Late Phase Of Differentiation Research Articles

Implication of Adipogenesis-Coupled CRMP2 Functional Profile in Metabolic Homeostasis and Imbalance

Our previous studies demonstrated that collapsin response mediator protein 2 (CRMP2) is associated with obesity and, in addition, that hyperglycemia-suppressed CRMP2 augments malignant traits of colorectal cancer and is associated with advanced tumor stage. Regulation of CRMP2 profile was further explored in this study using 3T3-L1 pre-adipocyte adipogenesis as a study model for illustrating the roles of CRMP2 in metabolic homeostasis. Hyperglycemia inhibited expression of CRMP2, adipogenic machinery and adipocyte markers. CRMP2 displayed f-CRMP2 (62~66 kDa) and s-CMRP2 (58 kDa) isoforms at the growth arrest phase. Expression of s-CRMP2 was coupled with the mitotic clonal expansion (MCE) phase to direct cell proliferation and rapidly down-regulated in post-mitotic cells. In the late differentiation phase, f-CRMP2 was co-localized with tubulin in the cortical area. Insulin-enhanced CRMP2-glucose transporter 4 (GLUT4) co-localization and CRMP2 puncta on lipid droplets (LDs) suggested participation of CRMP2 in GLUT4 translocation and LD fusion. Collectively, the CRMP2 functional profile must be finely controlled to adjust cytoskeletal stability for meeting dynamic cellular needs. Manipulating the s-CRMP2/f-CRMP2 ratio and thus the cytoskeleton dynamics is anticipated to improve glucose uptake and insulin sensitivity. In summary, our data provide molecular evidence explaining the functions of CRMP2 in physiological, pathological and disease progression in metabolic homeostasis and disorders related to metabolic abnormalities, including cancer.

The osteogenetic activities of mesenchymal stem cells in response to Mg2+ ions and inflammatory cytokines: a numerical approach using fuzzy logic controllers.

Magnesium (Mg2+) ions are frequently reported to regulate osteogenic activities of mesenchymal stem cells (MSCs). In this study, we propose a numerical model to study the regulatory importance of Mg2+ ions on MSCs osteoblastic differentiation in the presence of an inflammatory response. A fuzzy logic controller was formulated to receive the concentrations of Mg2+ ions and the inflammatory cytokines of TNF-α, IL-10, IL-1β, and IL-8 as cellular inputs and predict the cells’ early and late differentiation rates. Five sets of empirical data obtained from published cell culture experiments were used to calibrate the model. The model successfully reproduced the empirical data regarding the concentration- and phase-dependent effect of Mg2+ ions on the differentiation process. In agreement with the experiments, the model showed the stimulatory role of Mg2+ ions on the early differentiation phase, once administered at low concentration, and their inhibitory role on the late differentiation phase. The numerical approach used in this study suggested 6–8 mM as the most effective concentration of Mg2+ ions in promoting the early differentiation process. Also, the proposed model sheds light on the fundamental differences in the behavioral properties of cells cultured in different experiments, e.g. differentiation rate and the sensitivity of the cultured cells to stimulatory signals such as Mg2+ ions. Thus, it can be used to interpret and compare different empirical findings. Moreover, the model successfully reproduced the nonlinearities in the concentration-dependent role of the inflammatory cytokines in early and late differentiation rates. Overall, the proposed model can be employed in studying the osteogenic properties of Mg-based implants in the presence of an inflammatory response.

Generation and Staging of Human Retinal Organoids Based on Self-Formed Ectodermal Autonomous Multi-Zone System.

