Colored medaka and zebrafish: Transgenics with ubiquitous and strong transgene expression driven by the medaka β‐actin promoter

Conditional cell labeling, cell tracing, and genetic manipulation approaches are becoming increasingly important in developmental and regenerative biology. Such approaches in zebrafish research are hampered by the lack of an ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the medaka fish (Oryzias latipes) β‐actin (Olactb) promoter, which drives constitutive transgene expression during all developmental stages, and the analysis of adult organs except blood cell types. Taking advantage of the compact medaka promoter, we succeeded in generating a zebrafish transgenic (Tg) line with unprecedentedly strong and widespread transgene expression from embryonic to adult stages. Moreover, the Tg carries a pair of loxP sites, which enables the reporter fluorophore to switch from DsRed2 to enhanced green fluorescent protein (EGFP). We induced Cre/loxP recombination with Tg(hsp70l: mCherry‐t2a‐Cre^ERt2) in the double Tg embryo and generated a Tg line that constitutively expresses EGFP. We further demonstrate the powerful application of Olactb‐driven Tgs for cell lineage tracing using transplantation experiments with embryonic cells at the shield stage and adult cells of regenerating fin. Thus, the use of promoter elements from medaka is an alternative approach to generate Tgs with stronger and even novel expression patterns in zebrafish. The Olactb promoter and the Tg lines presented here represent an important advancement for the broader use of Cre/loxP‐based Tg applications in zebrafish.     

Pdgfrb‐Cre targets lymphatic endothelial cells of both venous and non‐venous origins

The Pdgfrb‐Cre line has been used as a tool to specifically target pericytes and vascular smooth muscle cells. Recent studies showed additional targeting of cardiac and mesenteric lymphatic endothelial cells (LECs) by the Pdgfrb‐Cre transgene. In the heart, this was suggested to provide evidence for a previously unknown nonvenous source of LECs originating from yolk sac (YS) hemogenic endothelium (HemEC). Here we show that Pdgfrb‐Cre does not, however, target YS HemEC or YS‐derived erythro‐myeloid progenitors (EMPs). Instead, a high proportion of ECs in embryonic blood vessels of multiple organs, as well as venous‐derived LECs were targeted. Assessment of temporal Cre activity using the R26‐mTmG double reporter suggested recent occurrence of Pdgfrb‐Cre recombination in both blood and lymphatic ECs. It thus cannot be excluded that Pdgfrb‐Cre mediated targeting of LECs is due to de novo expression of the Pdgfrb‐Cre transgene or their previously established venous endothelial origin. Importantly, Pdgfrb‐Cre targeting of LECs does not provide evidence for YS HemEC origin of the lymphatic vasculature. Our results highlight the need for careful interpretation of lineage tracing using constitutive Cre lines that cannot discriminate active from historical expression. The early vascular targeting by the Pdgfrb‐Cre also warrants consideration for its use in studies of mural cells. genesis 54:350–358, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Use of the Cre-loxP recombination system as an estimate for Agrobacterium-mediated co-transformation of tobacco leaves

Agrobacterium tumefaciens-mediated transformation of tobacco leaves (Nicotiana tabacum) is used to study gene expression in a heterologous genetic background. Here, the Cre-loxP recombination system was used to detect T-DNA transfer by two A. tumefaciens cells harboring different binary vectors. Cre, under the control of the CaMV 35S promoter, was cloned into one vector, and a loxP cassette into another vector. A mixture of A. tumefaciens, in which each cell contained either a Cre- or loxP-vector, was co-infiltrated into tobacco leaves. After two days, excision of loxP-flanked DNA was detected by PCR and used as an estimate for co-transformation events. Strongest excision (> 50%) was observed when the loxP cassette was cloned into vector pPZP112 and Cre into pISV2678. This fast and easy technique can be used to assess the co-transformation efficiency of tobacco cells in future studies.     

