NORMAL GENOMIC

• Gay DL et al.
• CD133 expression correlates with membrane beta-catenin and E-cadherin loss from human hair follicle placodes during morphogenesis.
• J Invest Dermatol. 2015; 135: 45-55
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• Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode "budding" is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions (AJs) is clearly required for budding. Snail-mediated downregulation of AJ component E-cadherin is important for placode budding in mice. Beta-catenin, another AJ component, has been more difficult to study owing to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes, and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent AJ dissolution.

• Meng Q, Jin C, Chen Y, Chen J, Medvedovic M, Xia Y
• Expression of signaling components in embryonic eyelid epithelium.
• PLoS One. 2014; 9: 87038-87038
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• Closure of an epithelium opening is a critical morphogenetic event for development. An excellent example for this process is the transient closure of embryonic eyelid. Eyelid closure requires shape change and migration of epithelial cells at the tip of the developing eyelids, and is dictated by numerous signaling pathways. Here we evaluated gene expression in epithelial cells isolated from the tip (leading edge, LE) and inner surface epithelium (IE) of the eyelid from E15.5 mouse fetuses by laser capture microdissection (LCM). We showed that the LE and IE cells are different at E15.5, such that IE had higher expression of muscle specific genes, while LE acquired epithelium identities. Despite their distinct destinies, these cells were overall similar in expression of signaling components for the "eyelid closure pathways". However, while the LE cells had more abundant expression of Fgfr2, Erbb2, Shh, Ptch1 and 2, Smo and Gli2, and Jag1 and Notch1, the IE cells had more abundant expression of Bmp5 and Bmpr1a. In addition, the LE cells had more abundant expression of adenomatosis polyposis coli down-regulated 1 (Apcdd1), but the IE cells had high expression of Dkk2. Our results suggest that the functionally distinct LE and IE cells have also differential expression of signaling molecules that may contribute to the cell-specific responses to morphogenetic signals. The expression pattern suggests that the EGF, Shh and NOTCH pathways are preferentially active in LE cells, the BMP pathways are effective in IE cells, and the Wnt pathway may be repressed in LE and IE cells via different mechanisms.

• Sun P et al.
• Pygo2 regulates beta-catenin-induced activation of hair follicle stem/progenitor cells and skin hyperplasia.
• Proc Natl Acad Sci U S A. 2014; 111: 10215-20
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• Understanding the epigenetic mechanisms that control the activation of adult stem cells holds the promise of tissue and organ regeneration. Hair follicle stem cells have emerged as a prime model to study stem cell activation. Wnt/beta-catenin signaling controls multiple aspects of skin epithelial regeneration, with its excessive activity promoting the hyperactivation of hair follicle stem/progenitor cells and tumorigenesis. The contribution of chromatin factors in regulating Wnt/beta-catenin pathway function in these processes is unknown. Here, we show that chromatin effector Pygopus homolog 2 (Pygo2) produced by the epithelial cells facilitates depilation-induced hair regeneration, as well as beta-catenin-induced activation of hair follicle stem/early progenitor cells and trichofolliculoma-like skin hyperplasia. Pygo2 maximizes the expression of Wnt/beta-catenin targets, but is dispensable for beta-catenin-mediated expansion of LIM/homeobox protein Lhx2(+) cells, in the stem/early progenitor cell compartment of the hair follicle. Moreover, beta-catenin and Pygo2 converge to induce the accumulation and acetylation of tumor suppressor protein p53 upon the cell cycle entry of hair follicle early progenitor cells and in cultured keratinocytes. These findings identify Pygo2 as an important regulator of Wnt/beta-catenin function in skin epithelia and p53 activation as a prominent downstream event of beta-catenin/Pygo2 action in stem cell activation.

• Ahmed MI et al.
• MicroRNA-214 controls skin and hair follicle development by modulating the activity of the Wnt pathway.
• J Cell Biol. 2014; 207: 549-67
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• Skin development is governed by complex programs of gene activation and silencing, including microRNA-dependent modulation of gene expression. Here, we show that miR-214 regulates skin morphogenesis and hair follicle (HF) cycling by targeting beta-catenin, a key component of the Wnt signaling pathway. miR-214 exhibits differential expression patterns in the skin epithelium, and its inducible overexpression in keratinocytes inhibited proliferation, which resulted in formation of fewer HFs with decreased hair bulb size and thinner hair production. The inhibitory effects of miR-214 on HF development and cycling were associated with altered activities of multiple signaling pathways, including decreased expression of key Wnt signaling mediators beta-catenin and Lef-1, and were rescued by treatment with pharmacological Wnt activators. Finally, we identify beta-catenin as one of the conserved miR-214 targets in keratinocytes. These data provide an important foundation for further analyses of miR-214 as a key regulator of Wnt pathway activity and stem cell functions during normal tissue homeostasis, regeneration, and aging.

• Motomura E et al.
• Cell-autonomous signal transduction in the Xenopus egg Wnt/beta-catenin pathway.
• Dev Growth Differ. 2014; 56: 640-52
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• Wnt proteins are thought to bind to their receptors on the cell surfaces of neighboring cells. Wnt8 likely substitutes for the dorsal determinants in Xenopus embryos to dorsalize early embryos via the Wnt/beta-catenin pathway. Here, we show that Wnt8 can dorsalize Xenopus embryos working cell autonomously. Wnt8 mRNA was injected into a cleavage-stage blastomere, and the subcellular distribution of Wnt8 protein was analyzed. Wnt8 protein was predominantly found in the endoplasmic reticulum (ER) and resided at the periphery of the cells; however, this protein was restricted to the mRNA-injected cellular region as shown by lineage tracing. A mutant Wnt8 that contained an ER retention signal (Wnt8-KDEL) could dorsalize Xenopus embryos. Finally, Wnt8-induced dorsalization occurred only in cells injected with Wnt8 mRNA. These experiments suggest that the Wnt8 protein acts within the cell, likely in the ER or on the cell surface in an autocrine manner for dorsalization.