Methods for stem cell-derived, three-dimensional retinal organoids induction have been established and shown great potential for retinal development modeling and drug screening. Herein, we reported an exogenous-factors-free and robust method to generate retinal organoids based on “self-formed ectodermal autonomous multi-zone” (SEAM) system, a two-dimensional induction scheme that can synchronously generate multiple ocular cell lineages. Characterized by distinct morphological changes, the differentiation of the obtained retinal organoids could be staged into the early and late differentiation phases. During the early differentiation stage, retinal ganglion cells, cone photoreceptor cells (PRs), amacrine cells, and horizontal cells developed; whereas rod PRs, bipolar cells, and Müller glial cells were generated in the late differentiation phase, resembling early-phase and late-phase retinogenesis in vivo. Additionally, we modified the maintenance strategy for the retinal organoids and successfully promoted their long-term survival. Using 3D immunofluorescence image reconstruction and transmission electron microscopy, the substantial mature PRs with outer segment, inner segment and ribbon synapse were demonstrated. Besides, the retinal pigment epithelium (RPE) was induced with distinct boundary and the formation of ciliary margin was observed by co-suspending retina organoids with the zone containing RPE. The obtained RPE could be expanded and displayed similar marker expression, ultrastructural feature and functional phagocytosis to native RPE. Thus, this research described a simple and robust system which enabled generation of retina organoids with substantial mature PRs, RPE and the ciliary margin without the need of exogenous factors, providing a new platform for research of retinogenesis and retinal translational application.

Temporal correlation of morphological and biochemical changes with the recruitment of different mechanisms of reactive oxygen species formation during human SW872 cell adipogenic differentiation.

Human SW872 preadipocyte conversion to mature adipocytes is associated with time‐dependent changes in differentiation markers' expression and with morphological changes accompanied by the accumulation of lipid droplets (LDs) as well as by increased mitochondriogenesis and mitochondrial membrane potential. Under identical conditions, the formation of reactive oxygen species (ROS) revealed with a general probe was significant at days 3 and 10 of differentiation and bearly detectable at day 6. NADPH oxidase (NOX)‐2 activity determined with an immunocytochemical approach followed a very similar pattern. There was no evidence of mitochondrial ROS (mROS), as detected with a selective fluorescence probe, at days 3 and 6, possibly due to the triggering of the Nrf‐2 antioxidant response. mROS were instead clearly detected at day 10, concomitantly with the accumulation of very large LDs, oxidation of both cardiolipin and thioredoxin 2, and decreased mitochondrial glutathione. In conclusion, the morphological and biochemical changes of differentiating SW872 cells are accompanied by the discontinuous formation of ROS derived from NOX‐2, increasingly implicated in adipogenesis and adipose tissue dysfunction. In addition, mROS formation was significant only in the late phase of differentiation and was associated with mitochondrial dysfunction.

MED1 is a lipogenesis coactivator required for postnatal adipose expansion

MED1 often serves as a surrogate of the general transcription coactivator complex Mediator for identifying active enhancers. MED1 is required for phenotypic conversion of fibroblasts to adipocytes in vitro but its role in adipose development and expansion in vivo has not been reported. Here we show that MED1 is dispensable for adipose development in mice. Instead, MED1 is required for postnatal adipose expansion and the induction of de novo lipogenesis (DNL) genes after pups switch diet from high-fat maternal milk to carbohydrate-based chow. During adipogenesis, MED1 is dispensable for induction of lineage-determining transcription factors (TFs) PPARγ and C/EBPα but is required for lipid accumulation in the late phase of differentiation. Mechanistically, MED1 controls the induction of DNL genes by facilitating lipogenic TF ChREBP-dependent recruitment of Mediator to active enhancers. Together, our findings identify a cell- and gene-specific regulatory role of MED1 as a lipogenesis coactivator required for postnatal adipose expansion.

MED1 is a lipogenesis coactivator required for postnatal adipose expansion.

MED1 often serves as a surrogate of the general transcription coactivator complex Mediator for identifying active enhancers. MED1 is required for phenotypic conversion of fibroblasts to adipocytes in vitro, but its role in adipose development and expansion in vivo has not been reported. Here, we show that MED1 is not generally required for transcription during adipogenesis in culture and that MED1 is dispensable for adipose development in mice. Instead, MED1 is required for postnatal adipose expansion and the induction of fatty acid and triglyceride synthesis genes after pups switch diet from high-fat maternal milk to carbohydrate-based chow. During adipogenesis, MED1 is dispensable for induction of lineage-determining transcription factors (TFs) PPARγ and C/EBPα but is required for lipid accumulation in the late phase of differentiation. Mechanistically, MED1 controls the induction of lipogenesis genes by facilitating lipogenic TF ChREBP- and SREBP1a-dependent recruitment of Mediator to active enhancers. Together, our findings identify a cell- and gene-specific regulatory role of MED1 as a lipogenesis coactivator required for postnatal adipose expansion.

