While cell division is essential for self-renewal and differentiation of stem cells and progenitors, dormancy is required to maintain the structure and function of the stem-cell niche. Here we use the hair follicle to show that during growth, the mesenchymal niche of the hair follicle, the dermal papilla (DP), is maintained quiescent by the activity of Hdac1 and Hdac2 in the DP that suppresses the expression of cell-cycle genes. Furthermore, Hdac1 and Hdac2 in the DP promote the survival of DP cells throughout the hair cycle. While during growth and regression this includes downregulation of p53 activity and the control of p53-independent programs, during quiescence, this predominantly involves p53-independent mechanisms. Remarkably, Hdac1 and Hdac2 in the DP during the growth phase also participate in orchestrating the hair cycle clock by maintaining physiological levels of Wnt signaling in the vicinity of the DP. Our findings not only provide insight into the molecular mechanism that sustains the function of the stem-cell niche in a persistently changing microenvironment, but also unveil that the same mechanism provides a molecular toolbox allowing the DP to affect and fine tune the microenvironment.

The mesenchymal components of the hair follicle—the dermal papilla (DP) and dermal sheath (DS)—are maintained by hair follicle dermal stem cells, but the position of this stem cell population throughout the hair cycle, its contribution to the maintenance of the dermis, and the existence of a migratory axis from the DP to the dermis remain unclear. In this study, we show that during homeostasis DP and DS cells are confined to their compartments, and during the regression phase of the hair cycle, some DP/DS cells undergo apoptosis and subsequently are internalized by nearby adipocytes. In contrast, during wound healing, DP/DS cells move toward the wound but do not directly participate in follicle neogenesis. Furthermore, hair follicle dermal stem cells, driving the cyclic renewal of the DS during the hair cycle, are heterogeneous and are housed during the growth phase within the most proximal part of the DS. Our analysis provides insight into the mechanisms of tissue maintenance and reveals a potential function of adipocytes in phagocytosis.

Tissue growth in the adult is an orchestrated process that often requires biological clocks to time stem cell and progenitor activity. Here, we employed the hair follicle, which cycles between growth and regression in a timely-restricted mode, to show that some components of the hair cycle clock reside within the mesenchymal niche of the hair follicle, the dermal papilla (DP), and both Fgf and Wnt signaling pathways interact within the DP to regulate the expression of these components that include Wnt agonists ( Rspondins ) and antagonists ( Dkk2 and Notum ). The levels of Wnt agonists and antagonists in the DP are progressively reduced and elevated during the growth phase, respectively. Consequently, Wnt signaling activity in the overlying epithelial progenitor cells decreases, resulting in the induction of the regression phase. Remarkably, DP properties allow Wnt activity in the DP to persist despite the Wnt-inhibiting milieu and consequently synchronize the induction and progression of the regression phase. This study provides insight into the importance of signaling crosstalk in coupling progenitors and their niche to regulate tissue growth.