• Deschene ER et al.
• beta-Catenin activation regulates tissue growth non-cell autonomously in the hair stem cell niche.
• Science. 2014; 343: 1353-6
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• Wnt/beta-catenin signaling is critical for tissue regeneration. However, it is unclear how beta-catenin controls stem cell behaviors to coordinate organized growth. Using live imaging, we show that activation of beta-catenin specifically within mouse hair follicle stem cells generates new hair growth through oriented cell divisions and cellular displacement. beta-Catenin activation is sufficient to induce hair growth independently of mesenchymal dermal papilla niche signals normally required for hair regeneration. Wild-type cells are co-opted into new hair growths by beta-catenin mutant cells, which non-cell autonomously activate Wnt signaling within the neighboring wild-type cells via Wnt ligands. This study demonstrates a mechanism by which Wnt/beta-catenin signaling controls stem cell-dependent tissue growth non-cell autonomously and advances our understanding of the mechanisms that drive coordinated regeneration.

• Zhang S, Li J, Lea R, Vleminckx K, Amaya E
• Fezf2 promotes neuronal differentiation through localised activation of Wnt/beta-catenin signalling during forebrain development.
• Development. 2014; 141: 4794-805
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• Brain regionalisation, neuronal subtype diversification and circuit connectivity are crucial events in the establishment of higher cognitive functions. Here we report the requirement for the transcriptional repressor Fezf2 for proper differentiation of neural progenitor cells during the development of the Xenopus forebrain. Depletion of Fezf2 induces apoptosis in postmitotic neural progenitors, with concomitant reduction in forebrain size and neuronal differentiation. Mechanistically, we found that Fezf2 stimulates neuronal differentiation by promoting Wnt/beta-catenin signalling in the developing forebrain. In addition, we show that Fezf2 promotes activation of Wnt/beta-catenin signalling by repressing the expression of two negative regulators of Wnt signalling, namely lhx2 and lhx9. Our findings suggest that Fezf2 plays an essential role in controlling when and where neuronal differentiation occurs within the developing forebrain and that it does so by promoting local Wnt/beta-catenin signalling via a double-repressor model.

• Kim BK, Lee HY, Kim I, Choi K, Park J, Yoon SK
• Increased expression of Dkk1 by HR is associated with alteration of hair cycle in hairpoor mice.
• J Dermatol Sci. 2014; 74: 81-7
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• BACKGROUND: Hairless (Hr), a transcriptional corepressor expressed mainly in the skin, regulates hair follicle (HF) morphogenesis and hair cycling. Recently, we reported a new Hr mutant mouse, "Hairpoor" (Hr(Hp)), that resembles the human hair disorder Marie Unna hereditary hypotrichosis (MUHH) in the heterozygous state. The Wnt/beta-catenin signaling pathway is critical for homeostasis in various adult tissues including skin and HFs. One of the Wnt inhibitors, Dickkopf (Dkk), inhibits hair growth during the hair cycle as a catagen inducer of apoptosis, resulting in HF reductions. OBJECTIVE: To investigate regulation of Dkk1 by HR and its effect on hair formation. METHODS: The relative expression of Dkk1 in (+)/Hr(HP) and Hr(Hp)/Hr(Hp) mice during the hair cycle was investigated using real time PCR and Western blot analysis. Immunohistochemistry was performed in order to confirm abnormal expression of Dkk1 in HFs of (+)/Hr(HP) and Hr(Hp)/Hr(Hp) mice. To determine whether Dkk1 expression was also regulated by HR in vitro, an Hr-transient transfection experiment was performed. Alteration of the hair cycle in Hr(HP) heterozygous mice was identified by determination of the hair cycle and measurement of HF length. RESULTS: Dkk1 expression was increased in the skin of (+)/Hr(HP) and Hr(Hp)/Hr(Hp) mice, as well as in Hr-overexpressing mouse keratinocytes. Additionally, an earlier entrance of HFs into catagen and shortened HF length in (+)/Hr(HP) mice compared to wild-type mice was observed. CONCLUSION: Study results suggested that up-regulation of Dkk1 by HR contributed to abnormal development of HFs and failure in regeneration of HFs in Hr(Hp)/Hr(Hp) mice. These findings also indicated that alteration of the hair cycle in (+)/Hr(HP) mice was related to the up-regulation of Dkk1 by HR.

• Lim YY et al.
• Potential relationship between the canonical Wnt signalling pathway and expression of the vitamin D receptor in alopecia.
• Clin Exp Dermatol. 2014; 39: 368-75
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• BACKGROUND: Vitamin D receptor (VDR) is expressed in dermal papilla cells and in the epidermis of the hair follicle. Patients with alopecia areata (AA) and alopecia universalis (AU) have VDR mutations. AIMS: We investigated expression of VDR in isolated hair follicles and epidermal keratinocytes from patients with AA or AU, and assessed changes in the expression of Wnt signalling regulation factors to determine the relationship between the occurrence of AA or AU and decreased expression of VDR. METHODS: Immunohistochemistry was performed for canonical Wnt signalling molecules, VDR, and proliferation and differentiation markers in the skin tissue of patients with AA or AU. After VDR small interfering RNA or Dickkopf-1 (DKK1) treatment of follicle dermal papilla (DP) cells, expression of VDR and Wnt signalling molecules was determined. DKK1 was used to treated keratinocytes and DP cells in a transwell coculture system. Western blotting analysis was performed to assay for VDR and beta-catenin. RESULTS: Expression of Wnt/beta-catenin signals and VDR was decreased in AA or AU lesions compared with nonlesional skin. Inhibition of Wnt/beta-catenin signals and VDR influenced differentiation and proliferation of epidermis and hair follicles. In the transwell coculture system, DKK1 reduced the expression of VDR and beta-catenin in cells in a dose-dependent manner. CONCLUSIONS: This study demonstrates that the decreased expression of VDR in AA and AU lesions is related to decreased expression of Wnt/beta-catenin signals, which inhibits proliferation and differentiation of hair follicles and epidermal cells.

