Notch1 is essential for postnatal hair follicle development and homeostasis

Notch genes encode evolutionarily conserved large, single transmembrane receptors, which regulate many cell fate decisions and differentiation processes during fetal and postnatal life. Multiple Notch receptors and ligands are expressed in both developing and adult epidermis and hair follicles. Proliferation and differentiation of these two ectodermal-derived structures have been proposed to be controlled in part by the Notch pathway. Whether Notch signaling is involved in postnatal hair homeostasis is currently unknown. Here, we investigate and compare the role of the Notch1 receptor during embryonic hair follicle development and postnatal hair homeostasis using Cre-loxP based tissue specific and inducible loss-of-function approaches. During embryonic development, tissue-specific ablation of Notch1 does not perturb formation and patterning of hair follicle placodes. However, Notch1 deficient hair follicles invaginate prematurely into the dermis. Embryonic as well as postnatal inactivation of Notch1 shortly after birth or in adult mice results in almost complete hair loss followed by cyst formation. The first hair cycle of Notch1 deficient mice is characterized by shortened anagen and a premature entry into catagen. These data show that Notch1 is essential for late stages of hair follicle development during embryogenesis as well as for post-natal hair follicle development and hair homeostasis.     

TGF-beta superfamily signaling is essential for tooth and hair morphogenesis and differentiation

Members of the transforming growth factor beta (TGF-beta) superfamily of signaling molecules are involved in the regulation of many developmental processes that involve the interaction between mesenchymal and epithelial tissues. Smad7 is a potent inhibitor of many members of the TGF-beta family, notably TGF-beta and activin. In this study, we show that embryonic overexpression of Smad7 in stratified epithelia using a keratin 5 promoter, results in severe morphogenetic defects in skin and teeth and leads to embryonic and perinatal lethality. To further analyze the functions of Smad7 in epithelial tissues of adult mice, we used an expression system that allowed a controlled overexpression of Smad7 in terms of both space and time. Skin defects in adult mice overexpressing Smad7 were characterized by hyper-proliferation and missing expression of early markers of keratinocyte differentiation. Upon Smad7-mediated blockade of TGF-beta superfamily signaling, ameloblasts failed to produce an enamel layer in incisor teeth. In addition, TGF-beta blockade in adult mice altered the pattern of thymic T cell differentiation and the number of thymic T cells was significantly reduced. This study shows that TGF-beta superfamily signaling is essential for development of hair, tooth and T-cells as well as differentiation and proliferation control in adult tissues.     

ALK5 is essential for tooth germ differentiation during tooth development

ABSTRACT

The TGFβ superfamily of proteins participates in tooth development. TGFβ1 and TGFβ3 regulate odontoblast differentiation and dentin extracellular matrix synthesis. Although the expression of TGFβ family member ligands is well-characterized during mammalian tooth development, less is known about the TGFβ receptor, which is a heteromeric complex consisting of a type I and type II receptors. The molecular mechanism of ALK5 (TGFβR1) in the dental mesenchyme is not clear. We investigated the role of ALK5 in tooth germ mesenchymal cells (TGMCs) from the lower first molar tooth germs of day 15.5 embryonic mice. Human recombinant TGFβ3 protein or an ALK5 inhibitor (SD208) was added to the cells. Cell proliferation was inhibited by SD208 and promoted by TGFβ3. We found that SD208 inhibited TGMCs osteogenesis and dentinogenesis. Both canonical and noncanonical TGFβ signaling pathways participated in the process. TAK1, P-TAK1, p38 and P-p38 showed greater expression and SMAD4 showed less expression when ALK5 was inhibited. Our findings contribute to understanding the role of TGFβ signaling for the differentiation of mesenchymal stem cells derived from dental germ and suggest possible targets for optimizing the use of stem cells of dental origin for tissue regeneration.     

Yes-associated protein is involved in proliferation and differentiation during postnatal liver development

