TMEM132A regulates mouse hindgut morphogenesis and caudal development.

Caudal developmental defects, including caudal regression, caudal dysgenesis and sirenomelia, are devastating conditions affecting the skeletal, nervous, digestive, reproductive and excretory systems. Defects in mesodermal migration and blood supply to the caudal region have been identified as possible causes of caudal developmental defects, but neither satisfactorily explains the structural malformations in all three germ layers. Here, we describe caudal developmental defects in transmembrane protein 132a (Tmem132a) mutant mice, including skeletal, posterior neural tube closure, genitourinary tract and hindgut defects. We show that, in Tmem132a mutant embryos, visceral endoderm fails to be excluded from the medial region of early hindgut, leading directly to the loss or malformation of cloaca-derived genitourinary and gastrointestinal structures, and indirectly to the neural tube and kidney/ureter defects. We find that TMEM132A mediates intercellular interaction, and physically interacts with planar cell polarity (PCP) regulators CELSR1 and FZD6. Genetically, Tmem132a regulates neural tube closure synergistically with another PCP regulator Vangl2. In summary, we have identified Tmem132a as a new regulator of PCP, and hindgut malformation as the underlying cause of developmental defects in multiple caudal structures.

A network of transient domains for breaking symmetry during anterior-posterior axis formation in the porcine embryo.

Breaking radial symmetry for anterior-posterior axis formation is one of the key developmental steps of vertebrate gastrulation and is established through a succession of transient domains defined by morphology or gene expression. Three such domains were interpreted recently in the rabbit to be part of a "three-anchor-point model" for axis formation. To answer the question as to whether the model is generally applicable to mammals, the dynamic expression patterns of four marker genes were analyzed in the pig, where gastrulating epiblast forms from half the inner cell mass: EOMES and PKDCC transcripts display decreasing expression intensities in the anterior hypoblast and-together with WNT3-increasing intensity in the anterior streak domain and the node; TBX6 expression changes from an initial central expression to exclusive expression in the posterior extremity of the primitive streak. The anterior streak domain has thus a molecular footprint similar to the one in the rabbit, the end node shares TBX6 between the species, while the anterior hypoblast-mirroring specific porcine epiblast derivation and fate-is marked by PKDCC instead of WNT3. The molecular similarities in transient domains point to conserved mechanisms for establishing the mammalian anterior-posterior axis and, possibly, breaking radial symmetry.

Transcriptional network governing extraembryonic endoderm cell fate choice.

The mechanism by which transcription factor (TF) network instructs cell-type-specific transcriptional programs to drive primitive endoderm (PrE) progenitors to commit to parietal endoderm (PE) versus visceral endoderm (VE) cell fates remains poorly understood. To address the question, we analyzed the single-cell transcriptional signatures defining PrE, PE, and VE cell states during the onset of the PE-VE lineage bifurcation. By coupling with the epigenomic comparison of active enhancers unique to PE and VE cells, we identified GATA6, SOX17, and FOXA2 as central regulators for the lineage divergence. Transcriptomic analysis of cXEN cells, an in vitro model for PE cells, after the acute depletion of GATA6 or SOX17 demonstrated that these factors induce Mycn, imparting the self-renewal properties of PE cells. Concurrently, they suppress the VE gene program, including key genes like Hnf4a and Ttr, among others. We proceeded with RNA-seq analysis on cXEN cells with FOXA2 knockout, in conjunction with GATA6 or SOX17 depletion. We found FOXA2 acts as a potent suppressor of Mycn while simultaneously activating the VE gene program. The antagonistic gene regulatory activities of GATA6/SOX17 and FOXA2 in promoting alternative cell fates, and their physical co-bindings at the enhancers provide molecular insights to the plasticity of the PrE lineage. Finally, we show that the external cue, BMP signaling, promotes the VE cell fate by activation of VE TFs and repression of PE TFs including GATA6 and SOX17. These data reveal a putative core gene regulatory module that underpins PE and VE cell fate choice.

Basal-type lumenogenesis in extraembryonic endoderm stem cells models the early visceral endoderm.