• Liu XH et al.
• Androgens up-regulate transcription of the Notch inhibitor Numb in C2C12 myoblasts via Wnt/beta-catenin signaling to T cell factor elements in the Numb promoter.
• J Biol Chem. 2013; 288: 17990-8
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• Androgen signaling via the androgen receptor is a key pathway that contributes to development, cell fate decisions, and differentiation, including that of myogenic progenitors. Androgens and synthetic steroids have well established anabolic actions on skeletal muscle. Wnt and Notch signaling pathways are also essential to myogenic cell fate decisions during development and tissue repair. However, the interactions among these pathways are largely unknown. Androgenic regulation of Wnt signaling has been reported. Nandrolone, an anabolic steroid, has been shown to inhibit Notch signaling and up-regulate Numb, a Notch inhibitor. To elucidate the mechanisms of interaction between nandrolone and Wnt/Notch signaling, we investigated the effects of nandrolone on Numb expression and Wnt signaling and determined the roles of Wnt signaling in nandrolone-induced Numb expression in C2C12 myoblasts. Nandrolone increased Numb mRNA and protein levels and T cell factor (Tcf) transcriptional activity via inhibition of glycogen synthase kinase 3beta. Up-regulation of Numb expression by nandrolone was blocked by the Wnt inhibitors, sFRP1 and DKK1, whereas Wnt3a increased Numb mRNA and protein expression. In addition, we observed that the proximal promoter of the Numb gene had functional Tcf binding elements to which beta-catenin was recruited in a manner enhanced by both nandrolone and Wnt3a. Moreover, site-directed mutagenesis indicated that the Tcf binding sites in the Numb promoter are required for the nandrolone-induced Numb transcriptional activation in this cell line. These results reveal a novel molecular mechanism underlying up-regulation of Numb transcription with a critical role for increased canonical Wnt signaling. In addition, the data identify Numb as a novel target gene of the Wnt signaling pathway by which Wnts would be able to inhibit Notch signaling.

• Park DS, Seo JH, Hong M, Choi SC
• Role of the Rap2/TNIK kinase pathway in regulation of LRP6 stability for Wnt signaling.
• Biochem Biophys Res Commun. 2013; 436: 338-43
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• The Wnt/beta-catenin signaling pathway plays critical roles in early embryonic development, stem cell biology and human diseases including cancers. Although Rap2, a member of Ras GTPase family, is essential for the Wnt/beta-catenin pathway during the body axis specification in Xenopus embryo, the mechanism underlying its regulation of Wnt signaling remains poorly understood. Here, we show that Rap2 is implicated in control of the stability of Wnt receptor, low-density lipoprotein receptor-related protein 6 (LRP6). Knockdown of Rap2 resulted in the proteasome and/or lysosome-dependent degradation of LRP6 both in the presence and absence of Wnt ligand stimulation. In line with this, constitutively active LRP6 lacking its extracellular domain, which is constitutively phosphorylated and resides in intracellular vesicles, was also degraded in the Rap2-silenced cells. In addition, Rap2 and LRP6 associated physically with each other. Furthermore, we found that TRAF2/Nck-interacting kinase (TNIK), a member of the Ste20 protein family, acts as a downstream effector of Rap2 in control of LRP6 stabilization. Consistently, TNIK could rescue the inhibitory effects of Rap2 depletion on Wnt-dependent gene transcription, reporter activation and neural crest induction. Taken together, these results suggest that Rap2 acts via TNIK to regulate the stability of LRP6 receptor for Wnt/beta-catenin signaling.

• Kwack MH, Kim MK, Kim JC, Sung YK
• Wnt5a attenuates Wnt/beta-catenin signalling in human dermal papilla cells.
• Exp Dermatol. 2013; 22: 229-31
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• Findings of recent studies have demonstrated modulation of Wnt/beta-catenin signalling by Wnt5a, which is highly expressed in hair follicular dermal papilla (DP) in vivo. Here, we investigated the question of whether Wnt5a can affect canonical Wnt/beta-catenin signalling in DP cells. Treatment with Wnt5a resulted in attenuation of Wnt3a-mediated elevation of beta-catenin signalling, which was increased by Wnt5a siRNA transfection in cultured DP cells, as examined by reporter assay. In addition, treatment with Wnt5a resulted in repressed Wnt3a-mediated expression of Axin2, EP2 and LEF1 in cultured DP cells, whereas Wnt5a siRNA transfection resulted in increased Wnt3a-mediated expression of the genes in isolated DPs of cultured hair follicles. Moreover, treatment with Wnt5a resulted in attenuation of Wnt3a-mediated accumulation of beta-catenin in the nucleus in DP cells. Our data strongly suggest that Wnt5a acts as an autocrine factor and attenuates canonical Wnt signalling pathway in human DP cells.

• Ullah M, Stich S, Haupl T, Eucker J, Sittinger M, Ringe J
• Reverse differentiation as a gene filtering tool in genome expression profiling of adipogenesis for fat marker gene selection and their analysis.
• PLoS One. 2013; 8: 69754-69754
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• BACKGROUND: During mesenchymal stem cell (MSC) conversion into adipocytes, the adipogenic cocktail consisting of insulin, dexamethasone, indomethacin and 3-isobutyl-1-methylxanthine not only induces adipogenic-specific but also genes for non-adipogenic processes. Therefore, not all significantly expressed genes represent adipogenic-specific marker genes. So, our aim was to filter only adipogenic-specific out of all expressed genes. We hypothesize that exclusively adipogenic-specific genes change their expression during adipogenesis, and reverse during dedifferentiation. Thus, MSC were adipogenic differentiated and dedifferentiated. RESULTS: Adipogenesis and reverse adipogenesis was verified by Oil Red O staining and expression of PPARG and FABP4. Based on GeneChips, 991 genes were differentially expressed during adipogenesis and grouped in 4 clusters. According to bioinformatic analysis the relevance of genes with adipogenic-linked biological annotations, expression sites, molecular functions, signaling pathways and transcription factor binding sites was high in cluster 1, including all prominent adipogenic genes like ADIPOQ, C/EBPA, LPL, PPARG and FABP4, moderate in clusters 2-3, and negligible in cluster 4. During reversed adipogenesis, only 782 expressed genes (clusters 1-3) were reverted, including 597 genes not reported for adipogenesis before. We identified APCDD1, CHI3L1, RARRES1 and SEMA3G as potential adipogenic-specific genes. CONCLUSION: The model system of adipogenesis linked to reverse adipogenesis allowed the filtration of 782 adipogenic-specific genes out of total 991 significantly expressed genes. Database analysis of adipogenic-specific biological annotations, transcription factors and signaling pathways further validated and valued our concept, because most of the filtered 782 genes showed affiliation to adipogenesis. Based on this approach, the selected and filtered genes would be potentially important for characterization of adipogenesis and monitoring of clinical translation for soft-tissue regeneration. Moreover, we report 4 new marker genes.

