopathy.

Methods/Results

Male C57BL/6 mice were fed with a standardized high-fat diet (obese) or regular diet (normal) for 16 months. Metabolic syndrome was evaluated testing plasma glucose, triglycerides, cholesterol, insulin, and glucose tolerance. Arterial pressure was measured using a sphygmomanometer (non invasive method) and by hemodynamic parameters (invasive method). Cardiac anatomy was described based on echocardiography and histological studies. Cardiac function was assessed by cardiac catheterization under a stress test. Cardiac remodelling and metabolic biomarkers were assessed by RT-qPCR and immunoblotting. As of month eight, the obese mice were overweight, hyperglycaemic, insulin resistant, hyperinsulinemic and hypercholesterolemic. At month 16, they also presented normal arterial pressure but altered vascular reactivity (vasoconstriction), and cardiac contractility reserve reduction, heart mass increase, cardiomyocyte hypertrophy, cardiac fibrosis, and heart metabolic compensations. By contrast, the normal mice remained healthy throughout the study.

Conclusions

Mice fed with a high-fat diet for prolonged time recapitulates the etiology, course and outcomes of the early phases of human diabetic cardiomyopathy.     

Long Term Metabolic Syndrome Induced by a High Fat High Fructose Diet Leads to Minimal Renal Injury in C57BL/6 Mice

Metabolic syndrome can induce chronic kidney disease in humans. Genetically engineered mice on a C57BL/6 background are highly used for mechanistic studies. Although it has been shown that metabolic syndrome induces cardiovascular lesions in C57BL/6 mice, in depth renal phenotyping has never been performed. Therefore in this study we characterized renal function and injury in C57BL/6 mice with long-term metabolic syndrome induced by a high fat and fructose diet (HFFD). C57BL/6 mice received an 8 months HFFD diet enriched with fat (45% energy from fat) and drinking water enriched with fructose (30%). Body weight, food/water consumption, energy intake, fat/lean mass ratio, plasma glucose, HDL, LDL, triglycerides and cholesterol levels were monitored. At 3, 6 and 8 months, renal function was determined by inulin clearance and measure of albuminuria. At sacrifice, kidneys and liver were collected. Metabolic syndrome in C57BL/6 mice fed a HFFD was observed as early 4 weeks with development of type 2 diabetes at 8 weeks after initiation of diet. However, detailed analysis of kidney structure and function showed only minimal renal injury after 8 months of HFFD. HFFD induced moderate glomerular hyperfiltration (436,4 µL/min vs 289,8 µL/min; p-value=0.0418) together with a 2-fold increase in albuminuria only after 8 months of HFFD. This was accompanied by a 2-fold increase in renal inflammation (p-value=0.0217) but without renal fibrosis or mesangial matrix expansion. In addition, electron microscopy did not show alterations in glomeruli such as basal membrane thickening and foot process effacement. Finally, comparison of the urinary peptidome of these mice with the urinary peptidome from humans with diabetic nephropathy also suggested absence of diabetic nephropathy in this model. This study provides evidence that the HFFD C57BL/6 model is not the optimal model to study the effects of metabolic syndrome on the development of diabetic kidney disease.     

Loss of CD24 in Mice Leads to Metabolic Dysfunctions and a Reduction in White Adipocyte Tissue

CD24 is a glycophosphatidylinositol (GPI)-linked cell surface receptor that is involved in regulating the survival or differentiation of several different cell types. CD24 has been used to identify pre-adipocytes that are able to reconstitute white adipose tissue (WAT) in vivo. Moreover, we recently found that the dynamic upregulation of CD24 in vitro during early phases of adipogenesis is necessary for mature adipocyte development. To determine the role of CD24 in adipocyte development in vivo, we evaluated the development of the inguinal and interscapular subcutaneous WAT and the epididymal visceral WAT in mice with a homozygous deletion of CD24 (CD24KO). We observed a significant decrease in WAT mass of 40% to 74% in WAT mass from both visceral and subcutaneous depots in male mice, with no significant effect in female mice, compared to wild-type (WT) sex- and age-matched controls. We also found that CD24KO mice had increased fasting glucose and free fatty acids, decreased fasting insulin, and plasma leptin. No major differences were observed in the sensitivity to insulin or glucose, or in circulating triglycerides, total cholesterol, HDL-cholesterol, or LDL-cholesterol levels between WT and CD24KO mice. Challenging the CD24KO mice with either high sucrose (35%) or high fat (45%) diets that promote increased adiposity, increased WAT mass and fasting insulin, adiponectin and leptin levels, as well as reduced the sensitivity to insulin and glucose, to the levels of WT mice on the same diets. The CD24-mediated reduction in fat pad size was due to a reduction in adipocyte cell size in all depots with no significant reduction pre-adipocyte or adipocyte cell number. Thus, we have clearly demonstrated that the global absence of CD24 affects adipocyte cell size in vivo in a sex- and diet-dependent manner, as well as causing metabolic disturbances in glucose homeostasis and free fatty acid levels.     

