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Historical tailings storage facilities (TSFs) are either abandoned or sparsely rehabilitated promoting acid mine drainage (AMD) formation and heavy metal release. To sustainably manage these sites, a geochemical audit coupled with numerical simulation to predict AMD flow paths and heavy metal migration are valuable. In this study, a 40-year-old TSF in Hokkaido, Japan was investigated. Tailings in this historical TSF contain pyrite (FeS

The repurposing of gold (Au) mine tailings from artisanal and small-scale mining (ASGM) operations via alkali activation technology is a promising strategy for waste reduction in developing countries. Direct activation of mine tailings, however, is challenging because these materials contain relatively low aluminum (Al)-bearing minerals. In this study, palm oil fuel ash (POFA) was elucidated as a high Al-bearing waste derived-admixture for the synthesis of an ASGM tailings-based geopolymer composite. Semi-quantitative XRD analysis showed that the tailings contained quartz (SiO2) (~58%), pyrite (FeS2) (~20%) and calcite (CaCO3) (~15%) with minor to trace amounts of aluminosilicates (~7%). Substantial amounts of environmentally regulated pollutants such as mercury (Hg) (40 mg/kg), lead (Pb) (8430 mg/kg) and arsenic (As) (300 mg/kg) were also found in the tailings. SEM-EDS, XRD and ATR-FTIR results showed the successful formation of a hybrid geopolymer-CASH matrix, which improved the unconfined compressive strength (UCS) of geopolymer composites from ~5 MPa to ~7 MPa. Furthermore, POFA did not significantly affect the thermal resistivity of geopolymer composites based on thermal analysis. Finally, the TCLP results showed that the Pb leaching concentrations from ASGM tailings exceeded environmental standards (~15,000 µg/L), which was suppressed after alkali activation to 300–500 µg/L. This means that POFA addition to ASGM tailings-based geopolymer composite improved not only its applicability as backfill, pavements and bricks but also its ability to immobilize toxic elements.

Scorodite is an important mineral not only for arsenic (As) removal from industrial wastewaters but also in the mobility and final fate of As in waste rocks, contaminated soils and sediments, and mine tailings. Because of the mineral's high As-loading capacity and stability, numerous studies have been done to understand its formation. Unfortunately, most of these studies were limited to elevated temperatures (>70 °C), so the processes involved in scorodite formation under ambient conditions remain unclear. This study provides evidence of the catalytic effects of hematite on the formation of scorodite at 25 °C in a pyrite-rich natural geologic material. Scorodite peaks were detected in the XRD patterns of the leaching residues with and without hematite, but those in the former were stronger and more pronounced than the latter. These results suggest that the formation of scorodite was catalysed by hematite, a generalisation that is further supported by strong characteristic IR absorption bands of scorodite at 819 cm–1 (As–O bending vibration), 785 and 725 cm–1 (As–O stretching vibrations), and 2990 cm−1 (OH-vibration) as well as the distinct XPS binding energies of Fe(III)–As (709.7 eV), As(V)–O (44.8, 44.31 and 43.7 eV), O2− (530.5 eV) and coordinated water (531.3 eV) in scorodite. This phenomenon could be attributed to three possible mechanisms: (1) more rapid precipitation promoted by the “seeding” effect of hematite particles, (2) additional supply of Fe3+ from hematite dissolution under acidic conditions, and (3) enhanced oxidations of Fe2+ to Fe3+ and As(III) to As(V) on the surface of hematite.

Tight junction complexes are involved in the establishment and maintenance of cell polarity and the regulation of signalling pathways, controlling biological processes such as cell differentiation and cell proliferation. MarvelD3 is a tight junction protein expressed in adult epithelial and endothelial cells. In Xenopus laevis, MarvelD3 morphants present differentiation defects of several ectodermal derivatives. In vitro experiments further revealed that MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behaviour and survival. In this work, we found that MarvelD3 is expressed from early developmental stages in the exocrine and endocrine compartments of the pancreas, as well as in endothelial cells of this organ. We thoroughly characterized MarvelD3 expression pattern in developing pancreas and evaluated its function by genetic ablation. Surprisingly, inactivation of MarvelD3 in mice did not alter development and differentiation of the pancreatic tissue. Moreover, tight junction formation and organization, cell polarization, and activity of the JNK-pathway were not impacted by the deletion of MarvelD3.