• Cruciat CM, Niehrs C
• Secreted and transmembrane wnt inhibitors and activators.
• Cold Spring Harb Perspect Biol. 2013; 5: 15081-15081
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• Signaling by the Wnt family of secreted glycoproteins plays important roles in embryonic development and adult homeostasis. Wnt signaling is modulated by a number of evolutionarily conserved inhibitors and activators. Wnt inhibitors belong to small protein families, including sFRP, Dkk, WIF, Wise/SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Their common feature is to antagonize Wnt signaling by preventing ligand-receptor interactions or Wnt receptor maturation. Conversely, the Wnt activators, R-spondin and Norrin, promote Wnt signaling by binding to Wnt receptors or releasing a Wnt-inhibitory step. With few exceptions, these antagonists and agonists are not pure Wnt modulators, but also affect additional signaling pathways, such as TGF-beta and FGF signaling. Here we discuss their interactions with Wnt ligands and Wnt receptors, their role in developmental processes, as well as their implication in disease.

• Chen D, Jarrell A, Guo C, Lang R, Atit R
• Dermal beta-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation.
• Development. 2012; 139: 1522-33
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• Dermal fibroblasts are required for structural integrity of the skin and for hair follicle development. Uniform Wnt signaling activity is present in dermal fibroblast precursors preceding hair follicle initiation, but the functional requirement of dermal Wnt signaling at early stages of skin differentiation and patterning remains largely uncharacterized. We show in mice that epidermal Wnt ligands are required for uniform dermal Wnt signaling/beta-catenin activity and regulate fibroblast cell proliferation and initiation of hair follicle placodes. In the absence of dermal Wnt signaling/beta-catenin activity, patterned upregulation of epidermal beta-catenin activity and Edar expression are absent. Conversely, forced activation of beta-catenin signaling leads to the formation of thickened dermis, enlarged epidermal placodes and dermal condensates that result in prematurely differentiated enlarged hair follicles. These data reveal functional roles for dermal Wnt signaling/beta-catenin in fibroblast proliferation and in the epidermal hair follicle initiation program.

• Pfister AS, Tanneberger K, Schambony A, Behrens J
• Amer2 protein is a novel negative regulator of Wnt/beta-catenin signaling involved in neuroectodermal patterning.
• J Biol Chem. 2012; 287: 1734-41
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• Wnt/beta-catenin signaling is negatively controlled by the adenomatous polyposis coli (APC) tumor suppressor, which induces proteasomal degradation of beta-catenin as part of the beta-catenin destruction complex. Amer2 (APC membrane recruitment 2; FAM123A) is a direct interaction partner of APC, related to the tumor suppressor Amer1/WTX, but its function in Wnt signaling is not known. Here, we show that Amer2 recruits APC to the plasma membrane by binding to phosphatidylinositol 4,5-bisphosphate lipids via lysine-rich motifs and that APC links beta-catenin and the destruction complex components axin and conductin to Amer2. Knockdown of Amer2 increased Wnt target gene expression and reporter activity in cell lines, and overexpression reduced reporter activity, which required membrane association of Amer2. In Xenopus embryos, Amer2 is expressed mainly in the dorsal neuroectoderm and neural tissues. Down-regulation of Amer2 by specific morpholino oligonucleotides altered neuroectodermal patterning, which could be rescued by expression of a dominant-negative mutant of Lef1 that interferes with beta-catenin-dependent transcription. Our data characterize Amer2 for the first time as a negative regulator of Wnt signaling both in cell lines and in vivo and define Amer proteins as a novel family of Wnt pathway regulators.

• Rankin SA, Gallas AL, Neto A, Gomez-Skarmeta JL, Zorn AM
• Suppression of Bmp4 signaling by the zinc-finger repressors Osr1 and Osr2 is required for Wnt/beta-catenin-mediated lung specification in Xenopus.
• Development. 2012; 139: 3010-20
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• Embryonic development of the respiratory system is regulated by a series of mesenchymal-epithelial interactions that are only partially understood. Mesenchymal FGF and Wnt2/Wnt2b signaling are implicated in specification of mammalian pulmonary progenitors from the ventral foregut endoderm, but their epistatic relationship and downstream targets are largely unknown. In addition, how wnt2 and wnt2b are regulated in the developing foregut mesenchyme is unknown. We show that the Odd-skipped-related (Osr) zinc-finger transcriptional repressors Osr1 and Osr2 are redundantly required for Xenopus lung specification in a molecular pathway linking foregut pattering by FGFs to Wnt-mediated lung specification and RA-regulated lung bud growth. FGF and RA signals are required for robust osr1 and osr2 expression in the foregut endoderm and surrounding lateral plate mesoderm (lpm) prior to respiratory specification. Depletion of both Osr1 and Osr2 (Osr1/Osr2) results in agenesis of the lungs, trachea and esophagus. The foregut lpm of Osr1/Osr2-depleted embryos fails to express wnt2, wnt2b and raldh2, and consequently Nkx2.1(+) progenitors are not specified. Our data suggest that Osr1/Osr2 normally repress bmp4 expression in the lpm, and that BMP signaling negatively regulates the wnt2b domain. These results significantly advance our understanding of early lung development and may impact strategies to differentiate respiratory tissue from stem cells.