Addition of Rapamycin to Anti-CD3 Antibody Improves Long-Term Glycaemia Control in Diabetic NOD Mice

Aims/Hypothesis

Non-Fc-binding Anti CD3 antibody has proven successful in reverting diabetes in the non-obese diabetes mouse model of type 1 diabetes and limited efficacy has been observed in human clinical trials. We hypothesized that addition of rapamycin, an mTOR inhibitor capable of inducing operational tolerance in allogeneic bone marrow transplantation, would result in improved diabetes reversal rates and overall glycemia.

Methods

Seventy hyperglycemic non-obese diabetic mice were randomized to either a single injection of anti CD3 alone or a single injection of anti CD3 followed by 14 days of intra-peritoneal rapamycin. Mice were monitored for hyperglycemia and metabolic control.

Results

Mice treated with the combination of anti CD3 and rapamycin had similar rates of diabetes reversal compared to anti CD3 alone (25/35 vs. 22/35). Mice treated with anti CD3 plus rapamycin had a significant improvement in glycemia control as exhibited by lower blood glucose levels in response to an intra-peritoneal glucose challenge; average peak blood glucose levels 30 min post intra-peritoneal injection of 2 gr/kg glucose were 6.9 mmol/L in the anti CD3 plus rapamycin group vs. 10 mmo/L in the anti CD3 alone (P<0.05).

Conclusions/Interpretation

The addition of rapamycin to anti CD3 results in significant improvement in glycaemia control in diabetic NOD mice.     

A Randomized Pilot Trial of a Moderate Carbohydrate Diet Compared to a Very Low Carbohydrate Diet in Overweight or Obese Individuals with Type 2 Diabetes Mellitus or Prediabetes

We compared the effects of two diets on glycated hemoglobin (HbA1c) and other health-related outcomes in overweight or obese adults with type 2 diabetes or prediabetes (HbA1c>6%). We randomized participants to either a medium carbohydrate, low fat, calorie-restricted, carbohydrate counting diet (MCCR) consistent with guidelines from the American Diabetes Association (n = 18) or a very low carbohydrate, high fat, non calorie-restricted diet whose goal was to induce nutritional ketosis (LCK, n = 16). We excluded participants receiving insulin; 74% were taking oral diabetes medications. Groups met for 13 sessions over 3 months and were taught diet information and psychological skills to promote behavior change and maintenance. At 3 months, mean HbA1c level was unchanged from baseline in the MCCR diet group, while it decreased 0.6% in the LCK group; there was a significant between group difference in HbA1c change favoring the LCK group (−0.6%, 95% CI, −1.1% to −0.03%, p = 0.04). Forty-four percent of the LCK group discontinued one or more diabetes medications, compared to 11% of the MCCR group (p = 0.03); 31% discontinued sulfonylureas in the LCK group, compared to 5% in the MCCR group (p = 0.05). The LCK group lost 5.5 kg vs. 2.6 kg lost in MCCR group (p = 0.09). Our results suggest that a very low carbohydrate diet coupled with skills to promote behavior change may improve glycemic control in type 2 diabetes while allowing decreases in diabetes medications.This clinical trial was registered with ClinicalTrials.gov, number {"type":"clinical-trial","attrs":{"text":"NCT01713764","term_id":"NCT01713764"}}NCT01713764.     

Immuno-Isolation of Pancreatic Islet Allografts Using Pegylated Nanotherapy Leads to Long-Term Normoglycemia in Full MHC Mismatch Recipient Mice