High-mobility group box 2 protein is essential for the early phase of adipogenesis

Understanding of the mechanism of adipogenesis is essential for the control of obesity, which predisposes toward numerous health problems. High-mobility group box protein 2 (HMGB2) is a non-histone chromosomal protein that facilitates DNA replication, transcription, recombination, and repair. Here, we studied the role of HMGB2 in adipogenic differentiation. The expression of HMGB2 was measured at the mRNA and protein levels in cultured 3T3-L1 pre-adipocyte cells and during the process of adipogenic differentiation induced bya cocktail of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone. This increased in the early phase and decreased in the late phase of differentiation. However, 3T3-L1 pre-adipocyte cells did not differentiate into adipocytes after the knockdown of HMGB2 expression by small interfering RNA (siRNA). Similarly, mesenchymal stem cells (MSCs) isolated from Hmgb2−/− mice did not express peroxisome proliferator-activated receptor gamma (PPARγ) in response to the adipocyte differentiation cocktail and did not differentiate. Wnt/β-catenin signaling is a negative regulator of adipogenic differentiation. We found that β-catenin expression was downregulated during 3T3-L1 adipogenic differentiation, as expected, but not when endogenous HMBG2 expression was knocked down using siRNA. These results indicate that HMGB2 plays an essential role in the early phase of the differentiation of pre-adipocytes and MSCs, and probably interacts with other regulators, such as PPARγ and Wnt/β-catenin signaling.

Gain-of-Function STIM1 L96V Mutation Causes Myogenesis Alteration in Muscle Cells From a Patient Affected by Tubular Aggregate Myopathy.

Tubular Aggregate Myopathy (TAM) is a hereditary ultra-rare muscle disorder characterized by muscle weakness and cramps or myasthenic features. Biopsies from TAM patients show the presence of tubular aggregates originated from sarcoplasmic reticulum due to altered Ca2+ homeostasis. TAM is caused by gain-of-function mutations in STIM1 or ORAI1, proteins responsible for Store-Operated-Calcium-Entry (SOCE), a pivotal mechanism in Ca2+ signaling. So far there is no cure for TAM and the mechanisms through which STIM1 or ORAI1 gene mutation lead to muscle dysfunction remain to be clarified. It has been established that post-natal myogenesis critically relies on Ca2+ influx through SOCE. To explore how Ca2+ homeostasis dysregulation associated with TAM impacts on muscle differentiation cascade, we here performed a functional characterization of myoblasts and myotubes deriving from patients carrying STIM1 L96V mutation by using fura-2 cytofluorimetry, high content imaging and real-time PCR. We demonstrated a higher resting Ca2+ concentration and an increased SOCE in STIM1 mutant compared with control, together with a compensatory down-regulation of genes involved in Ca2+ handling (RyR1, Atp2a1, Trpc1). Differentiating STIM1 L96V myoblasts persisted in a mononuclear state and the fewer multinucleated myotubes had distinct morphology and geometry of mitochondrial network compared to controls, indicating a defect in the late differentiation phase. The alteration in myogenic pathway was confirmed by gene expression analysis regarding early (Myf5, Mef2D) and late (DMD, Tnnt3) differentiation markers together with mitochondrial markers (IDH3A, OGDH). We provided evidences of mechanisms responsible for a defective myogenesis associated to TAM mutant and validated a reliable cellular model usefull for TAM preclinical studies.