• Samuelov L, Sprecher E, Tsuruta D, Biro T, Kloepper JE, Paus R
• P-cadherin regulates human hair growth and cycling via canonical Wnt signaling and transforming growth factor-beta2.
• J Invest Dermatol. 2012; 132: 2332-41
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• P-cadherin is a key component of epithelial adherens junctions, and it is prominently expressed in the hair follicle (HF) matrix. Loss-of-function mutations in CDH3, which encodes P-cadherin, result in hypotrichosis with juvenile macular dystrophy (HJMD), an autosomal recessive disorder featuring sparse and short hair. Here, we attempted to recapitulate some aspects of HJMD in vitro by transfecting normal, organ-cultured human scalp HFs with lipofectamine and CDH3-specific or scrambled control siRNAs. As in HJMD patients, P-cadherin silencing inhibited hair shaft growth, prematurely induced HF regression (catagen), and inhibited hair matrix keratinocyte proliferation. In situ, membrane beta-catenin expression and transcription of the beta-catenin target gene, axin2, were significantly reduced, whereas glycogen synthase kinase 3 beta (GSK3beta) and phospho-beta-catenin immunoreactivity were increased. These effects were partially reversed by inhibiting GSK3beta. P-cadherin silencing reduced the expression of the anagen-promoting growth factor, IGF-1, whereas that of transforming growth factor beta 2 (TGFbeta2; catagen promoter) was enhanced. Neutralizing TGFbeta antagonized the catagen-promoting effects of P-cadherin silencing. In summary, we introduce human HFs as an attractive preclinical model for studying the functions of P-cadherin in human epithelial biology and pathology. This model demonstrates that cadherins can be successfully knocked down in an intact human organ in vitro, and shows that P-cadherin is needed for anagen maintenance by regulating canonical Wnt signaling and suppressing TGFbeta2.

• Qu Q et al.
• Orphan nuclear receptor TLX activates Wnt/beta-catenin signalling to stimulate neural stem cell proliferation and self-renewal.
• Nat Cell Biol. 2010; 12: 31-40
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• The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/beta-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/beta-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active beta-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a beta-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active beta-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active beta-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active beta-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/beta-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode.

• Janssens S, Denayer T, Deroo T, Van Roy F, Vleminckx K
• Direct control of Hoxd1 and Irx3 expression by Wnt/beta-catenin signaling during anteroposterior patterning of the neural axis in Xenopus.
• Int J Dev Biol. 2010; 54: 1435-42
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• During and after gastrulation, the neural axis in vertebrates is patterned along the antero-posterior axis by the combined activity of signaling factors secreted in the neural ectoderm and the underlying mesoderm. These signals divide the neural axis into four major divisions: the forebrain, midbrain, hindbrain and spinal chord. Among the signals that pattern the neural axis, Wnts play a prominent role and many patterning genes have been found to be direct Wnt/beta-catenin target genes, including several homeobox domain-containing transcription factors. Here we show that HoxD1 and Irx3 are transcriptionally induced by the Wnt pathway during neurulation. Using induction in the presence of the translation blocking drug cycloheximide and chromatin immunoprecipitation assays, we confirm that HoxD1 and Irx3 are both direct Wnt target genes. In addition, we identified Crabp2 (cellular retinoic acid binding protein 2) as an indirect target that potentially links the activities of Wnt and retinoic acid during antero-posterior patterning.

• Mii Y, Taira M
• Secreted Frizzled-related proteins enhance the diffusion of Wnt ligands and expand their signalling range.
• Development. 2009; 136: 4083-8
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• Secreted Frizzled-related proteins (sFRPs) are thought to negatively modulate Wnt signalling. Although Wnt proteins are thought to diffuse extracellularly and act as morphogens, little is known about the diffusibility of either Wnts or sFRPs. Here we show that Frzb and Crescent (Cres), which are members of the sFRP family, have the ability to regulate the diffusibility and signalling areas of the Wnt ligands Wnt8 and Wnt11. We found, using the Xenopus embryo, that Wnts do not diffuse effectively, whereas Frzb and Cres spread very widely. Interestingly, Frzb and Cres substantially promoted the diffusion of Wnt8 and Wnt11 through extracellular interactions. Importantly, we show that Wnt8 conveyed by sFRPs can activate canonical Wnt signalling despite the function of sFRPs as Wnt inhibitors, suggesting a novel regulatory system for Wnts by sFRPs.

• Louie SH et al.
• Modulation of the beta-catenin signaling pathway by the dishevelled-associated protein Hipk1.
• PLoS One. 2009; 4: 4310-4310
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• BACKGROUND: Wnts are evolutionarily conserved ligands that signal through beta-catenin-dependent and beta-catenin-independent pathways to regulate cell fate, proliferation, polarity, and movements during vertebrate development. Dishevelled (Dsh/Dvl) is a multi-domain scaffold protein required for virtually all known Wnt signaling activities, raising interest in the identification and functions of Dsh-associated proteins. METHODOLOGY: We conducted a yeast-2-hybrid screen using an N-terminal fragment of Dsh, resulting in isolation of the Xenopus laevis ortholog of Hipk1. Interaction between the Dsh and Hipk1 proteins was confirmed by co-immunoprecipitation assays and mass spectrometry, and further experiments suggest that Hipk1 also complexes with the transcription factor Tcf3. Supporting a nuclear function during X. laevis development, Myc-tagged Hipk1 localizes primarily to the nucleus in animal cap explants, and the endogenous transcript is strongly expressed during gastrula and neurula stages. Experimental manipulations of Hipk1 levels indicate that Hipk1 can repress Wnt/beta-catenin target gene activation, as demonstrated by beta-catenin reporter assays in human embryonic kidney cells and by indicators of dorsal specification in X. laevis embryos at the late blastula stage. In addition, a subset of Wnt-responsive genes subsequently requires Hipk1 for activation in the involuting mesoderm during gastrulation. Moreover, either over-expression or knock-down of Hipk1 leads to perturbed convergent extension cell movements involved in both gastrulation and neural tube closure. CONCLUSIONS: These results suggest that Hipk1 contributes in a complex fashion to Dsh-dependent signaling activities during early vertebrate development. This includes regulating the transcription of Wnt/beta-catenin target genes in the nucleus, possibly in both repressive and activating ways under changing developmental contexts. This regulation is required to modulate gene expression and cell movements that are essential for gastrulation.