Two major hurdles need to be surmounted for cell therapy for diabetes: (i) allo-immune rejection of grafted pancreatic islets, or stem/precursor cell-derived insulin-secreting cells; and (ii) continuing auto-immunity against the diabetogenic endogenous target antigen. Nanotherapeutics offer a novel approach to overcome these problems and here we ask if creation of “stealth” islets encapsulated within a thin cage of pegylated material of 100–200 nanometers thick provides a viable option for islet transplantation. The aims of this study were to test islet viability and functionality following encapsulation within the pegylated cage, and functional efficacy in vivo in terms of graft-derived control of normoglycemia in diabetic mice. We first demonstrated that pegylation of the islet surface, plus or minus nanoparticles, improved long-term islet viability in vitro compared to non-pegylated (naked) control islets. Moreover, pegylation of the islets with nanoparticles was compatible with glucose-stimulated insulin secretion and insulin biogenesis. We next looked for functionality of the created “stealth” DBA/2 (H-2^d) islets in vivo by comparing glycemic profiles across 4 groups of streptozotozin-induced diabetic C57BL/6 (H-2^b) recipients of (i) naked islets; (ii) pegylated islets; (iii) pegylated islets with nanoparticles (empty); and (iv) pegylated islets with nanoparticles loaded with a cargo of leukemia inhibitory factor (LIF), a factor both promotes adaptive immune tolerance and regulates pancreatic β cell mass. Without any other treatment, normoglycemia was lost after 17 d (+/−7.5 d) in control group. In striking contrast, recipients in groups (ii), (iii), and (iv) showed long-term (>100 d) normoglycemia involving 30%; 43%, and 57% of the recipients in each respective group. In conclusion, construction of “stealth” islets by pegylation-based nanotherapeutics not only supports islet structure and function, but also effectively isolates the islets from immune-mediated destruction. The added value of nanoparticles to deliver immune modulators plus growth factors such as LIF expands the potential of this novel therapeutic approach to cell therapy for diabetes.     

Maternal Magnesium Deficiency in Mice Leads to Maternal Metabolic Dysfunction and Altered Lipid Metabolism with Fetal Growth Restriction

Inadequate magnesium (Mg) intake is a widespread problem, with over 50% of women of reproductive age consuming less than the Recommended Dietary Allowance (RDA). Because pregnancy increases the requirement for Mg and the beneficial effects of magnesium sulfate for preeclampsia/eclampsia and fetal neuroprotection are well described, we examined the outcomes of Mg deficiency during pregnancy. Briefly, pregnant Swiss Webster mice were fed either control or Mg-deficient diets starting on gestational day (GD) 6 through euthanasia on GD17. Mg-deficient dams had significantly reduced weight gain and higher plasma adipokines, in the absence of inflammation. Livers of Mg-deficient dams had significantly higher saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) and lower polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) (P < 0.0001) and arachidonic acid (AA) (P < 0.0001). Mechanistically, Mg deficiency was accompanied by enhanced desaturase and elongase mRNA expression in maternal livers along with higher circulating insulin and glucose concentrations (P < 0.05) and increased mRNA expression of Srebf1 and Chrebp, regulators of fatty acid synthesis (P < 0.05). Fetal pups exposed to Mg deficiency were growth-restricted and exhibited reduced survival. Mg-deficient fetal livers showed lower MUFAs and higher PUFAs, with lower desaturase and elongase mRNA expression than controls. In addition, DHA concentrations were lower in Mg-deficient fetal brains (P < 0.05). These results indicate that Mg deficiency during pregnancy influences both maternal and fetal fatty acid metabolism, fetal growth and fetal survival, and support better understanding maternal Mg status before and during pregnancy.     

ALKBH4 Depletion in Mice Leads to Spermatogenic Defects

ALKBH4, an AlkB homologue in the 2-oxoglutarate and Fe²⁺ dependent hydroxylase family, has previously been shown to regulate the level of monomethylated lysine-84 in actin and thereby indirectly influences the ability of non-muscular myosin II to bind actin filaments. ALKBH4 modulates fundamental processes including cytokinesis and cell motility, and its depletion is lethal during early preimplantation embryo stage. The aim of this study was to investigate the effect of ALKBH4 deficiency in a physiological context, using inducible Alkbh4 knockout mice. Here, we report that ALKBH4 is essential for the development of spermatocytes during the prophase of meiosis, and that ALKBH4 depletion leads to insufficient establishment of the synaptonemal complex. We also show that ALKBH4 is localized in nucleolar structures of Sertoli cells, spermatogonia and primary spermatocytes.     

Dll1 Haploinsufficiency in Adult Mice Leads to a Complex Phenotype Affecting Metabolic and Immunological Processes

Background

The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligands is less well understood. Loss-of-function of Dll1 leads to embryonic lethality, but reduction of Delta-like 1 protein levels has not been studied in adult stage.