Artisanal and small-scale gold mining (ASGM) operations are major contributors to the Philippines’ annual gold (Au) output (at least 60%). Unfortunately, these ASGM activities lacked adequate tailings management strategies, so contamination of the environment is prevalent. In this study, soil contamination with copper (Cu), lead (Pb), zinc (Zn) and arsenic (As) due to ASGM activities in Nabunturan, Davao de Oro, Philippines was investigated. The results showed that ASGM-impacted soils had Cu, Pb, Zn and As up to 3.6, 83, 73 and 68 times higher than background levels, respectively and were classified as ‘extremely’ polluted (CD = 30–228; PLI = 5.5–34.8). Minerals typically found in porphyry copper-gold ores like pyrite, chalcopyrite, malachite, galena, sphalerite and goethite were identified by XRD and SEM-EDS analyses. Furthermore, sequential extraction results indicate substantial Cu (up to 90%), Pb (up to 50%), Zn (up to 65%) and As (up to 48%) partitioned with strongly adsorbed, weak acid soluble, reducible and oxidisable fractions, which are considered as ‘geochemically mobile’ phases in the environment. Although very high Pb and Zn were found in ASGM-impacted soils, they were relatively immobile under oxidising conditions around pH 8.5 because of their retention via adsorption to hydrous ferric oxides (HFOs), montmorillonite and kaolinite. In contrast, Cu and As release from the historic ASGM site samples exceeded the environmental limits for Class A and Class C effluents, which could be attributed to the removal of calcite and dolomite by weathering. The enhanced desorption of As at around pH 8.5 also likely contributed to its release from these soils., Highlights • ASGM-impacted soils from Davao de Oro, Philippines were evaluated. • ASGM-impacted soils were highly contaminated with As, Pb and Zn. • Cu, Pb, Zn and As in the contaminated soil came from sulphide minerals. • Pb and Zn were strongly retained in the soils via adsorption to HFOs and clays. • Cu and As release from the historic ASGM site exceeded environmental standards.

The continuous accumulation of artisanal and small-scale gold mining (ASGM) tailings in the Philippines without adequate storage and disposal facility could lead to human health and environmental disasters in the long run. In this study, ASGM tailings was simultaneously stabilized and repurposed as construction material via geopolymerization using coal fly ash, palm oil fuel ash and a powder-based alkali activator. Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS) identified iron sulfides in the tailings containing arsenic (As), cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn), which could be released via weathering. The average unconfined compressive strengths (UCS) of tailings-based geopolymers at 14 days curing were 7.58 MPa and 7.7 MPa with fly ash and palm oil fuel ash, respectively. The tailings-based geopolymers with palm oil fuel ash had higher UCS most likely due to CASH reaction product formation that improved strength formation. The toxicity characteristic leaching procedure (TCLP) results showed very low leachabilities of As, Pb and Fe in the geopolymer materials suggesting ASGM tailings was effectively encapsulated within the geopolymer matrix. Overall, the geopolymerization of ASGM tailings is a viable and promising solution to simultaneously stabilize mining and industrial wastes and repurpose them into construction materials., Geopolymerization; Mine tailings; Sugar mill lime sludge; Fly ash; Palm oil fuel ash; Powder activator

Massive and ambitious underground space development projects are being undertaken by many countries around the world to decongest megacities, improve the urban landscapes, upgrade outdated transportation networks, and expand modern railway and road systems. A number of these projects, however, reported that substantial portions of the excavated debris are oftentimes naturally contaminated with hazardous elements, which are readily released in substantial amounts once exposed to the environment. These contaminated excavation debris/spoils/mucks, loosely referred to as "naturally contaminated rocks", contain various hazardous and toxic inorganic elements like arsenic (As), selenium (Se), boron (B), and heavy metals like lead (Pb), cadmium (Cd), copper (Cu), and zinc (Zn). If left untreated, these naturally contaminated rocks could pose very serious problems not only to the surrounding ecosystem but also to people living around the construction and disposal sites. Several incidents of soil and ground/surface water contamination, for example, have been documented due to the false assumption that excavated materials are non-hazardous because they only contain background levels of environmentally regulated elements. Naturally contaminated rocks are hazardous wastes, but they still remain largely unregulated. In fact, standard leaching tests for their evaluation and classification are not yet established. In this review, we summarized all available studies in the literature about the factors and processes crucial in the enrichment, release, and migration of the most commonly encountered hazardous and toxic elements in naturally contaminated geological materials. Although our focus is on naturally contaminated rocks, analogue systems like contaminated soils, sediments, and other hazardous wastes that have been more widely studied will also be discussed. Classification schemes and leaching tests to properly identify and regulate excavated rocks that may potentially pose environmental problems will be examined. Finally, management and mitigation strategies to limit the negative effects of these hazardous wastes are introduced.