• Lehman JM, Laag E, Michaud EJ, Yoder BK
• An essential role for dermal primary cilia in hair follicle morphogenesis.
• J Invest Dermatol. 2009; 129: 438-48
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• The primary cilium is a microtubule-based organelle implicated as an essential component of a number of signaling pathways. It is present on cells throughout the mammalian body; however, its functions in most tissues remain largely unknown. Herein we demonstrate that primary cilia are present on cells in murine skin and hair follicles throughout morphogenesis and during hair follicle cycling in postnatal life. Using the Cre-lox system, we disrupted cilia assembly in the ventral dermis and evaluated the effects on hair follicle development. Mice with disrupted dermal cilia have severe hypotrichosis (lack of hair) in affected areas. Histological analyses reveal that most follicles in the mutants arrest at stage 2 of hair development and have small or absent dermal condensates. This phenotype is reminiscent of that seen in the skin of mice lacking Shh or Gli2. In situ hybridization and quantitative RT-PCR analysis indicates that the hedgehog pathway is downregulated in the dermis of the cilia mutant hair follicles. Thus, these data establish cilia as a critical signaling component required for normal hair morphogenesis and suggest that this organelle is needed on cells in the dermis for reception of signals such as sonic hedgehog.

• Steinbeisser H, Swain RK
• Wnt-frizzled interactions in Xenopus.
• Methods Mol Biol. 2008; 469: 451-63
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• The Wnt signaling cascades are regulatory modules which are involved in embryonic patterning, cell differentiation, morphogenesis, and diseases. The Wnt pathways are activated when secreted Wnt ligands interact with 7-trans-membrane receptors of the Frizzled (Fz) family. Specific readouts are determined by the ligand/receptor combinations and the cellular context. Here we describe two methods for the analysis of Wnt/Frizzled interactions in Xenopus embryos. Physical interaction of ligand and receptor are demonstrated by co-immunoprecipitation assays. The activation of Wnt targets in Xenopus animal cap tissue provides a versatile test system for activating and inhibitory components of the Wnt/beta -catenin pathway.

• Qiang YW, Barlogie B, Rudikoff S, Shaughnessy JD Jr
• Dkk1-induced inhibition of Wnt signaling in osteoblast differentiation is an underlying mechanism of bone loss in multiple myeloma.
• Bone. 2008; 42: 669-80
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• Expression of the Wnt signaling inhibitor, DKK1 by multiple myeloma cells is correlated with lytic bone disease in multiple myeloma. However, the mechanism(s) by which DKK1 contributes to this process is not clear. Herein, we analyzed the functional role of canonical Wnt signaling and Dkk1 inhibition of this pathway in bone morphogenic protein (BMP)-2-induced osteoblast differentiation. Osteoblast differentiation was measured by alkaline phosphatase (ALP) activity in murine (C2C12) and human pre-osteoblast (hFOB1.19) and osteoblast-like (Saos-2 and MG63) cell lines. Cytoplasmic beta-catenin protein was separated by E-cadherin-GST pull-down assay and analyzed by Western blotting. A dominant negative form of beta-catenin, Dkk1 and TCF reporter constructs were transfected into C2C12 cells. C2C12 cells were also transfected with siRNA specific to LRP5/6 to knockdown receptor expression. Canonical Wnt signaling was activated in these cell lines in response to Wnt3a as assessed by increased cytoplasmic, non-phosphorylated beta-catenin and TCF/LEF transcription activity. Recombinant Dkk1 and plasma from MM patients containing high levels of Dkk1 blocked Wnt3a-induced beta-catenin accumulation. Importantly, Dkk1 abrogated BMP-2 mediated osteoblast differentiation. The requirement for Wnt signaling in osteoblast differentiation was confirmed by the following observations: 1) overexpression of Dkk1 decreased endogenous beta-catenin and ALP activity; 2) silencing of Wnt receptor mRNAs blocked ALP activity; and 3) a dominant negative form of beta-catenin eliminated BMP-2-induced ALP activity. Furthermore, Wnt3a did not increase ALP activity nor did BMP-2 treatment result in beta-catenin stabilization indicating that cooperation between these two pathways is required, but they are not co-regulated by either ligand. These studies have revealed that autocrine Wnt signaling in osteoblasts is necessary to promote BMP-2-mediated differentiation of pre-osteoblast cells, while Wnt signaling alone is not capable of inducing such differentiation. Dkk1 inhibits this process and may be a key factor regulating pre-osteoblast differentiation and myeloma bone disease.

• Andreu P et al.
• A genetic study of the role of the Wnt/beta-catenin signalling in Paneth cell differentiation.
• Dev Biol. 2008; 324: 288-96
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• Wnt/beta-catenin signalling plays a key role in the homeostasis of the intestinal epithelium. Whereas its role in the maintenance of the stem cell compartment has been clearly demonstrated, its role in the Paneth cell fate remains unclear. We performed genetic studies to elucidate the functions of the Wnt/beta-catenin pathway in Paneth cell differentiation. We analysed mice with inducible gain-of-function mutations in the Wnt/beta-catenin pathway and mice with a hypomorphic beta-catenin allele that have not been previously described. We demonstrated that acute activation of Wnt/beta-catenin signalling induces de novo specification of Paneth cells in both the small intestine and colon and that colon cancers resulting from Apc mutations expressed many genes involved in Paneth cell differentiation. This suggests a key role for the Wnt/beta-catenin pathway in Paneth cell differentiation. We also showed that a slight decrease in beta-catenin gene dosage induced a major defect in Paneth cell differentiation, but only a modest effect on crypt morphogenesis. Overall, our findings show that a high level of beta-catenin activation is required to determine Paneth cell fate and that fine tuning of beta-catenin signalling is critical for correct Paneth cell lineage.