Cultured rat primitive extraembryonic endoderm (pXEN) cells easily form free-floating multicellular vesicles de novo, exemplifying a poorly studied type of morphogenesis. Here, we reveal the underlying mechanism and the identity of the vesicles. We resolve the morphogenesis into vacuolization, vesiculation and maturation, and define the molecular characteristics and requirements of each step. Vacuolization is fueled by macropinocytosis and occurs by default if not blocked by high cell density or matrix proteins. Fine-tuned cell-cell contact then forms nascent three-cell vesicles with vacuole-derived lumina. In maturation, the vesicles complete epithelialization, expand via mitosis and continued fluid uptake, and differentiate further. The mature vesicles consist of a simple squamous epithelium with an apical-outside/basal-inside polarity that we trace back to the single cell stage. The polarity and gene expression pattern of the vesicles are similar to those of the early visceral endoderm. pXEN cells provide a useful in vitro model for study of matrix-independent, basal-type lumenogenesis and the physiology of the visceral endoderm.This article has an associated First Person interview with the first author of the paper.

ROSA26 reporter mouse lines and image analyses reveal distinct region-specific cell behaviors in the visceral endoderm.

The early post-implantation mouse embryo changes dramatically in both size and shape. These morphological changes are based on characteristic cellular behaviors, including cell growth and allocation. To perform clonal analysis, we established a Cre/loxP-based reporter mouse line, R26R-ManGeKyou, that enables clonal labeling with multiple colors. We also developed a novel ImageJ plugin, LP-Clonal, for quantitative measurement of the tilt angle of clonal cluster shape, enabling identification of the direction of cluster expansion. We carried out long-term and short-term lineage tracking. We also performed time-lapse imaging to characterize cellular behaviors using R26-PHA7-EGFP and R26R-EGFP These images were subjected to quantitative image analyses. We found that the proximal visceral endoderm overlying the extra-embryonic ectoderm shows coherent cell growth in a proximal-anterior to distal-posterior direction. We also observed that directional cell migration is coupled with cell elongation in the anterior region. Our observations suggest that the behaviors of visceral endoderm cells vary between regions during peri-implantation stages.

The T-box transcription factor Eomesodermin is essential for AVE induction in the mouse embryo.

Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior-posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.

Concerted cell divisions in embryonic visceral endoderm guide anterior visceral endoderm migration.

Migration of Anterior Visceral Endoderm (AVE) is a critical symmetry breaking event in the early post-implantation embryo development and is essential for establishing the correct body plan. Despite much effort, cellular and molecular events influencing AVE migration are only partially understood. Here, using time-lapse live imaging of mouse embryos, we demonstrate that cell division in the embryonic visceral endoderm is coordinated with AVE migration. Moreover, we demonstrate that temporal inhibition of FGF signalling during the pre-implantation specification of embryonic visceral endoderm perturbs cell cycle progression, thus affecting AVE migration. These findings demonstrate that coordinated cell cycle progression during the implantation stages of development is important for post-implantation morphogenesis in the mouse embryo.

TAK1/Map3k7 enhances differentiation of cardiogenic endoderm from mouse embryonic stem cells.

Specification of the primary heart field in mouse embryos requires signaling from the anterior visceral endoderm (AVE). The nature of these signals is not known. We hypothesized that the TGFß-activated kinase (TAK1/Map3k7) may act as a cardiogenic factor, based on its expression in heart-inducing endoderm and its requirement for cardiac differentiation of p19 cells. To test this, mouse embryonic stem (ES) cells overexpressing Map3k7 were isolated and differentiated as embryoid bodies (EBs). Map3k7-overexpressing EBs showed increased expression of AVE markers but interestingly, showed little effect on mesoderm formation and had no impact on overall cardiomyocyte formation. To test whether the pronounced expansion of endoderm masks an expansion of cardiac lineages, chimeric EBs were made consisting of Map3k7-overexpressing ES and wild type ES cells harboring a cardiac reporter transgene, MHCα::GFP, allowing cardiac differentiation to be assessed specifically in wild type ES cells. Wild type ES cells co-cultured with Map3k7-overexpressing cells had a 4-fold increase in expression of the cardiac reporter, supporting the hypothesis that Map3k7 increases the formation of cardiogenic endoderm. To further examine the role of Map3k7 in early lineage specification, other endodermal markers were examined. Interestingly, markers that are expressed in both the VE and later in gut development were expanded, whereas transcripts that specifically mark the early definitive (streak-derived) endoderm (DE) were not. To determine if Map3k7 is necessary for endoderm differentiation, EBs were grown in the presence of the Map3k7 specific inhibitor 5Z-7-oxozeaenol. Endoderm differentiation was dramatically decreased in these cells. Western blot analysis showed that known downstream targets of Map3k7 (Jnk, Nemo-like kinase (NLK) and p38 MAPK) were all inhibited. By contrast, transcripts for another TGFß target, Sonic Hedgehog (Shh) were markedly upregulated, as were transcripts for Gli2 (but not Gli1 and Gli3). Together these data support the hypothesis that Map3k7 governs the formation, or proliferation of cardiogenic endoderm.