Bimetallic materials—well-known for their strong reducing properties—are promising tools to recover battery/critical elements from waste streams for resources conservation and decontamination. To date, however, their synthesis has been limited to the use of pure monometallic elements. In this study, we synthesized magnetic bimetallic materials from aluminum wastes (Al-scrap) using a two-stage mechanical-chemical method composed of (1) polishing to remove protective coatings, and (2) etching-cementation for zero-valent iron (ZVI) deposition. Argon-etching using X-ray photoelectron spectroscopy (XPS) showed the effective removal of anodized/polymeric coatings on Al-scrap but the exposed Al0 was rapidly passivated by an Al-oxyhydroxide/oxide film composed of corundum, bayerite, and boehmite. In the etching-cementation stage, the Al-oxyhydroxide/oxide film was removed using NaCl–HCl and Fe3+ was deposited in corrosion pits on Al-scrap. Strong “magnetic susceptibility” was observed in 1.0 M Fe3+/3.5 M Cl− synthesized products but not in 0.5 M Fe3+/2 M Cl−. This difference could be attributed to the prevalence of ZVI formation in the former but akaganeite precipitation in the latter. The results also showed “targeted” recovery of copper dissolved from Al-scrap on ZVI regions of synthesized product. Finally, two pathways are proposed for Fe3+ reduction: (i) direct cementation (Fe3+→ZVI), and (ii) sequential reduction (Fe3+→Fe2+→ZVI).

Porphyry ores and E-wastes/WEEE are two of the most important copper-bearing materials on the planet. Over 60% of world copper output comes from porphyry copper ores while E-waste(s) is globally the largest copper-bearing waste category since the 1980s. They also contain critical elements for low-carbon technologies essential in the clean energy transition's success. In this review, a critical analysis of ore distribution/processing, metal extraction, E-waste generation and E-waste recycling is presented, focusing on identifying challenges and how to address them with emerging technologies and sustainable socio-environmental strategies. Access to ore deposits is a major hurdle for mine development while the absence of a consistent E-waste classification and legislation, including poor collection rates, remains serious problems in E-waste recycling. As lower grade porphyry ores are exploited, difficulties in processing/extraction due to mineralogical complexities, very fine particles and the generation of “dirty” concentrates will become more prevalent. For E-wastes recycling, current trends are to develop smaller, more mobile, and eco-friendly hydrometallurgical alternatives to pyrometallurgy that can handle localised compositional and feed variabilities. Finally, more sustainable mine waste management strategies, including better LCIA tools with spatial and temporal dimensions, are needed to limit socio-environmental impacts of resources exploitation and maintain the sector's SLO.

Acid mine drainage (AMD) formation and toxic arsenic (As) contamination are serious environmental problems encountered worldwide. In this study, we investigated the crucial roles played by common secondary mineral phases formed during the natural weathering of pyrite-bearing wastes—soluble salts (melanterite, FeSO4·7H2O) and metal oxides (hematite, Fe2O3)—on AMD formation and As mobility under acidic conditions (pH 1.5–4) prevalent in historic tailings storage facilities, pyrite-bearing rock dumps and AMD-contaminated soils and sediments. Our results using a pyrite-rich natural geologic material containing arsenopyrite (FeAsS) showed that melanterite and hematite both directly—by supplying H+ and/or oxidants (Fe3+)—and indirectly—via changes in the nature of oxidation layer formed on pyrite—influenced pyrite oxidation dynamics. Based on SEM-EDS, DRIFT spectroscopy and XPS results, the oxidation layer on pyrite was mainly composed of ferric arsenate and K-Jarosite when melanterite was abundant with/without hematite but changed to Fe-oxyhydroxide/oxide and scorodite when melanterite was low and hematite was present. This study also observed the formation of a mechanically ‘strong’ coating on pyrite that suppressed the mineral’s oxidation. Finally, As mobility under acidic conditions was limited by its precipitation as ferric arsenate, scorodite, or a Fe/Al arsenate phase, including its strong adsorption to Fe-oxyhydroxides/oxides.