LATE BREAKING NEWS E-POSTER PRESENTATION: LBP 9 Constitutive Stim1 activation impairs myogenesis in Tubular Aggregate Myopathy: defective late differentiation of patient-derived myoblasts and potential druggable targets

Tubular aggregate myopathy (TAM) is a dysfunctional inherited muscle disorder characterized by the presence of tubular aggregates (TAs) originating from sarcoplasmic reticulum. It is caused by gain-of-function mutations in STIM1 or ORAI1, proteins responsible for Store-Operated-Calcium-Entry (SOCE). The insight into molecular mechanisms is pivotal to understand clinical phenotypes and identify therapeutic targets. Considering the role of calcium homeostasis in postnatal myogenesis we investigated for the first time if Ca2+ alteration via SOCE in TAM impacts muscle myogenesis and differentiation efficacy. We characterized myoblasts and myotubes derived from a TAM patient's biopsy carrying Leu96Val STIM1 mutant. By cytofluorimetry we confirmed a significantly higher resting Ca2+ concentration and an increased SOCE activity vs controls. By automated fluorescence microscopy, we found that differentiating Leu96Val STIM1 myoblasts persisted in a mononuclear state and resulted in a reduced myotubes multinucleation with distinct morphology and different geometry of mitochondrial network compared to controls, suggesting a defect in fusion process. RealTime-PCR analysis showed a compensatory down-regulation of genes involved in Ca2+handling (RyR1,SERCA1/Atp2a1,Trpc1) in STIM1 mutants vs controls. Moreover, early differentiation markers (Myf5,Mef2D) were increased in mutated myoblasts, while a reduction in late differentiation markers (Dystrophin/DMD, Troponin/Tnnt3) were found in mutated myoblasts and myotubes indicating an altered myogenesis in the late differentiation phase associated to TAM. Finally, the mitochondrial genes Isocitrate-Dehydrogenases (IDH3A) and 2-Oxoglutarate-Dehydrogenase (OGDH) were reduced in Leu96Val myoblasts, with defect of OGDH persisting in mutated myotubes. We provided novel evidences of a defective myogenesis in TAM; SOCE or mitochondrial biogenesis can be druggable targets to be exploited.

Activation of GPR40 induces hypothalamic neurogenesis through p38- and BDNF-dependent mechanisms

Hypothalamic adult neurogenesis provides the basis for renewal of neurons involved in the regulation of whole-body energy status. In addition to hormones, cytokines and growth factors, components of the diet, particularly fatty acids, have been shown to stimulate hypothalamic neurogenesis; however, the mechanisms behind this action are unknown. Here, we hypothesized that GPR40 (FFAR1), the receptor for medium and long chain unsaturated fatty acids, could mediate at least part of the neurogenic activity in the hypothalamus. We show that a GPR40 ligand increased hypothalamic cell proliferation and survival in adult mice. In postnatal generated neurospheres, acting in synergy with brain-derived neurotrophic factor (BDNF) and interleukin 6, GPR40 activation increased the expression of doublecortin during the early differentiation phase and of the mature neuronal marker, microtubule-associated protein 2 (MAP2), during the late differentiation phase. In Neuro-2a proliferative cell-line GPR40 activation increased BDNF expression and p38 activation. The chemical inhibition of p38 abolished GPR40 effect in inducing neurogenesis markers in neurospheres, whereas BDNF immunoneutralization inhibited GPR40-induced cell proliferation in the hypothalamus of adult mice. Thus, GPR40 acts through p38 and BDNF to induce hypothalamic neurogenesis. This study provides mechanistic advance in the understating of how a fatty acid receptor regulates adult hypothalamic neurogenesis.

Inhibition of cardiomyocyte differentiation of human induced pluripotent stem cells by Ribavirin: Implication for its cardiac developmental toxicity

Ribavirin has been proven to be an antiviral treatment, whereas there are still risks of hemolysis and congenital malformation. Abnormal cardiac development contributes to the occurrence and development of many heart diseases. However, there is so far no evidence that ribavirin induces human cardiac developmental toxicity. Herein, we employed the cardiac differentiation model of human induced pluripotent stem cells (hiPSCs) to determine the impact of ribavirin on heart development. Our data showed that ribavirin at clinically high concentrations (5 and 10 μM) significantly inhibited the proliferation and differentiation of hiPSCs from mesoderm to cardiac progenitor cells and cardiac progenitor cells to cardiomyocytes, but not from pluripotent status to mesoderm. Meanwhile, DCFH-DA staining revealed that ribavirin could increase ROS content in the mid-phase of differentiation. In addition, ribavirin treatment (1, 5 and 10 μM) remarkably caused DNA damage which was shown by the increase of γH2AX-positive cells and upregulation of the p53 during the differentiation of hiPSCs from mesoderm to cardiac progenitor cells. Moreover, exposuring to ribavirin (5 and 10 μM) markedly upregulated the expression of lncRNAs Gas5 in both mid-phase and late phase of differentiation and HBL1 in the mid-phase. In conclusion, our results suggest that ribavirin is detrimental in cardiac differentiation of hiPSCs, which may be associated with DNA damage, upregulated p53 and increased Gas5. It may provide the evidence for the rational clinical application of ribavirin.