It is known that the liver undergoes size increase and differentiation simultaneously during the postnatal period. Cells in the liver undergo a period of well-controlled proliferation to achieve the adult liver-to-body weight ratio. The postnatal liver growth is also accompanied by simultaneous hepatic differentiation. However, the mechanisms of liver size regulation and differentiation are not completely clear. Herein we report that yes-associated protein (Yap), the downstream effector of the Hippo Kinase signaling pathway, plays a role in liver size regulation and differentiation during the postnatal liver growth period. Postnatal liver growth was studied in C57BL/6 mice over a time course of postnatal days (PND) 0-30. Analysis of nuclear Yap by Western blot indicated peak Yap activation between PND15-20, which coincided with increased cyclin D1 expression and liver cell proliferation. Analysis of postnatal liver development in Yap(+/-) mice revealed a significant decrease in the liver-to-body weight ratio compared with Yap(+/+) mice at PND15 and -30. Yap(+/-) mice exhibited a significant decrease in postnatal liver cell proliferation, but no change in apoptosis was observed. Furthermore, global gene expression analysis of Yap(+/-) livers revealed a role of Yap in regulation of genes involved in bile acid metabolism, retinoic acid metabolism, ion transport, and extracellular matrix proteins. Taken together, these data indicate that Yap plays a role in both cell proliferation and possibly in hepatic differentiation during postnatal liver development.     

Notch signaling regulates late-stage epidermal differentiation and maintains postnatal hair cycle homeostasis

Background

Notch signaling involves ligand-receptor interactions through direct cell-cell contact. Multiple Notch receptors and ligands are expressed in the epidermis and hair follicles during embryonic development and the adult stage. Although Notch signaling plays an important role in regulating differentiation of the epidermis and hair follicles, it remains unclear how Notch signaling participates in late-stage epidermal differentiation and postnatal hair cycle homeostasis.

Methodology and Principal Findings

We applied Cre/loxP system to generate conditional gene targeted mice that allow inactivation of critical components of Notch signaling pathway in the skin. Rbpj, the core component of all four Notch receptors, and Pofut1, an essential factor for ligand-receptor interactions, were inactivated in hair follicle lineages and suprabasal layer of the epidermis using the Tgfb3-Cre mouse line. Rbpj conditional inactivation resulted in granular parakeratosis and reactive epidermal hyperplasia. Pofut1 conditional inactivation led to ultrastructural abnormalities in the granular layer and altered filaggrin processing in the epidermis, suggesting a perturbation of the granular layer differentiation. Disruption of Pofut1 in hair follicle lineages resulted in aberrant telogen morphology, a decrease of bulge stem cell markers, and a concomitant increase of K14-positive keratinocytes in the isthmus of mutant hair follicles. Pofut1-deficent hair follicles displayed a delay in anagen re-entry and dysregulation of proliferation and apoptosis during the hair cycle transition. Moreover, increased DNA double stand breaks were detected in Pofut1-deficent hair follicles, and real time PCR analyses on bulge keratinocytes isolated by FACS revealed an induction of DNA damage response and a paucity of DNA repair machinery in mutant bulge keratinocytes.

Significance

our data reveal a role for Notch signaling in regulating late-stage epidermal differentiation. Notch signaling is required for postnatal hair cycle homeostasis by maintaining proper proliferation and differentiation of hair follicle stem cells.     

TGF-β superfamily signaling is essential for tooth and hair morphogenesis and differentiation

Members of the transforming growth factor β (TGF-β) superfamily of signaling molecules are involved in the regulation of many developmental processes that involve the interaction between mesenchymal and epithelial tissues. Smad7 is a potent inhibitor of many members of the TGF-β family, notably TGF-β and activin. In this study, we show that embryonic overexpression of Smad7 in stratified epithelia using a keratin 5 promoter, results in severe morphogenetic defects in skin and teeth and leads to embryonic and perinatal lethality. To further analyze the functions of Smad7 in epithelial tissues of adult mice, we used an expression system that allowed a controlled overexpression of Smad7 in terms of both space and time. Skin defects in adult mice overexpressing Smad7 were characterized by hyper-proliferation and missing expression of early markers of keratinocyte differentiation. Upon Smad7-mediated blockade of TGF-β superfamily signaling, ameloblasts failed to produce an enamel layer in incisor teeth. In addition, TGF-β blockade in adult mice altered the pattern of thymic T cell differentiation and the number of thymic T cells was significantly reduced. This study shows that TGF-β superfamily signaling is essential for development of hair, tooth and T-cells as well as differentiation and proliferation control in adult tissues.     