Acid mine drainage (AMD), the very acidic and highly contaminated leachate generated in closed/abandoned mines, is commonly managed by neutralization to raise the pH and precipitate most of the heavy metals. Although effective, this approach does not generate any product of economic value, so it is very costly and unsustainable in the long-term. Unfortunately, there are currently no effective alternatives to neutralization, and one way to improve the sustainability of this process is to reduce the volume of AMD generated and/or the concentration of heavy metals. The tailings dam investigated in this study is located in northern Hokkaido, Japan. Detailed characterization of borehole core samples showed that even after almost 40 years of exposure to the environment, the tailings still contain pyrite (FeS2) and substantial amounts of copper (Cu) and zinc (Zn). Reactive-transport modeling using Visual MODFLOW predicted that AMD quality would likely continue to deteriorate with time and that treatment should be continued for at least 1,000 years. The model also predicted that a barrier with low permeability installed downstream of the tailings dam or ground sealing techniques for recharge reduction could lower the volume of AMD and concentration of Zn from the site.

Acid mine drainage (AMD) is one of the most serious environmental problems encountered in mining areas worldwide. When released into the environment without treatment, AMD pollutes the surrounding water bodies and soils with hazardous and toxic elements like arsenic (As), selenium (Se) and heavy metals such as lead (Pb), cadmium (Cd), copper (Cu) and zinc (Zn) that rapidly destroy affected ecosystems. The most commonly used method to treat AMD is chemical neutralization, a technique whereby basic materials like limestone or lime are mixed with AMD to raise its pH and remove most of the contaminants via precipitation. Although effective, this approach requires the continuous supply of chemicals, energy, and manpower, which makes it unsustainable because AMD generation has been documented to continue for a very long time (up to several centuries to millennia). One promising alternative to chemical neutralization is (micro) encapsulation, a technique that directly treats pyrite, the main mineral responsible for AMD formation, and renders it unreactive by encapsulating the mineral with protective coatings. In this study, we introduced two advanced pyrite passivation techniques to limit AMD formation called “carrier-microencapsulation” (CME) and “galvanic microencapsulation” (GME). CME uses a redox-reactive organic carrier to deliver the coating material on the surface of pyrite. Because the carrier only decomposes oxidatively, the primary strength of this technique is its high selectivity for pyrite even in complex systems like mine tailings and pyrite-rich waste rocks. Meanwhile, GME is based on galvanic interactions between pyrite and metals with lower rest potentials so this technique could be applied directly in a ball mill during ore processing or coal cleaning.

The Wnt/β-catenin signaling is essential for various organogenesis and is often implicated during tumorigenesis. Dysregulated β-catenin signaling is associated with the formation of endometrial adenocarcinomas (EACs), which is considered as the common form of endometrial cancer in women. In the current study, we investigate the downstream target of Wnt/β-catenin signaling in the uterine epithelia and the mechanism leading to the formation of endometrial hyperplasia. We report that conditional ablation and activation of β-catenin in the uterine epithelia lead to aberrant epithelial structures and endometrial hyperplasia formation, respectively. We demonstrate that β-catenin regulates Foxa2 with its candidate upstream region for the uterine epithelia. Furthermore, knockdown of Foxa2 leads to defects in cell cycle regulation, suggesting a possible function of Foxa2 in the control of cell proliferation. We also observe that β-catenin and Foxa2 expression levels are augmented in the human specimens of complex atypical endometrial hyperplasia, which is considered to have a greater risk of progression to EACs. Thus, our study indicates that β-catenin regulates Foxa2 expression, and this interaction is possibly essential to control cell cycle progression during endometrial hyperplasia formation. Altogether, the augmented expression levels of β-catenin and Foxa2 are essential features during the formation of endometrial hyperplasia.