• Zhang C et al.
• Inhibition of Wnt signaling by the osteoblast-specific transcription factor Osterix.
• Proc Natl Acad Sci U S A. 2008; 105: 6936-41
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• The recent identification of the genes responsible for several human genetic diseases affecting bone homeostasis and the characterization of mouse models for these diseases indicated that canonical Wnt signaling plays a critical role in the control of bone mass. Here, we report that the osteoblast-specific transcription factor Osterix (Osx), which is required for osteoblast differentiation, inhibits Wnt pathway activity. First, in calvarial cells of embryonic day (E)18.5 Osx-null embryos, expression of the Wnt antagonist Dkk1 was abolished, and that of Wnt target genes c-Myc and cyclin D1 was increased. Moreover, our studies demonstrated that Osx bound to and activated the Dkk1 promoter. In addition, Osx inhibited beta-catenin-induced Topflash reporter activity and beta-catenin-induced secondary axis formation in Xenopus embryos. Importantly, in calvaria of E18.5 Osx-null embryos harboring the TOPGAL reporter transgene, beta-galactosidase activity was increased, suggesting that Osx inhibited the Wnt pathway in osteoblasts in vivo. Our data further showed that Osx disrupted binding of Tcf to DNA, providing a likely mechanism for the inhibition by Osx of beta-catenin transcriptional activity. We also showed that Osx decreased osteoblast proliferation. Indeed, E18.5 Osx-null calvaria showed greater BrdU incorporation than wild-type calvaria and that Osx overexpression in C2C12 mesenchymal cells inhibited cell growth. Because Wnt signaling has a major role in stimulating osteoblast proliferation, we speculate that Osx-mediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/beta-catenin activity. Our results add a layer of control to Wnt/beta-catenin signaling in bone.

• Nakamura T, Nakamura T, Matsumoto K
• The functions and possible significance of Kremen as the gatekeeper of Wnt signalling in development and pathology.
• J Cell Mol Med. 2008; 12: 391-408
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• Kremen (Krm) was originally discovered as a novel transmembrane protein containing the kringle domain. Both Krm1 (the first identified Krm) and its relative Krm2 were later identified to be the high-affinity receptors for Dickkopf (Dkk), the inhibitor of Wnt/beta-catenin signalling. The formation of a ternary complex composed of Krm, Dkk, and Lrp5/6 (the coreceptor of Wnt) inhibits Wnt/beta-catenin signalling. In Xenopus gastrula embryos, Wnt/beta-catenin signalling regulates anterior-posterior patterning, with low-signalling in anterior regions. Inhibition of Krm1/2 induces embryonic head defects. Together with anterior localization of Krms and Dkks, the inhibition of Wnt signalling by Dkk-Krm action seems to allow anterior embryonic development. During mammalian development, krm1 mRNA expression is low in the early stages, but gradually and continuously increases with developmental progression and differentiation. In contrast with the wide, strong expression of krm1 mRNA in mature tissues, expression of krm1 is diminished in a variety of human tumor cells. Since stem cells and undifferentiated cells rely on Wnt/beta-catenin signalling for maintenance in a low differentiation state, the physiological shutdown of Wnt/beta-catenin signalling by Dkk-Krm is likely to set cells on a divergent path toward differentiation. In tumour cells, a deficit of Krm may increase the susceptibility to tumourigenic transformation. Both positive and negative regulation of Wnt/beta-catenin signalling definitively contributes to diverse developmental and physiological processes, including cell-fate determination, tissue patterning and stem cell regulation. Krm is quite significant in these processes as the gatekeeper of the Wnt/beta-catenin signalling pathway.

• Maini PK, Baker RE, Chuong CM
• Developmental biology. The Turing model comes of molecular age.
• Science. 2006; 314: 1397-8

• Kobayashi M et al.
• Nuclear localization of Duplin, a beta-catenin-binding protein, is essential for its inhibitory activity on the Wnt signaling pathway.
• J Biol Chem. 2002; 277: 5816-22
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• Duplin binds to beta-catenin and inhibits the Wnt signaling pathway, thereby leading to repression of the beta-catenin-mediated transactivation and Xenopus axis formation. To find an additional function of Duplin, yeast two-hybrid screening was carried out. Importin alpha was isolated as a binding protein of Duplin. Importin alpha bound directly to basic amino acid clusters of Duplin. Although Duplin was present in the nucleus, deletion of the basic amino acid clusters (Duplin(Delta 500-584)) retained Duplin in the cytoplasm. Duplin(Delta 500-584) bound to beta-catenin as efficiently as wild-type Duplin, but it neither repressed Wnt-dependent Tcf transcriptional activation in mammalian cells nor showed ventralization in Xenopus embryos. The Duplin mutant without a beta-catenin-binding region lost the ability to inhibit the Wnt-dependent Tcf activation, but retained its ventralizing activity. Furthermore, Duplin not only suppressed beta-catenin-dependent axis duplication and expression of siamois, a Wnt-regulated gene, but also inhibited siamois-dependent axis duplication. These results indicate that Duplin is translocated to the nucleus by interacting with importin alpha, and that nuclear localization is essential for the function of Duplin. Moreover, Duplin has an additional activity of inhibiting the Wnt signaling pathway by affecting the downstream beta-catenin target genes.

• Kishimoto J, Burgeson RE, Morgan BA
• Wnt signaling maintains the hair-inducing activity of the dermal papilla.
• Genes Dev. 2000; 14: 1181-5
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• The formation of the hair follicle and its cyclical growth, quiescence, and regeneration depend on reciprocal signaling between its epidermal and dermal components. The dermal organizing center, the dermal papilla (DP), regulates development of the epidermal follicle and is dependent on signals from the epidermis for its development and maintenance. GFP specifically expressed in DP cells of a transgenic mouse was used to purify this population and study the signals required to maintain it. We demonstrate that specific Wnts, but not Sonic hedgehog (Shh), maintain anagen-phase gene expression in vitro and hair inductive activity in a skin reconstitution assay.

• Farr GH 3rd, Ferkey DM, Yost C, Pierce SB, Weaver C, Kimelman D
• Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification.
• J Cell Biol. 2000; 148: 691-702
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• Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.