Methodology/Principal Findings

Here we present the haploinsufficient phenotype of Dll1 and a missense mutant Dll1 allele (Dll1^C413Y). Haploinsufficiency leads to a complex phenotype with several biological processes altered. These alterations reveal the importance of Dll1 mainly in metabolism, energy balance and in immunology. The animals are smaller, lighter, with altered fat to lean ratio and have increased blood pressure and a slight bradycardia. The animals have reduced cholesterol and triglyceride levels in blood. At the immunological level a subtle phenotype is observed due to the effect and fine-tuning of the signaling network at the different levels of differentiation, proliferation and function of lymphocytes. Moreover, the importance of the proteolytic regulation of the Notch signaling network emphasized.

Conclusions/Significance

In conclusion, slight alterations in one player of Notch signaling alter the entire organism, emphasizing the fine-tuning character of this pathway in a high number of processes.     

Dissecting Long-Term Glucose Metabolism Identifies New Susceptibility Period for Metabolic Dysfunction in Aged Mice

Metabolic disorders, like diabetes and obesity, are pathogenic outcomes of imbalance in glucose metabolism. Nutrient excess and mitochondrial imbalance are implicated in dysfunctional glucose metabolism with age. We used conplastic mouse strains with defined mitochondrial DNA (mtDNA) mutations on a common nuclear genomic background, and administered a high-fat diet up to 18 months of age. The conplastic mouse strain B6-mt^FVB, with a mutation in the mt-Atp8 gene, conferred β-cell dysfunction and impaired glucose tolerance after high-fat diet. To our surprise, despite of this functional deficit, blood glucose levels adapted to perturbations with age. Blood glucose levels were particularly sensitive to perturbations at the early age of 3 to 6 months. Overall the dynamics consisted of a peak between 3–6 months followed by adaptation by 12 months of age. With the help of mathematical modeling we delineate how body weight, insulin and leptin regulate this non-linear blood glucose dynamics. The model predicted a second rise in glucose between 15 and 21 months, which could be experimentally confirmed as a secondary peak. We therefore hypothesize that these two peaks correspond to two sensitive periods of life, where perturbations to the basal metabolism can mark the system for vulnerability to pathologies at later age. Further mathematical modeling may perspectively allow the design of targeted periods for therapeutic interventions and could predict effects on weight loss and insulin levels under conditions of pre-diabetic obesity.     

Disruption of Smad4 Expression in T Cells Leads to IgA Nephropathy-Like Manifestations

The link between glomerular IgA nephropathy (IgAN) and T helper 2 (Th2) response has been implicated, however, the mechanisms are poorly defined because of the lack of an appropriate model. Here we report a novel murine model characterized by lineage-restricted deletion of the gene encoding MAD homologue 4 (Smad4) in T cells (Smad4^{co/co;Lck-cre}). Loss of Smad4 expression in T cells results in overproduction of Th2 cytokines and high serum IgA levels. We found that Smad4^{co/co;Lck-cre} mice exhibited massive glomerular IgA deposition, increased albumin creatinine ratio, aberrant glycosylated IgA, IgA complexed with IgG1 and IgG2a, and polymeric IgA, all known features of IgAN in humans. Furthermore, we examined the β1, 4-galactosyltransferases (β4GalT) enzyme which is involved in the synthesis of glycosylated murine IgA, and we found reduced β4GalT2 and β4GalT4 mRNA levels in B cells. These findings indicate that Smad4^{co/co;Lck-cre} mice could be a useful model for studying the mechanisms between IgAN and Th2 response, and further, disruption of Smad4-dependent signaling in T cells may play an important role in the pathogenesis of human IgAN and contributing to a Th2 T cell phenotype.     

Olfactory Bulbectomy Leads to the Development of Epilepsy in Mice

There is a clear link between epilepsy and depression. Clinical data demonstrate a 30–35% lifetime prevalence of depression in patients with epilepsy, and patients diagnosed with depression have a three to sevenfold higher risk of developing epilepsy. Traditional epilepsy models partially replicate the clinical observations, with the demonstration of depressive traits in epileptic animals. Studies assessing pro-epileptogenic changes in models of depression, however, are more limited. Here, we examined whether a traditional rodent depression model—bilateral olfactory bulbectomy—predisposes the animals towards the development of epilepsy. Past studies have demonstrated increased neuronal excitability after bulbectomy, but continuous seizure monitoring had not been conducted. For the present study, we monitored control and bulbectomized animals by video-EEG 24/7 for approximately two weeks following the surgery to determine whether they develop spontaneous seizures. All seven bulbectomized mice exhibited seizures during the monitoring period. Seizures began about one week after surgery, and occurred in clusters with severity increasing over the monitoring period. These results suggest that olfactory bulbectomy could be a useful model of TBI-induced epilepsy, with advantages of relatively rapid seizure onset and a high number of individuals developing the disease. The model may also be useful for investigating the mechanisms underlying the bidirectional relationship between epilepsy and depression.     