The bulbourethral gland (BUG) is a male-specific organ, which secretes part of the semen fluid. As the BUG is located in the deep pelvic floor, its developmental process is still unclear. Bone morphogenetic protein (Bmp) signaling plays pivotal roles in various organs. However, the function of Bmp signaling for BUG development is still unclear. The present study aimed to elucidate the role of Bmp signaling in the development of the BUG. We observed the prominent nuclear accumulation of phosphorylated (p) SMAD1/5/8, the downstream molecules of Bmp signaling, during BUG epithelial development. These results suggest that Bmp signaling contributes to BUG development. Bmp receptor1a (Bmpr1a) is known as the major type 1 signal transducer in some organogeneses. To analyze the Bmp signaling function for BUG development, we examined epithelial cell-specific Bmpr1a gene conditional mutant mice utilizing the tamoxifen-inducible Cre recombinase system. We observed cystic dilation and epithelial hyperplasia of the BUG in the Bmpr1a conditional knockout mice. The mutant cystic BUG specimens also showed inflammatory lesions. These BUG abnormalities resembled some of the BUG malformations observed in human congenital syndromes. The current study suggests that Bmp signaling possesses an essential role in BUG development and homeostasis. This would be the first report showing that the mutation of the Bmpr1a gene in the BUG epithelia phenocopied some abnormalities of human congenital syndromes affecting the BUG duct.

Thyroid follicles, the functional units of the thyroid gland, are delineated by a monolayer of thyrocytes resting on a continuous basement membrane. Developmental mechanisms whereby follicles are formed by reorganization of a non-structured mass of non-polarized epithelial cells (folliculogenesis) largely unknown. Here we show that assembly of the epithelial basement membrane is critical for folliculogenesis and is controlled by endothelial cell invasion and by BMP-Smad signaling in thyrocytes. Thyroid-specific double Smad1 and Smad5 knockout mice (Smad1/5(dKO)) displayed growth retardation, hypothyroidism and defective follicular architecture. In Smad1/5(dKO)embryonic thyroids, epithelial cells remained associated in large clusters and formed small follicles. Although similar follicular defects are found in Vegfa(KO)thyroids, Smad1/5(dKO)thyroids had normal endothelial cell density yet impaired endothelial differentiation. Interestingly, both Vegfa(KO)and Smad1/5(dKO)thyroids displayed impaired basement membrane assembly. Furthemore, conditioned medium (CM) from embryonic endothelial progenitor cells (eEPC) rescued the folliculogenic defects of both Smad1/5(dKO)and Vegfa(KO)thyroids. Laminin α1β1γ1, abundantly released by eEPC into CM, was critically required for folliculogenesis. Thus, epithelial Smad signaling and endothelial cell invasion promote folliculogenesis via assembly of the basement membrane. ispartof: Development vol:143 issue:11 pages:1958-1970 ispartof: location:England status: published

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the "angio-follicular" units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood. This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo. Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR. In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the "angio-follicular" units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood. This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo. Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR. In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

The bulbourethral gland (BUG) is a male-specific organ, which secretes part of the semen fluid. As the BUG is located in the deep pelvic floor, its developmental process is still unclear. Bone morphogenetic protein (Bmp) signaling plays pivotal roles in various organs. However, the function of Bmp signaling for BUG development is still unclear. The present study aimed to elucidate the role of Bmp signaling in the development of the BUG. We observed the prominent nuclear accumulation of phosphorylated (p) SMAD1/5/8, the downstream molecules of Bmp signaling, during BUG epithelial development. These results suggest that Bmp signaling contributes to BUG development. Bmp receptor1a (Bmpr1a) is known as the major type 1 signal transducer in some organogeneses. To analyze the Bmp signaling function for BUG development, we examined epithelial cell-specific Bmpr1a gene conditional mutant mice utilizing the tamoxifen-inducible Cre recombinase system. We observed cystic dilation and epithelial hyperplasia of the BUG in the Bmpr1a conditional knockout mice. The mutant cystic BUG specimens also showed inflammatory lesions. These BUG abnormalities resembled some of the BUG malformations observed in human congenital syndromes. The current study suggests that Bmp signaling possesses an essential role in BUG development and homeostasis. This would be the first report showing that the mutation of the Bmpr1a gene in the BUG epithelia phenocopied some abnormalities of human congenital syndromes affecting the BUG duct.