Osteogenic activity of resveratrol in human fetal osteoblast cells

Background: Resveratrol (RSV) is a polyphenolic phytomolecule naturally present in the skin of grapes fruit. It is reported to be phytoestrogen because of its estrogenic potentials, hence it can be explored to the treatment of osteoporosis. There was no conclusive evidence for the estrogenic potential of RSV in osteoporosis. In this study, RSV is evaluated for its effects on human osteoblast cells. Objective: The main objective of the study was to evaluate RSV on the proliferation and differentiation of immortalized human fetal osteoblastic cells 1.19 (hFOB). Materials and Methods: The osteoblastic cell proliferation and differentiation potentials of RSV were tested by cell viability assay, alkaline phosphatase (ALP) activity, total protein content, and alizarin staining for the mineralization assays. Results: The cell viability assay indicated that RSV was found to be safe at a wider concentration range and the EC50 was 72.05 μM. The therapeutic concentrations as 500 nM and 1 μM were selected for the further assays. RSV at 500-nM and 1-μM concentrations treatment on immortalized human fetal osteoblastic cells 1.19 (hFOB) did not show a significant effect on ALP activity. The total cellular proteins in hFOB increased in a dose-dependent manner on RSV treatment (P ≤ 0.05). The significant staining and the color intensity of the calcium crystals by Alizarin staining assay indicate a stimulatory effect on the mineralization phase of bone formation. Conclusion: In mature osteoblasts, ALP activity is expressed in early stage, whereas mineralized nodules are formed in the late stage of differentiation. Therefore, the present study suggests that RSV stimulates the process of bone formation through activation of late differentiation phase and may have positive effects on osteoblastic differentiation potential.

Oleanolic acid suppresses resistin induction in adipocytes by modulating Tyk-STAT signaling

Oleanolic acid, a naturally occurring triterpenoid widely distributed in foods and medicinal plants, has anticancer, antioxidant, and antiaging properties. We hypothesized that oleanolic acid would suppress the production of the inflammatory adipokine resistin during adipogenic differentiation of 3T3-L1 adipocytes. 3T3-L1 adipocytes were cultured in adipogenic media with and without 1 to 25 μM oleanolic acid for up to 8 days to stimulate adipocyte differentiation. Adipocyte production of resistin was markedly enhanced during differentiation and was dose dependently attenuated by 1 to 25 μM oleanolic acid. This study further investigated whether Tyk2-Stat1/3 signaling was responsible for cellular production of resistin. Signal transducer and activator of transcription factor (STAT) 1 and STAT3 were activated during differentiation in a disparate temporal fashion; STAT1 was maximally phosphorylated on day 5 after initiating differentiation, whereas STAT3 was rapidly activated within 1 day of differentiation. When oleanolic acid was supplied to differentiating adipocytes, STAT1 and STAT3 phosphorylation was substantially suppressed. Upstream Tyk2 was rapidly activated in a manner similar to STAT3 and reactivated on days 3 to 5 after initiating differentiation, which was attenuated by incubating adipocytes with oleanolic acid. In addition, cellular expression of suppressor of cytokine signaling 3 (SOCS3), which inhibits Tyk2 activity, was markedly promoted from day 5 of adipocyte differentiation. Oleanolic acid attenuated SOCS3 expression, which was highly enhanced during the late phase of differentiation. Taken together, oleanolic acid suppressed adipocyte differentiation-associated resistin and adipogenesis production by disturbing the Tyk2-STAT1/3 signaling pathway and promoting SOCS3 expression. Therefore, oleanolic acid may be a possible bioactive agent that blunts adipogenesis and adipokine inflammation.