Expression of the Intermediate Filament Keratin Gene,K15,in the Basal Cell Layers of Epithelia and the Hair Follicle

The intermediate filament keratin, K15, is present in variable abundance in stratified epithelia. In this study we have isolated and characterized the sheepK15gene, focusing on its expression in the follicles of sheep and mice. We show thatK15is expressed throughout the hair cycle in the basal layer of the outer root sheath that envelops the follicle. Strikingly, however, in large medullated wool follicles, a small group of basal outer root sheath cells located in the region thought to contain hair follicle stem cells areK15-negative. In the follicle bulbK15is expressed in cells situated next to the dermal papilla but not in the inner bulb cells. Elsewhere,K15is expressed at a low, variable level in the basal layer of the epidermis and sebaceous gland, often in a punctate pattern. In the esophagus of the sheepK15expression is restricted to the basal layer, in contrast to human esophagus where it is expressed throughout the epithelium. Transgenic mouse lines established with a 15-kb sheepK15gene construct exhibited faithful expression and showed no phenotypic consequences ofK15overexpression. An investigation of transgene expression showed thatK15is continuously expressed in outer root sheath cells during the hair cycle. Given its expression in the mitotically active basal cell layers of diverse epithelia and the follicle,K15expression appears to signal an early stage in the pathway of keratinocyte differentiation that precedes the decision of a cell to become epidermal or hair-like.     

Generation and validation of mice carrying a conditional allele of the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is important for normal homeostasis in a variety of tissues and, when abnormally expressed or mutated, contributes to the development of many diseases. However, in vivo functional studies are hindered by the lack of adult mice lacking EGFR because of the pre‐ and postnatal lethality of EGFR deficient mice. We generated a conditional allele of Egfr (Egfr^tm1Dwt) by flanking exon 3 with loxP sites in order to investigate tissue‐specific functions of this widely expressed receptor tyrosine kinase. The activity of the Egfr^tm1Dwt allele is indistinguishable from wildtype Egfr. Conversely, the Egfr^Δ allele, generated by Cre‐mediated deletion of exon 3 using the germline EIIa‐Cre transgenic line, functions as a null allele. Egfr^Δ/Δ embryos that have complete ablation of EGFR activity and die at mid‐gestation with placental defects identical to those reported for mice homozygous for the Egfr^tm1Mag null allele. We also inactivated the Egfr^tm1Dwt allele tissue‐specifically in the skin epithelium using the K14‐Cre transgenic line. These mice were viable but exhibited wavy coat hair remarkably similar to mice homozygous for the Egfr^wa2 hypomorphic allele or heterozygous for the Egfr^Wa5 antimorphic allele. These results suggest that the hairless phenotype of Egfr nullizygous mice is not solely due to absence of EGFR in the epithelium, but that EGFR activity is required also in skin stromal cells for normal hair morphogenesis. This new mouse model should have wide utility to inactivate Egfr conditionally for functional analysis of EGFR in adult tissues and disease states. genesis 47:85–92, 2009. © 2008 Wiley‐Liss, Inc.     

The cell biology of human hair follicle pigmentation

Although we have made significant progress in understanding the regulation of the UVR‐exposed epidermal‐melanin unit, we know relatively little about how human hair follicle pigmentation is regulated. Progress has been hampered by gaps in our knowledge of the hair growth cycle’s controls, to which hair pigmentation appears tightly coupled. However, pigment cell researchers may have overly focused on the follicular melanocytes of the nocturnal and UVR‐shy mouse as a proxy for human epidermal melanocytes. Here, I emphasize the epidermis‐follicular melanocyte pluralism of human skin, as research models for vitiligo, alopecia areata and melanoma, personal care/cosmetics innovation. Further motivation could be in finding answers to why hair follicle and epidermal pigmentary units remain broadly distinct? Why melanomas tend to originate from epidermal rather than follicular melanocytes? Why multiple follicular melanocyte sub‐populations exist? Why follicular melanocytes are more sensitive to aging influences? In this perspective, I attempt to raise the status of the human hair follicle melanocyte and highlight some species‐specific issues involved which the general reader of the pigmentation literature (with its substantial mouse‐based data) may not fully appreciate.     