Digit and interdigit (D/ID) development is one of the important research fields in molecular developmental biology. Interdigital cell death (ICD) is a morphogenetic event which has been considered as an essential process for D/ID formation. Although some growth factors including Bmp and Fgf signaling can modulate ICD, growth factor crosstalk regulating ICD is Poorly understood. Wnt canonical pathway and Bmp signal crosstalk has been considered as the essential growth factor crosstalk in organogenesis. To elucidate the crosstalk to regulate the D/ID formation, we analyzed conditional mutant mice with limb bud ectoderm expressing constitutively activated beta-catenin signaling. We showed that modulation of Wnt/beta-catenin signal in the limb ectoderm including the AER regulates ID apoptosis. We also demonstrated that Wnt/beta-catenin signaling in the ectoderm call positively regulate Fgf8 possibly antagonizing the epithelial derived Bmp signaling. Human birth defects for digit abnormalities have been known to be affected by multiple parameters. Elucidation of the potential mechanisms underlying such D/ID development is all urgent medical issue to be solved. This work would be one of the first Studies showing essential growth factor cascades in the D/ID formation. (C) 2009 Elsevier Inc. All rights reserved.

Recent, molecular biology techniques have accomplished a large contribution to developmental biology. Especially, gene transduction techniques are indispensable to study the roles of regulatory genes underlying embryogenesis. Replication-competent retroviruses, transfection with lipofection and an in ovo electroporation have been established as gene transduction techniques for embryos. (Yamada et al., 1997; Muramatsu et al., 1998; Fukuda et al., 2000; Iba, 2000; Nakamura et al., 2000). Particularly, in ovo electroporation for chick embryos has been recognized as a powerful method to efficiently induce exogenous genes into target cells or tissues, and it has been widely utilized to investigate their functions during embryonic development. Because the electric current tend to affect mainly the epithelial cells of the embryo, in ovo electroporation is suitable for the studies of neurogenesis and neuronal differentiation. In fact, large number of information relating to neuronal development has been brought from the studies using in ovo electroporation (Okafuji et al., 1999; Nakamura and Funahashi, 2001). However, gene transduction using in ovo electroporation for mesenchymal tissues, e.g., somite, limb mesenchyme generally show lower efficiency. Hence, it is necessary to design appropriate electrodes to set up conditions of the electric pulses (voltage, pulse length, number of pulses) for each tissue in order to avoid tissue damages for embryos.

beta-catenin signaling is one of the key factors regulating the fate of hair follicles (HFs). To elucidate the regulatory mechanism of embryonic HF fate determination during epidermal development/differentiation, we analyzed conditional mutant mice with keratinocytes expressing constitutively active beta-catenin (K5-Cre Catnb(ex3)fl/+). The mutant mice developed scaly skin with a thickened epidermis and showed impaired epidermal stratification. The hair shaft keratins were broadly expressed in the epidermis but there was no expression of the terminal differentiation markers K1 and loricrin. Hair placode markers (Bmp2 and Shh) and follicular dermal condensate markers (noggin, patched 1 and Pdgfra) were expressed throughout the epidermis and the upper dermis, respectively. These results indicate that the embryonic epidermal keratinocytes have switched extensively to the HF fate. A series of genetic studies demonstrated that the epidermal switching to HF fate was suppressed by introducing the conditional mutation K5-Cre Catnb(ex3)fl/+Shhfl/- (with additional mutation of Shh signaling) or K5-Cre Catnb(ex3)fl/+BmprIAfl/fl (with additional mutation of Bmp signaling). These results demonstrate that Wnt/beta-catenin signaling relayed through Shh and Bmp signals is the principal regulatory mechanism underlying the HF cell fate change. Assessment of Bmp2 promoter activities suggested a putative regulation by beta-catenin signaling relayed by Shh signaling towards Bmp2. We also found that Shh protein expression was increased and expanded in the epidermis of K5-Cre Catnb(ex3)fl/+BmprIAfl/fl mice. These results indicate the presence of growth factor signal cross-talk involving beta-catenin signaling, which regulates the HF fate.