Adenomatous Polyposis Coli Mutation Leads to Myopia Development in Mice

Myopia incidence in China is rapidly becoming a very serious sight compromising problem in a large segment of the general population. Therefore, delineating the underlying mechanisms leading to myopia will markedly lessen the likelihood of other sight compromising complications. In this regard, there is some evidence that patients afflicted with familial adenomatous polyposis (FAP), havean adenomatous polyposis coli (APC) mutation and a higher incidence of myopia. To clarify this possible association, we determined whether the changes in pertinent biometric and biochemical parameters underlying postnatal refractive error development in APC^Min mice are relevant for gaining insight into the pathogenesis of this disease in humans. The refraction and biometrics in APC^Min mice and age-matched wild-type (WT) littermates between postnatal days P28 and P84 were examined with eccentric infrared photorefraction (EIR) and customized optical coherence tomography (OCT). Compared with WT littermates, the APC^Min mutated mice developed myopia (average -4.64 D) on P84 which was associated with increased vitreous chamber depth (VCD). Furthermore, retinal and scleral changes appear in these mice along with: 1) axial length shortening; 2) increased retinal cell proliferation; 3) and decreased tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of DA synthesis. Scleral collagen fibril diameters became heterogeneous and irregularly organized in the APC^Min mice. Western blot analysis showed that scleral alpha-1 type I collagen (col1α1) expression also decreased whereas MMP2 and MMP9 mRNA expression was invariant. These results indicate that defective APC gene function promotes refractive error development. By characterizing in APC^Min mice ocular developmental changes, this approach provides novel insight into underlying pathophysiological mechanisms contributing to human myopia development.     

Mice Lacking Natural Killer T Cells Are More Susceptible to Metabolic Alterations following High Fat Diet Feeding

Current estimates suggest that over one-third of the adult population has metabolic syndrome and three-fourths of the obese population has non-alcoholic fatty liver disease (NAFLD). Inflammation in metabolic tissues has emerged as a universal feature of obesity and its co-morbidities, including NAFLD. Natural Killer T (NKT) cells are a subset of innate immune cells that abundantly reside within the liver and are readily activated by lipid antigens. There is general consensus that NKT cells are pivotal regulators of inflammation; however, disagreement exists as to whether NKT cells exert pathogenic or suppressive functions in obesity. Here we demonstrate that CD1d^−/− mice, which lack NKT cells, were more susceptible to weight gain and fatty liver following high fat diet (HFD) feeding. Compared with their WT counterparts, CD1d^−/− mice displayed increased adiposity and greater induction of inflammatory genes in the liver suggestive of the precursors of NAFLD. Calorimetry studies revealed a significant increase in food intake and trends toward decreased metabolic rate and activity in CD1d^−/− mice compared with WT mice. Based on these findings, our results suggest that NKT cells play a regulatory role that helps to prevent diet-induced obesity and metabolic dysfunction and may play an important role in mechanisms governing cross-talk between metabolism and the immune system to regulate energy balance and liver health.     

Early-Life Antibiotic-Driven Dysbiosis Leads to Dysregulated Vaccine Immune Responses in Mice

1. Download high-res image (160KB)
2. Download full-size image

     

iRhom2 Mutation Leads to Aberrant Hair Follicle Differentiation in Mice

iRhom1 and iRhom2 are inactive homologues of rhomboid intramembrane serine proteases lacking essential catalytic residues, which are necessary for the maturation of TNFα-converting enzyme (TACE). In addition, iRhoms regulate epidermal growth factor family secretion. The functional significance of iRhom2 during mammalian development is largely unclear. We have identified a spontaneous single gene deletion mutation of iRhom2 in Uncv mice. The iRhom2^Uncv/Uncv mice exhibit hairless phenotype in a BALB/c genetic background. In this study, we observed dysplasia hair follicles in iRhom2^Uncv/Uncv mice from postnatal day 3. Further examination found decreased hair matrix proliferation and aberrant hair shaft and inner root sheath differentiation in iRhom2^Uncv/Uncv mutant hair follicles. iRhom2 is required for the maturation of TACE. Our data demonstrate that iRhom2^Uncv cannot induce the maturation of TACE in vitro and the level of mature TACE is also significantly reduced in the skin of iRhom2^Uncv/Uncv mice. The activation of Notch1, a substrate of TACE, is disturbed, associated with dramatically down-regulation of Lef1 in iRhom2^Uncv/Uncv hair follicle matrix. This study identifies iRhom2 as a novel regulator of hair shaft and inner root sheath differentiation.