Dexamethasone modulates osteogenesis and adipogenesis with regulation of osterix expression in rat calvaria‐derived cells

Osteoblasts and adipocytes originate from common mesenchymal progenitor cells and although a number of compounds can induce osteoblastic and adipogenic differentiation from progenitor cells, the underlying mechanisms have not been elucidated. The present study examined the synergistic effects of dexamethasone (Dex) and bone morphogenetic protein (BMP)-2 on the differentiation of clonal mesenchymal progenitor cells isolated from rat calvaria into osteoblasts and adipocytes, as well as the effects of the timing of treatment. Cells were cultured for various periods of time in the presence of Dex and/or BMP-2. When cells were treated with Dex+BMP-2 during the early phase of differentiation, they differentiated into adipocytes. However, when cells were treated with Dex+BMP-2 during the late phase of differentiation, a synergistic effect on in vitro matrix mineralization was observed. To examine differences between the early and late phases of differentiation, ALP activity was measured in the presence of BMP-2. ALP activity increased markedly on Day 9, corresponding to the onset of the synergistic effect of Dex. Dex treatment inhibited osterix (OSX) expression in cells committed to adipogenic differentiation, but not in cells committed to osteogenic differentiation following BMP-2 treatment. The isoform2 OSX promoter region was found to be involved in the effects of Dex on cells during the early phase of differentiation. Furthermore, cells stably expressing OSX (isoform2) formed mineralized nodules even though they had been treated with Dex+BMP-2 during the early phase of differentiation. It appears that Dex modulates osteogenesis and adipogenesis in mesenchymal stem cells by regulating OSX expression.

Molecular mechanisms of the inhibitory effect of lipopolysaccharide (LPS) on osteoblast differentiation

Osteoblasts express Toll like receptor (TLR) 4 and produce osteoclast-activating cytokines in response to the stimulation by lipopolysaccharide (LPS). It has recently been reported that LPS exerts an inhibitory effect on osteoblast differentiation into osteocytes. However, the molecular mechanisms of this inhibitory effect remain ambiguous. The downstream signals of TLR4 are mediated by adaptor molecules including myeloid differentiation factor 88 (MyD88), leading to the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases (ERKs), whose activation by LPS requires the upstream serine/threonine kinase, Cot/Tpl2. To determine the signal molecules responsible for the inhibitory effects of LPS on osteoblast differentiation, we examined the in vitro differentiation of the primary osteoblasts from myd88−/− and cot/tpl2−/− mice. The matrix mineralization by the wild-type and cot/tpl2−/− osteoblasts was significantly inhibited by LPS, whereas that of myd88−/− was not affected. During differentiation, LPS suppressed the mRNA expression of runt related transcription factor 2 (Runx2), osterix (Sp7), and activating transcription factor 4 (ATF4) in the wild-type, but not in the myd88−/− osteoblasts. The inhibitory effect of LPS on the mRNA expression of these transcription factors was absent in the early phase but partially impaired in the late phase of differentiation in the cot/tpl2−/− osteoblasts. Thus, the inhibitory effect of LPS on osteoblast differentiation is Myd88-dependent, whereas the degree of its requirement for Cot/Tpl2 varies depending on the differentiation phase.

SHP-2 positively regulates adipogenic differentiation in 3T3-L1 cells

The Src homology domain 2 (SH2)-containing tyrosine phosphatase SHP-2 has been implicated in the regulation of proliferation and differentiation in various cell types. Here, we investigated the ability of SHP-2 to mediate insulin-induced adipogenic differentiation of mouse 3T3-L1 cells. We found that the expression of SHP-2 was increased along with adipogenic differentiation. Overexpression of wild-type SHP-2 in 3T3-L1 cells resulted in enhanced adipocyte differentiation. Furthermore, insulin-stimulated adipogenic differentiation of 3T3-L1 cells was abolished by down-regulating SHP-2 expression using short interfering RNA. These results suggest that SHP-2 is a positive effector in signal transduction pathways necessary for adipocyte differentiation. In SHP-2 knockdown cells, the expression of peroxisome proliferator-activated receptor gamma, a master regulator of adipogenesis, was entirely suppressed even in the late phase of differentiation, whereas the expression level of C/EBPdelta was unchanged. These results highlight a novel role of SHP-2 in the signal transduction pathways regulating adipocyte differentiation.