Akt1 is essential for postnatal mammary gland development, function, and the expression of Btn1a1

Akt1, a serine-threonine protein kinase member of the PKB/Akt gene family, plays critical roles in the regulation of multiple cellular processes, and has previously been implicated in lactation and breast cancer development. In this study, we utilized Akt1+/+ and Akt1−/− C57/Bl6 female mice to assess the role that Akt1 plays in normal mammary gland postnatal development and function. We examined postnatal morphology at multiple time points, and analyzed gene and protein expression changes that persist into adulthood. Akt1 deficiency resulted in several mammary gland developmental defects, including ductal outgrowth and defective terminal end bud formation. Adult Akt1−/− mammary gland composition remained altered, exhibiting fewer alveolar buds coupled with increased epithelial cell apoptosis. Microarray analysis revealed that Akt1 deficiency altered expression of genes involved in numerous biological processes in the mammary gland, including organismal development, cell death, and tissue morphology. Of particular importance, a significant decrease in expression of Btn1a1, a gene involved in milk lipid secretion, was observed in Akt1−/− mammary glands. Additionally, pseudopregnant Akt1−/− females failed to induce Btn1a1 expression in response to hormonal stimulation compared to their wild-type counterparts. Retroviral-mediated shRNA knockdown of Akt1 and Btn1a1 in MCF-7 human breast epithelial further illustrated the importance of Akt1 in mammary epithelial cell proliferation, as well as in the regulation of Btn1a1 and subsequent expression of ß-casein, a gene that encodes for milk protein. Overall these findings provide mechanistic insight into the role of Akt1 in mammary morphogenesis and function.     

Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

We investigated the expression of P2X₅, P2X₇, P2Y₁ and P2Y₂ receptor subtypes in adult human anagen hair follicles and in relation to markers of proliferation [proliferating cell nuclear antigen (PCNA) and Ki-67], keratinocyte differentiation (involucrin) and apoptosis (anticaspase-3). Using immunohistochemistry, we showed that P2X₅, P2Y₁ and P2Y₂ receptors were expressed in spatially distinct zones of the anagen hair follicle: P2Y₁ receptors in the outer root sheath and bulb, P2X₅ receptors in the inner and outer root sheaths and medulla and P2Y₂ receptors in living cells at the edge of the cortex/medulla. P2X₇ receptors were not expressed. Colocalisation experiments suggested different functional roles for these receptors: P2Y₁ receptors were associated with bulb and outer root sheath keratinocyte proliferation, P2X₅ receptors were associated with differentiation of cells of the medulla and inner root sheaths and P2Y₂ receptors were associated with early differentiated cells in the cortex/medulla that contribute to the formation of the hair shaft. The therapeutic potential of purinergic agonists and antagonists for controlling hair growth is discussed.     

Site-1 protease is essential to growth plate maintenance and is a critical regulator of chondrocyte hypertrophic differentiation in postnatal mice

Site-1 protease (S1P) is a proprotein convertase with essential functions in lipid homeostasis and unfolded protein response pathways. We previously studied a mouse model of cartilage-specific knock-out of S1P in chondroprogenitor cells. These mice exhibited a defective cartilage matrix devoid of type II collagen protein (Col II) and displayed chondrodysplasia with no endochondral bone formation even though the molecular program for endochondral bone development appeared intact. To gain insights into S1P function, we generated and studied a mouse model in which S1P is ablated in postnatal chondrocytes. Postnatal ablation of S1P results in chondrodysplasia. However, unlike early embryonic ablations, the growth plates of these mice exhibit a lack of Ihh, PTHrP-R, and Col10 expression indicating a loss of chondrocyte hypertrophic differentiation and thus disruption of the molecular program required for endochondral bone development. S1P ablation results in rapid growth plate disruption due to intracellular Col II entrapment concomitant with loss of chondrocyte hypertrophy suggesting that these two processes are related. Entrapment of Col II in the chondrocytes of the prospective secondary ossification center precludes its development. Trabecular bone formation is dramatically diminished in the primary spongiosa and is eventually lost. The primary growth plate is eradicated by apoptosis but is gradually replaced by a fully functional new growth plate from progenitor stem cells capable of supporting new bone growth. Our study thus demonstrates that S1P has fundamental roles in the preservation of postnatal growth plate through chondrocyte differentiation and Col II deposition and functions to couple growth plate maturation to trabecular bone development in growing mice.     