• Gat U, DasGupta R, Degenstein L, Fuchs E
• De Novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin.
• Cell. 1998; 95: 605-14
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• An effector of intercellular adhesion, beta-catenin also functions in Wnt signaling, associating with Lef-1/Tcf DNA-binding proteins to form a transcription factor. We report that this pathway operates in keratinocytes and that mice expressing a stabilized beta-catenin controlled by an epidermal promoter undergo a process resembling de novo hair morphogenesis. The new follicles formed sebaceous glands and dermal papilla, normally established only in embryogenesis. As in embryologically initiated hair germs, transgenic follicles induce Lef-1, but follicles are disoriented and defective in sonic hedgehog polarization. Additionally, proliferation continues unchecked, resulting in two types of tumors also found in humans. Our findings suggest that transient beta-catenin stabilization may be a key player in the long-sought epidermal signal leading to hair development and implicate aberrant beta-catenin activation in hair tumors.

• Brannon M, Gomperts M, Sumoy L, Moon RT, Kimelman D
• A beta-catenin/XTcf-3 complex binds to the siamois promoter to regulate dorsal axis specification in Xenopus.
• Genes Dev. 1997; 11: 2359-70
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• The Wnt pathway regulates the early dorsal-ventral axis in Xenopus through a complex of beta-catenin and HMG box transcription factors of the Lef/Tcf family. We show that the promoter of the dorsalizing homeo box gene siamois is a direct target for the beta-catenin/XTcf-3 complex, establishing a link between the Wnt pathway and the activation of genes involved in specifying the dorsal axis. By injecting siamois reporter constructs into the animal pole of Xenopus embryos, we show that a 0.8-kb fragment of the siamois promoter is strongly activated by beta-catenin. The proximal 0.5 kb, which is also activated by beta-catenin, contains three Lef/Tcf-binding sites. Mutations in these sites eliminate the beta-catenin-mediated activation of siamois and show that siamois is regulated by the beta-catenin/XTcf-3 complex, in combination with additional transcriptional activators. When expressed at the equator of the embryo, the siamois promoter is activated to much higher levels on the dorsal side than the ventral side. Ectopic ventral expression of beta-catenin raises the ventral expression of the siamois promoter to the dorsal levels. Conversely, ectopic dorsal expression of dominant-negative XTcf-3 abolishes the dorsal activation of the siamois promoter. Furthermore, elimination of the Lef/Tcf sites elevates the ventral expression of siamois, revealing a repressive role for XTcf-3 in the absence of beta-catenin. Finally, we find that the endogenous siamois activator, although present throughout the dorsal side of the embryo, is most potent in the dorsal vegetal region. We propose that the dorsal activation of siamois by the beta-catenin/XTcf-3 complex combined with the ventral repression of siamois by XTcf-3 results in the restriction of endogenous siamois expression to the dorsal side of Xenopus embryos.

• Taylor MF, Paulauskis JD, Weller DD, Kobzik L
• In vitro efficacy of morpholino-modified antisense oligomers directed against tumor necrosis factor-alpha mRNA.
• J Biol Chem. 1996; 271: 17445-52
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• Chemical modification of antisense oligonucleotides to increase nuclease resistance may improve their efficacy within enzyme-rich cellular targets (e.g. macrophages). We evaluated a panel of morpholino antisense oligomers (M-AS) for their ability to inhibit macrophage tumor necrosis factor-alpha (TNF-alpha) release and compared them to phosphodiester (O-AS) and phosphorothioate (S-AS) types of oligonucleotides. M-AS inhibited translation in vitro (rabbit reticulocyte lysate) of target mRNA at concentrations as low as 200 nM (e.g. percent inhibition by M-AS 2 at 0.2, 1.0, and 2.0 microM was 40.9 +/- 5.3%, 50.2 +/- 4.6%, and 57.7 +/- 3.6%, respectively, n = 4, p

• Wilson PA, Melton DA
• Mesodermal patterning by an inducer gradient depends on secondary cell-cell communication.
• Curr Biol. 1994; 4: 676-86
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• BACKGROUND: Gradients of inducing molecules, or morphogens, could impose pattern on early embryos. Although there are candidates for morphogens in several systems, it is not well understood how cells might translate differences in extracellular inducer concentration into an orderly arrangement of cell types. With this question in mind, we have re-examined mesodermal patterning in Xenopus in response to the secreted growth factor activin. Previous work has shown that activin can initiate the formation of a variety of mesodermal tissues in a concentration-dependent fashion. We have sought to disentangle the roles played by individual cell responses to activin and subsequent interactions among induced cells in producing this outcome. RESULTS: We find that the initial response of dispersed cells to activin concentration is unexpectedly simple, showing neither the thresholds of activin concentration nor the distinct domains of gene expression that characterize the later response. The eventual emergence of an ordered series of coherent differentiation steps requires the reaggregation of the induced cells, implying that secondary interactions occur. Furthermore, when cells induced at different doses of activin are mixed, the final response apparently represents a consensus, rather than a mosaic, of the mixed populations. CONCLUSIONS: We conclude that communication among responding cells underlies much of the remarkable patterning influence of activin. Moreover, we suggest that these findings can inform thinking about how inducer gradients might act in other systems, shifting emphasis from the initial response of cells to inducer concentration toward the elaboration of complex pattern by secondary interactions.

• Schaeren-Wiemers N, Gerfin-Moser A
• A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes.
• Histochemistry. 1993; 100: 431-40
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• We have developed a simple non-radioactive in situ hybridization procedure for tissue sections and cultured cells using digoxigenin-labelled cRNA probes. This protocol can be applied for the detection of various transcripts present at a wide range of expression levels in the central nervous system. Cerebellar hybridization signals for transcripts estimated to be expressed at high (MBP, myelin basic protein), moderate (GluR1, subunit of AMPA/kainate sensitive glutamate receptors) and low (inositol polyphosphate-5-phosphatase) levels of abundance are demonstrated as examples. The sensitivity and cellular resolution were significantly improved by avoiding any ethanol treatment commonly used in other procedures. The localization of a labelled cell with respect to its environment is shown to be more easily assessed by counterstaining of the tissue with the nuclear dye Hoechst 33258. The present protocol can be combined with immunocytochemistry as demonstrated for glial fibrillary acidic protein (GFAP). All steps of the procedure, including preparation and labelling of the cRNA probes, pretreatment of tissue, hybridization and visualization of the labelled transcripts, are described in detail.