Up-Regulation of Heme Biosynthesis during Differentiation of Neuro2a Cells

Heme is an iron-containing tetrapyrrole molecule that functions as a prosthetic group for proteins such as mitochondrial respiratory enzymes. Several studies have suggested that heme has essential functions in the construction and maintenance of the nervous system. In this study, the contents of three biologically important forms of heme (types a, b, and c) and the expression of heme biosynthetic enzymes were examined in differentiating Neuro2a cells. During neuronal differentiation, there were increases in the cellular heme levels and increases in the mRNA levels for the rate-limiting enzymes of heme biosynthesis, such as aminolevulinic acid synthase (ALAS; EC 2.3.1.37) and coproporphyrinogen oxidase (EC 1.3.3.3). With respect to heme contents, heme b increased in the late phase of differentiation, but no apparent increase in heme a or b was observed in the early phase. In contrast, heme c (cytochrome c) markedly increased during the early phase of differentiation. This change preceded the increase in heme b and the up-regulation of the mRNA levels for heme biosynthetic enzymes. This study suggests the up-regulation of heme biosynthesis and differential regulation of the heme a, b, and c levels during neuronal differentiation.

Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.

Microarray analysis reveals expression regulation of Wnt antagonists in differentiating osteoblasts

Wnt signaling has been implicated in regulating bone formation by controlling osteoblast proliferation and function. Although stabilization of β-catenin by Wnt has been shown to increase alkaline phosphatase expression and osteoblast differentiation, the precise role of Wnt signaling during the process of osteoblast differentiation is largely unknown. In this study, we used microarray technology to investigate expression regulation of Wnt signaling components during in vitro osteoblast differentiation. Expression was analyzed during bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation of murine C2C12 and MC3T3 cells and data were compared with expression in BMP2-treated NIH3T3 fibroblasts. During osteoblast differentiation, particularly strong expression regulation of the Wnt antagonists Sfrp2 (secreted frizzled related protein 2) and Wif1 (Wnt inhibitory factor 1) was observed in the late phase of differentiation. In situ expression analysis in murine tail vertebrae supported Wif1 expression during late phase bone cell differentiation, since Wif1 was found to be expressed in vivo in trabecular, but not in cortical bone. We further analyzed the effects of continuous activation of Wnt signaling by lithium chloride and observed that osteoblast differentiation was reduced, as measured by expression of osteoblast marker genes encoding alkaline phosphatase, osteocalcin, and osterix, as well as by the amount of calcium release. Taken together, our data indicate that endogenous expression of Wnt antagonists by osteoblasts provides a negative Wnt feedback loop which is essential in controlling osteoblast maturation.

Differential distribution of MAP1A isoforms in the adult mouse barrel cortex and comparison with the serotonin 5-HT 2A receptor

Microtubule-associated protein 1A (MAP1A) is essential during the late differentiation phase of neuronal development. Here, we demonstrated the presence of two MAP1A isoforms with a differential spatial distribution in the adult mouse barrel cortex. Antibody A stained MAP1A in pyramidal and stellate cells, including dendrites that crossed layer IV in the septa between barrels. The other antibody, BW6 recognized a MAP1A isoform that was mainly confined to the barrel hollow and identified smaller caliber dendrites. Previously, an interaction of MAP1A and the serotonin 5-hydroxytryptamine 2A (5-HT 2A) receptor was shown in the rat cortex. Here, we identified, by double-immunofluorescent labeling, MAP1A isoform and serotonin 5-HT 2A receptor distribution. MAP1A co-localized mainly with 5-HT 2A receptor in larger apical dendrites situated in septa. This differential staining of MAP1A and a serotonin receptor in defined barrel compartments may be due to changes in the expression or processing of MAP1A during dendritic transport as a consequence of functional differences in processing of whisker-related sensory input.