Platelet sonicates activate hair follicle stem cells and mediate enhanced hair follicle regeneration

An increasing number of studies show that platelet‐rich plasma (PRP) is effective for androgenic alopecia (AGA). However, the underlying cellular and molecular mechanisms along with its effect on hair follicle stem cells are poorly understood. In this study, we designed to induce platelets in PRP to release factors by calcium chloride (PC) or by sonication where platelet lysates (PS) or the supernatants of platelet lysate (PSS) were used to evaluate their effect on the hair follicle activation and regeneration. We found that PSS and PS exhibited a superior effect in activating telogen hair follicles than PC. In addition, PSS injection into the skin activated quiescent hair follicles and induced K15⁺ hair follicle stem cell proliferation in K14‐H2B‐GFP mice. Moreover, PSS promoted skin‐derived precursor (SKP) survival in vitro and enhanced hair follicle formation in vivo. In consistence, protein array analysis of different PRP preparations revealed that PSS contained higher levels of 16 growth factors (out of 41 factors analysed) than PC, many of them have been known to promote hair follicle regeneration. Thus, our data indicate that sonicated PRP promotes hair follicle stem cell activation and de novo hair follicle regeneration.     

Maintaining hair follicle stem cell identity in a dish

Identifying and mimicking the signals that regulate stem cell self‐renewal, differentiation and maintenance in a petri dish is crucial to faithfully recapitulate stem cell behaviour in vitro. In this issue, Chacón‐Martínez et al ( 2017 ) describe novel culture conditions that allow the long‐term expansion and maintenance of functional murine hair follicle stem cells. This exciting discovery provides a faithful platform to study hair follicle stem cells in vitro and potentially perform drug screening for skin and hair follicle disorders.     

Embryonic expression of an Nkx2‐5/Cre gene using ROSA26 reporter mice

Summary: Nkx2‐5, one of the earliest cardiac‐specific markers in vertebrate embryos, was used as a genetic locus to knock in the Cre recombinase gene by homologous recombination. Offspring resulting from heterozygous Nkx2‐5/Cre mice mated to ROSA26 (R26R) reporter mice provided a model system for following Nkx2‐5 gene activity by β‐galactosidase (β‐gal) activity. β‐gal activity was initially observed in the early cardiac crescent, cardiomyocytes of the looping heart tube, and in the epithelium of the first pharyngeal arch. In later stage embryos (10.5–13.5 days postcoitum, dpc), β‐gal activity was observed in the stomach and spleen, the dorsum of the tongue, and in the condensing primordium of the tooth. The Nkx2‐5/Cre mouse model should provide a useful genetic resource to elucidate the role of loxP manipulated genetic targets in cardiogenesis and other developmental processes. genesis 31:176–180, 2001. © 2001 Wiley‐Liss, Inc.     

Generation and characterization of a conditional allele of Interferon Regulatory Factor 6

Interferon Regulatory Factor 6 (IRF6) is a critical regulator of differentiation, proliferation, and migration of keratinocytes. Mutations in IRF6 cause two autosomal dominant disorders characterized by cleft lip with or without cleft palate. In addition, DNA variation in IRF6 confers significant risk for non‐syndromic cleft lip and palate. IRF6 is also implicated in adult onset development and disease processes, including mammary gland development and squamous cell carcinoma. Mice homozygous for a null allele of Irf6 die shortly after birth due to severe skin, limb, and craniofacial defects, thus impeding the study of gene function after birth. To circumvent this, a conditional allele of Irf6 was generated. To validate the functionality of the conditional allele, we used three “deleter” Cre strains: Gdf9‐Cre, CAG‐Cre, and Ella‐Cre. When Cre expression was driven by the Gdf9‐Cre or CAG‐Cre transgenes, 100% recombination was observed as indicated by DNA genotyping and phenotyping. In contrast, use of the Ella‐Cre transgenic line resulted in incomplete recombination, despite expression at the one‐cell stage. In sum, we generated a novel tool to delete Irf6 in a tissue specific fashion, allowing for study of gene function past perinatal stages. However, recombination efficiency of this allele was dictated by the Cre‐driver used.     