The production and deposition of pigment in mouse skin are confined to the hair follicle and hair shaft, respectively. Hair pigmentation in mice involves the deposition of two types of pigment, black (eumelanin) and yellow (pheomelanin), in a very specific pattern (Barsh et al., 2000Barsh G. Gunn T. He L. Schlossman S. Duke-Cohan J. Biochemical and genetic studies of pigment-type switching.Pigment Cell Res. 2000; 13: 48-53Crossref PubMed Scopus (65) Google Scholar). Pigment, either black or yellow, is synthesized during the growth phase (anagen) of the hair cycle by melanocytes that reside in the hair bulb surrounding the upper half of the dermal papilla (DP), a specialized mesenchymal compartment that plays important roles in regulating different aspects of hair follicle biology. Keratinocytes in the hair bulb that undergo differentiation to form the building blocks of the hair shaft take up pigment from nearby melanocytes, leading to the formation of pigmented hair. Activity of Mc1r receptor in melanocytes results in black pigment production (Jackson et al., 2007Jackson I.J. Budd P.S. Keighren M. McKie L. Humanized MC1R transgenic mice reveal human specific receptor function.Hum Mol Genet. 2007; 16: 2341-2348Crossref PubMed Scopus (34) Google Scholar, Slominski et al., 2005Slominski A. Plonka P.M. Pisarchik A. Smart J.L. Tolle V. Wortsman J. et al.Preservation of eumelanin hair pigmentation in proopiomelanocortin-deficient mice on a nonagouti (a/a) genetic background.Endocrinology. 2005; 146: 1245-1253Crossref PubMed Scopus (104) Google Scholar, Smart and Low, 2003Smart J.L. Low M.J. Lack of proopiomelanocortin peptides results in obesity and defective adrenal function but normal melanocyte pigmentation in the murine C57BL/6 genetic background.Ann NY Acad Sci. 2003; 994: 202-210Crossref PubMed Scopus (54) Google Scholar). Binding of Agouti to Mc1r reduces Mc1r signaling and switches the production from black pigment to yellow (Ollmann et al., 1998Ollmann M.M. Lamoreux M.L. Wilson B.D. Barsh G.S. Interaction of agouti protein with the melanocortin 1 receptor in vitro and in vivo.Genes Dev. 1998; 12: 316-330Crossref PubMed Scopus (189) Google Scholar). During early anagen, a sharp peak of Agouti expression in DP cells is observed (Millar et al., 1995Millar S.E. Miller M.W. Stevens M.E. Barsh G.S. Expression and transgenic studies of the mouse agouti gene provide insight into the mechanisms by which mammalian coat color patterns are generated.Development. 1995; 121: 3223-3232Crossref PubMed Google Scholar). This peak generates a short and provisional period in which Mc1r activity is suppressed by Agouti and temporarily switches the melanocytes to produce pheomelanin. This creates a subapical yellow band in an otherwise black hair, resulting in an overall appearance of a mottled brown hair coat. The interaction between Mc1r and Agouti is modified by Corin (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease corin is a novel modifier of the agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (92) Google Scholar). Corin encodes a type II transmembrane serine protease that is expressed specifically in the DP and adjusts Agouti inhibition by narrowing the window of effective Agouti activity. In the absence of Corin, Agouti activity is prolonged and the yellow band is extended, leading to lighter coat color. Because Corin is a type II transmembrane serine protease and its single-pass transmembrane domain resides in close proximity to the N-terminus, most of Corin is extracellular (Figure 1a). In addition to regulating pigment type switching, Corin plays important role in blood pressure regulation. The protease activity of Corin in the heart cleaves the prohormone Nppa to its active form and thus activates the natriuretic peptide pathway to control blood tension (Chan et al., 2005Chan J.C. Knudson O. Wu F. Morser J. Dole W.P. Wu Q. Hypertension in mice lacking the proatrial natriuretic peptide convertase corin.Proc Natl Acad Sci USA. 2005; 102: 785-790Crossref PubMed Scopus (207) Google Scholar, Yan et al., 2000Yan W. Wu F. Morser J. Wu Q. Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme.Proc Natl Acad Sci USA. 2000; 97: 8525-8529Crossref PubMed Scopus (381) Google Scholar). In the uterus, Corin activates Nppa to regulate blood pressure during pregnancy by augmenting trophoblast invasion and remodeling spiral arteries, thus preventing preeclampsia (Cui et al., 2012Cui Y. Wang W. Dong N. Lou J. Srinivasan D.K. Cheng W. et al.Role of corin in trophoblast invasion and uterine spiral artery remodelling in pregnancy.Nature. 2012; 484: 246-250Crossref PubMed Scopus (220) Google Scholar). In contrast, little is known about the molecular mechanism by which Corin acts to inhibit Agouti activity and regulate pigment type switching. It was speculated that the protease activity of Corin mediates the Corin role in pigment type switching, but direct evidence for such a mechanism is lacking (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease corin is a novel modifier of the agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (92) Google Scholar). In contrast to Corin’s role in blood pressure regulation, Corin may regulate Agouti activity by a different mode of action that is unrelated to its serine protease activity. In such model, Corin may act as a receptor that transduces signaling into DP cells to modify Agouti activity or as extracellular inhibitor that sequesters the activity of Agouti. To explore whether the serine protease activity of Corin is required in the regulation of pigment type switching, the serine protease activity of Corin was abolished while preserving its complex structure. Using a gene targeting approach, a missense mutation that substitutes the serine (S) residue of the catalytic triad to alanine (A) was introduced (Figure 1b and c). This alteration has been previously shown in vitro to diminish the catalytic activity of Corin without affecting its surface localization and stability (Yan et al., 2000Yan W. Wu F. Morser J. Wu Q. Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme.Proc Natl Acad Sci USA. 2000; 97: 8525-8529Crossref PubMed Scopus (381) Google Scholar). Furthermore, this substitution was also designed to introduce the SgrA I restriction site to allow routine genotyping. A targeting construct was generated to include the S/A substitution and transfected into embryonic stem cells. Chimeric mice were derived from correctly targeted embryonic stem cell clones and used to establish a mouse line that harbors the S/A substitution. Wild-type, heterozygous, and homozygous mice for the missense mutation were designated CorinS/S, CorinS/A, and CorinA/A, respectively. Primers that flank the SgrA I site were designed to PCR-amplify a fragment of 390 base pairs (Figure 1b). In the case of the CorinA allele, digestion of the PCR product resulted in two fragments of 118 base pairs and 272 base pairs. This way, the CorinS and CorinA alleles were easily distinguished in agarose gel electrophoresis after digestion with SgrA I (Figure 1d). Similar to mice with a null allele of Corin (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease corin is a novel modifier of the agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (92) Google Scholar), mice homozygous for the CorinA allele were viable, fertile, and recovered at expected Mendelian frequencies. Furthermore, as expected, follicle morphology and the hair cycle appeared normal. Homozygous mutants were also assessed in vivo for the presence of the Corin mutant in the DP of the hair follicle in skin sections using immunostaining with anti-Corin antibodies (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease corin is a novel modifier of the agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (92) Google Scholar). Because the missense mutation is not expected to affect Corin levels and structure, these antibodies detected apparently normal levels of Corin in the DP of homozygous CorinA/A mutant mice (Figure 2a and b). Phenotypic analysis of coat color on normal Agouti background showed that mice homozygous for the CorinA allele display a distinctively lighter coat color (Figure 2c–e). Similar to the null phenotype (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease corin is a novel modifier of the agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (92) Google Scholar), this coat color phenotype is most pronounced in juveniles (Figure 2c). Hair shafts were plucked from the back skin of wild-type and mutant mice at the end of the first hair cycle and analyzed microscopically (Figure 2e). Mice homozygous for the CorinA allele exhibit extended subapical yellow band. This clearly illustrates that the serine protease activity of Corin is required for inhibiting Agouti activity during pigment type switching. A missense mutation in the Corin gene of the golden tiger has been recently identified (Xu et al., 2017Xu X. Dong G.X. Schmidt-Kuntzel A. Zhang X.L. Zhuang Y. Fang R. et al.The genetics of tiger pelage color variations.Cell Res. 2017; 27: 954-957Crossref PubMed Scopus (15) Google Scholar). This mutation results in the substitution of a histidine residue into tyrosine in the sixth LDLR domain of Corin and therefore does not interfere with its protease activity. However, this genetic alteration results in extremely elongated yellow band and, consequently, a light-colored tiger. Although the ability of Corin to proteolytically cleave Agouti has not been directly tested in that study and therefore precluded the assessment of Agouti as a direct substrate of Corin, this analysis may suggest that the underlying mechanism by which Corin interacts with Agouti is similar to the way Corin regulates proANP in the heart (Knappe et al., 2004Knappe S. Wu F. Madlansacay M.R. Wu Q. Identification of domain structures in the propeptide of corin essential for the processing of proatrial natriuretic peptide.J Biol Chem. 2004; 279: 34464-34471Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Initially, a direct protein-protein interaction between the LDLR domains of Corin and Agouti recruits Agouti to the vicinity of the protease domain, and subsequently, the serine protease activity of Corin inactivates Agouti by proteolytic cleavage. Unfortunately, the low and variable level of Agouti protein in the skin and the limited sensitivity of available antisera prevent the in vivo assessment of direct interaction between Agouti and Corin and the proteolytic consequences of this interaction on Agouti. Only future development of novel tools will allow us to molecularly address the mechanism by which Corin and Agouti interact. Mice were housed according to Federation of Laboratory Animal Science Associations guidelines. All mice were bred and maintained in a temperature-controlled room, on a 12-hour light-dark cycle, and with food and water available ad libitum. All experimental protocols were approved by the Animal Care and Use Committee of Bar Ilan University. The authors state no conflict of interest. This research was funded by the United States-Israel Binational Science Foundation (BSF: 2013375) and the Israel Science Foundation (ISF: 1304/14). We thank Hofesh Haruach for technical support. Download .pdf (.07 MB) Help with pdf files Supplementary Material