Evidence that autocrine signaling through Bmpr1a regulates the proliferation, survival and morphogenetic behavior of distal lung epithelial cells

Lung development requires reciprocal epithelial/mesenchymal interactions, mediated by signaling factors such as Bmps made in both cell populations. To address the role of Bmp signaling in the epithelium, we have exploited the fact that Bmp receptor type Ia (Alk3) is expressed in the epithelium during branching morphogenesis. Deletion of Bmpr1a in the epithelium with an Sftpc-cre transgene leads to dramatic defects in lung development. There is reduced epithelial proliferation, extensive apoptosis, changes in cell morphology and extrusion of cells into the lumen. By E18.5, there are fewer Type II cells than normal, and the lung contains large fluid-filled spaces. If cell death is prevented by making embryos homozygous null for the proapoptotic gene, Bax, the epithelial cells that are rescued can apparently differentiate, but normal morphogenesis is not restored. To determine whether Bmps made by the epithelium can function in an autocrine manner, mesenchyme-free endoderm was cultured in Matrigel with Fgfs. Under these conditions, the mutant epithelium fails to undergo secondary budding. Abnormal development was also seen when Bmp4 was specifically deleted in the epithelium using the Sftpc-cre transgene. Our results support a model in which Bmp signaling primarily regulates the proliferation, survival and morphogenetic behavior of distal lung epithelial cells.     

Rac1 regulates cardiovascular development and postnatal function of endothelium

Rac1 is a member of the small Rho GTPase family, which controls actin cytoskeleton and focal adhesion dynamics in cellular protrusions. While Rac1 therefore contributes to regulation of endothelial cell-cell and cell-matrix interactions, a detailed understanding of its role in endothelium function is lacking. Recently, the role of Rac1 in development and postnatal regulation of the cardiovascular system has been investigated in murine models lacking Rac1 specifically in endothelium. Homozygous endothelial deletion was lethal, primarily due to defects in angiogenesis. Rac1-deficient endothelial cells were unable to form cellular protrusions/lamellipodia, leading to impaired cell-cell and cell-matrix interactions, and resulting in dysfunctional adhesion, motility, permeability and capillary morphogenesis. Development was normal in the heterozygous model, however a hypertensive phenotype was observed as a result of reduced nitric oxide signalling. Nitric oxide synthase activity was regulated by Rac1 at multiple levels; expression, mRNA stability and uptake of the nitric oxide synthase substrate L-arginine. Therefore, Rac1 activity is essential in regulating developmental and postnatal angiogenesis and cardiovascular function, by controlling nitric oxide production, and formation of endothelial cell protrusions.     

BMPR1A signaling is necessary for hair follicle cycling and hair shaft differentiation in mice

Interactions between ectodermal and mesenchymal extracellular signaling pathways regulate hair follicle (HF) morphogenesis and hair cycling. Bone morphogenetic proteins (BMPs) are known to be important in hair follicle development by affecting the local cell fate modulation. To study the role of BMP signaling in the HF, we disrupted Bmpr1a, which encodes the BMP receptor type IA (BMPR1A) in an HF cell-specific manner, using the Cre/loxP system. We found that the differentiation of inner root sheath, but not outer root sheath, was severely impaired in mutant mice. The number of HFs was reduced in the dermis and subcutaneous tissue, and cycling epithelial cells were reduced in mutant mice HFs. Our results strongly suggest that BMPR1A signaling is essential for inner root sheath differentiation and is indispensable for HF renewal in adult skin.     

BTB-Kelch protein Krp1 regulates proliferation and differentiation of myoblasts

The BTB-Kelch protein Krp1 is highly and specifically expressed in skeletal muscle, where it is proposed to have a role in myofibril formation. We observed significant upregulation of Krp1 in C2 cells early in myoblast differentiation, well before myofibrillogenesis. Krp1 has a role in cytoskeletal organization and cell motility; since myoblast migration and elongation/alignment are important events in early myogenesis, we hypothesized that Krp1 is involved with earlier regulation of differentiation. Krp1 protein levels were detectable by 24 h after induction of differentiation in C2 cells and were significantly upregulated by 48 h, i.e., following the onset myogenin expression and preceding myosin heavy chain (MHC) upregulation. Upregulation of Krp1 required a myogenic stimulus as signaling derived from increased myoblast cell density was insufficient to activate Krp1 expression. Examination of putative Krp1 proximal promoter regions revealed consensus E box elements associated with myogenic basic helix-loop-helix binding. The activity of a luciferase promoter-reporter construct encompassing this 2,000-bp region increased in differentiating C2 myoblasts and in C2 cells transfected with myogenin and/or MyoD. Knockdown of Krp1 via short hairpin RNA resulted in increased C2 cell number and proliferation rate as assessed by bromodeoxyuridine incorporation, whereas overexpression of Krp1-myc had the opposite effect; apoptosis was unchanged. No effects of changed Krp1 protein levels on cell migration were observed, either by scratch wound assay or live cell imaging. Paradoxically, both knockdown and overexpression of Krp1 inhibited myoblast differentiation assessed by expression of myogenin, MEF2C, MHC, and cell fusion.