Polyclonal origin and hair induction ability of dermal papillae in neonatal and adult mouse back skin

Hair follicle development and growth are regulated by Wnt signalling and depend on interactions between epidermal cells and a population of fibroblasts at the base of the follicle, known as the dermal papilla (DP). DP cells have a distinct gene expression signature from non-DP dermal fibroblasts. However, their origins are largely unknown. By generating chimeric mice and performing skin reconstitution assays we show that, irrespective of whether DP form during development, are induced by epidermal Wnt activation in adult skin or assemble from disaggregated cells, they are polyclonal in origin. While fibroblast proliferation is necessary for hair follicle formation in skin reconstitution assays, mitotically inhibited cells readily contribute to DP. Although new hair follicles do not usually develop in adult skin, adult dermal fibroblasts are competent to contribute to DP during hair follicle neogenesis, irrespective of whether they originate from skin in the resting or growth phase of the hair cycle or skin with β-catenin-induced ectopic follicles. We propose that during skin reconstitution fibroblasts may be induced to become DP cells by interactions with hair follicle epidermal cells, rather than being derived from a distinct subpopulation of cells.     

Transthyretin mouse transgenes direct RFP expression or Cre‐mediated recombination throughout the visceral endoderm

Transthyretin (Ttr) is a thyroid hormone transport protein secreted by cells of the visceral yolk sac and fetal liver in developing embryos, and by hepatocytes and the choroid plexus epithelium of the brain in adult mice. Spatiotemporal localization of Ttr mRNA during embryogenesis suggested that Ttr regulatory elements might drive transgene expression throughout the visceral endoderm of early mouse embryos. We use Ttr cis‐regulatory elements to generate Ttr::RFP and Ttr::Cre strains of mice, driving red fluorescent protein (RFP) and a nuclear‐localized Cre recombinase, respectively. Visualization of RFP fluorescence in Ttr::RFP transgenics confirms reporter localization throughout the visceral endoderm in early embryos and in the visceral yolk sac and fetal liver of later stage embryos. Using both GFP‐based and LacZ‐based Cre reporter strains, we demonstrate that in Ttr::Cre transgenics, Cre‐mediated recombination occurs throughout the visceral endoderm. The Ttr::Cre strain can therefore be used as a tool for genetic modifications within the visceral endoderm lineage. genesis 47:447–455, 2009. © 2009 Wiley‐Liss, Inc.     

Generation of a KOR‐Cre knockin mouse strain to study cells involved in kappa opioid signaling

The kappa opioid receptor (KOR) has numerous important roles in the nervous system including the modulation of mood, reward, pain, and itch. In addition, KOR is expressed in many non‐neuronal tissues. However, the specific cell types that express KOR are poorly characterized. Here, we report the development of a KOR‐Cre knockin allele, which provides genetic access to cells that express KOR. In this mouse, Cre recombinase (Cre) replaces the initial coding sequence of the Opkr1 gene (encoding the kappa opioid receptor). We demonstrate that the KOR‐Cre allele mediates recombination by embryonic day 14.5 (E14.5). Within the brain, KOR‐Cre shows expression in numerous areas including the cerebral cortex, nucleus accumbens and striatum. In addition, this allele is expressed in epithelium and throughout many regions of the body including the heart, lung, and liver. Finally, we reveal that KOR‐Cre mediates recombination of a subset of bipolar and amacrine cells in the retina. Thus, the KOR‐Cre mouse line is a valuable new tool for conditional gene manipulation to enable the study of KOR. genesis 54:29–37, 2016. © 2015 Wiley Periodicals, Inc.     

Adam10 haploinsufficiency causes freckle-like macules in Hairless mice

The Hairless nuclear receptor co‐repressor is required for hair follicle regeneration during the hair cycle. The classical Hairless^Hr/Hairless^Hr mouse mutant loses all hair between 2 and 3 weeks of age. As the mice age, their trunk skin develops epidermal pigmentation, a feature of human skin which is not found in normal haired mice. In this report, we present a new, dominant mouse mutation, Pied, which arose within a colony of Hairless^Hr/Hairless^Hr mice and causes freckle‐like macules on the skin. The Pied macules require Hairless^Hr homozygosity to form and are composed of localized clusters of epidermal melanocytes. Through linkage analysis, we find that the Pied mutation is a 1914 base pair loss‐of‐function deletion in the Adam10 zinc metalloprotease gene. The pathways that specifically maintain long‐term pigmentation patterns in adults are not well understood. We have identified Adam10 as an inhibitor of melanocyte expansion in adult skin.