The mechanisms by which stem cell (SC) quiescence is regulated to allow normal regeneration are poorly understood. Here, we show that the mesenchymal niche of the hair follicle, the dermal papilla (DP), governs the properties of quiescent SCs in the bulge despite its relatively distant location. The DP induces regeneration by downregulating bulge-dependent inhibitory effects that restrain the intrinsic proliferation features of primed progenitors. Once regeneration initiates, the DP orchestrates Shh expression in primed-progenitor descendants by an autoregulatory circuit to restrict Shh expression to the DP vicinity and to confine Shh levels to act only on nearby cells. As the DP moves away from the bulge, quiescent SCs are exposed to Shh transiently. This ensures a short period of quiescent SC activation required for normal regeneration. Furthermore, our findings show that Shh signaling in the DP fine-tunes Wnt signaling activity and reveal the importance of signaling cross talk in coordinating regeneration pace.

Some bacterial toxins and viruses have evolved the capacity to bind mammalian glycosphingolipids to gain access to the cell interior, where they can co-opt the endogenous mechanisms of cellular trafficking and protein translocation machinery to cause toxicity. Cholera toxin (CT) is one of the best-studied examples, and is the virulence factor responsible for massive secretory diarrhea seen in cholera. CT enters host cells by binding to monosialotetrahexosylganglioside (GM1 gangliosides) at the plasma membrane where it is transported retrograde through the trans-Golgi network (TGN) into the endoplasmic reticulum (ER). In the ER, a portion of CT, the CT-A1 polypeptide, is unfolded and then "retro-translocated" to the cytosol by hijacking components of the ER associated degradation pathway (ERAD) for misfolded proteins. CT-A1 rapidly refolds in the cytosol, thus avoiding degradation by the proteasome and inducing toxicity. Here, we highlight recent advances in our understanding of how the bacterial AB5 toxins induce disease. We highlight the molecular mechanisms by which these toxins use glycosphingolipid to traffic within cells, with special attention to how the cell senses and sorts the lipid receptors. We also discuss several new studies that address the mechanisms of toxin unfolding in the ER and the mechanisms of CT A1-chain retro-translocation to the cytosol.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase protein localized to regions called focal adhesions, which are contact points between cells and the extracellular matrix. FAK protein acts as a scaffold to transfer adhesion-dependent and growth factor signals into the cell. Increased FAK expression is linked to aggressive metastatic and invasive tumors. However, little is known about its normal embryonic function. FAK protein knockdown during early Xenopus laevis development anteriorizes the embryo. Morphant embryos express increased levels of anterior neural markers, with reciprocally reduced posterior neural marker expression. Posterior neural plate folding and convergence-extension is also inhibited. This anteriorized phenotype resembles that of embryos knocked down zygotically for canonical Wnt signaling. FAK and Wnt3a genes are both expressed in the neural plate, and Wnt3a expression is FAK dependent. Ectopic Wnt expression rescues this FAK morphant anteriorized phenotype. Wnt3a thus acts downstream of FAK to balance anterior-posterior cell fate specification in the developing neural plate. Wnt3a gene expression is also FAK dependent in human breast cancer cells, suggesting that this FAK-Wnt linkage is highly conserved. This unique observation connects the FAK- and Wnt-signaling pathways, both of which act to promote cancer when aberrantly activated in mammalian cells.

Kupffer's vesicle (KV), a ciliated fluid‐filled sphere in the zebrafish embryo with a critical role in laterality determination, is derived from a group of superficial cells in the organizer region of the gastrula named the dorsal forerunner cells (DFC). We have examined the role of the expression of sox17 and chordin ( chd ) in the DFC in KV formation and laterality determination. Whereas sox17 was known to be expressed in DFC, its function in these cells was not studied before. Further, expression of chd in these cells has not been reported previously. Targeted knockdown of Sox17 and Chd in DFC led to aberrant Left‐Right (L‐R) asymmetry establishment, as visualized by the expression of southpaw and lefty , and heart and pancreas placement in the embryo. These defects correlated with the formation of small KVs with apparently diminished cilia, consistent with the known requirement for ciliary function in the laterality organ for the establishment of L‐R asymmetry. Developmental Dynamics 239:2980–2988, 2010. Published 2010 Wiley‐Liss, Inc.

The zebrafish pineal gland (epiphysis) is a site of melatonin production, contains photoreceptor cells, and functions as a circadian clock pace maker. Here, we have used microarray technology to study the zebrafish pineal transcriptome. Analysis of gene expression at three larval and two adult stages revealed a highly dynamic transcriptional profile, revealing many genes that are highly expressed in the zebrafish pineal gland. Statistical analysis of the data based on Gene Ontology annotation indicates that many transcription factors are highly expressed during larval stages, whereas genes dedicated to phototransduction are preferentially expressed in the adult. Furthermore, several genes were identified that exhibit day/night differences in expression. Among the multiple candidate genes suggested by these data, we note the identification of a tissue-specific form of the unc119 gene with a possible role in pineal development.

Protocadherin-18a (Pcdh18a) belongs to the δ2-protocadherins, which constitute the largest subgroup within the cadherin superfamily. Here we present isolation of a full-length zebrafish cDNA that encodes a protein highly similar to human and mouse Pcdh18. Zebrafish pcdh18a is expressed in a complex and dynamic pattern in the nervous system from gastrula stages onward, with lesser expression in mesodermal derivatives. Pcdh18a-eGFP fusion protein is expressed in a punctate manner on the membranes between cells. Overexpression of pcdh18a in embryos caused cyclopia, mislocalization of hatching gland tissue, and duplication or splitting of the neural tube. Most neural markers tested were expressed in an approximately correct A–P pattern. By cell transplantation we showed that overexpression of pcdh18a causes diminished cell migration and reduced cell protrusions, resulting in a tendency of cells to stay more firmly aggregated, probably due to increased cell adhesion. In contrast, knockdown of pcdh18a by a morpholino oligonucleotide caused defects in epiboly, and led to reduced cell adhesion as shown by cell dissociation, sorting and transplantation experiments. These results suggest a role for Pcdh18a in cell adhesion, migration and behavior but not cell specification during gastrula and segmentation stages of development.

In this report, we present the isolation and identification of a zebrafish homolog of the winged helix\forkhead transcription factor Foxj1. Foxj1 was identified in other species but not in zebrafish. Foxj1 was shown in mice to be required in ciliogenesis and left-right asymmetry establishment. Here we present a spatio-temporal expression pattern of zebrafish foxj1, showing that this gene is expressed in dorsal forerunner cells, Kupffer's vesicle, the floor plate, pronephric ducts and kidney. This expression pattern is overlapping but different from that of the foxj1.2, the closest related gene in zebrafish. Foxj1 in zebrafish appears to have similar functions as those reported in other species connected to ciliogenesis and left-right asymmetry.

Knockdown studies in Xenopus demonstrated that the XMeis3 gene is required for proper hindbrain formation. An explant assay was developed to distinguish between autonomous and inductive activities of XMeis3 protein. Animal cap explants caudalized by XMeis3 were recombined with explants neuralized by the BMP dominant-negative receptor protein. XMeis3-expressing cells induced convergent extension cell elongations in juxtaposed neuralized explants. Elongated explants expressed hindbrain and primary neuron markers, and anterior neural marker expression was extinguished. Cell elongation was dependent on FGF/MAP-kinase and Wnt-PCP activities. XMeis3 activates FGF/MAP-kinase signaling, which then modulates the PCP pathway. In this manner, XMeis3 protein establishes a hindbrain-inducing center that determines anteroposterior patterning in the brain.

Fibroblast growth factor (FGF) has been proposed to be involved in the specification and patterning of the developing vertebrate nervous system. There is conflicting evidence, however, concerning the requirement for FGF signaling in these processes. To provide insight into the signaling mechanisms that are important for neural induction and anterior–posterior neural patterning, we have employed the dominant negative Ras mutant, N17Ras, in addition to a truncated FGF receptor (XFD). Both N17Ras and XFD, when expressed in Xenopus laevis animal cap ectoderm, inhibit the ability of FGF to generate neural pattern. They also block induction of posterior neural tissue by XBF2 and XMeis3. However, neither XFD nor N17Ras inhibits noggin, neurogenin, or XBF2 induction of anterior neural markers. MAP kinase activation has been proposed to be necessary for neural induction, yet N17Ras inhibits the phosphorylation of MAP kinase that usually follows explantation of explants. In whole embryos, Ras-mediated FGF signaling is critical for the formation of posterior neural tissues but is dispensable for neural induction.