Apical constriction in distal visceral endoderm cells initiates global, collective cell rearrangement in embryonic visceral endoderm to form anterior visceral endoderm.

The behavior of visceral endoderm cells was examined as the anterior visceral endoderm (AVE) formed from the distal visceral endoderm (DVE) using the mouse lines R26-H2B-EGFP and R26-PHA7-EGFP to visualize cell nuclei and adherens junction, respectively. The analysis using R26-H2B-EGFP demonstrated global cell rearrangement that was not specific to the DVE cells in the monolayer embryonic visceral endoderm sheet; each population of the endoderm cells moved collectively in a swirling movement as a whole. Most of the AVE cells at E6.5 were not E5.5 DVE cells but were E5.5 cells that were located caudally behind them, as previously reported (Hoshino et al., 2015; Takaoka et al., 2011). In the rearrangement, the posterior embryonic visceral endoderm cells did not move, as extraembryonic visceral endoderm cells did not, and they constituted a distinct population during the process of anterior-posterior axis formation. The analysis using R26-PHA7-EGFP suggested that constriction of the apical surfaces of the cells in prospective anterior portion of the DVE initiated the global cellular movement of the embryonic visceral endoderm to drive AVE formation.

The embryo reunited with its membranes in Göttingen.

An EMBO workshop entitled 'Embryonic-Extraembryonic Interfaces' took place in Göttingen, Germany, in May 2015. It showcased the enormous breadth of this area not only by touching on the molecular and cellular mechanisms of development, but also because of its coverage of particularly interesting evolutionary questions and of several medically related aspects. This Meeting Review discusses some highlights from the workshop and the emerging themes in the field.

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage.

The ability to follow and modify cell behaviour with accurate spatiotemporal resolution is a prerequisite to study morphogenesis in developing organisms. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has been used with this aim in different model systems. However, current localised electroporation strategies suffer from insufficient reproducibility and mediocre survival when applied to small and delicate organisms such as early post-implantation mouse embryos. We introduce here a microdevice to achieve localised electroporation with high efficiency and reduced cell damage. In silico simulations using a simple electrical model of mouse embryos indicated that a dielectric guide-based design would improve on existing alternatives. Such a device was microfabricated and its capacities tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Transfection was efficiently and reproducibly restricted to fewer than four visceral endoderm cells without compromising cell behaviour and embryo survival. Combining targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos revealed that, like leading DVE cells, non-leading ones send long basal projections and intercalate during their migration. Finally, we show that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation stages to organ explants. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in the mouse embryo. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices.

Vertebrate Axial Patterning: From Egg to Asymmetry.

The emergence of the bilateral embryonic body axis from a symmetrical egg has been a long-standing question in developmental biology. Historical and modern experiments point to an initial symmetry-breaking event leading to localized Wnt and Nodal growth factor signaling and subsequent induction and formation of a self-regulating dorsal "organizer." This organizer forms at the site of notochord cell internalization and expresses primarily Bone Morphogenetic Protein (BMP) growth factor antagonists that establish a spatiotemporal gradient of BMP signaling across the embryo, directing initial cell differentiation and morphogenesis. Although the basics of this model have been known for some time, many of the molecular and cellular details have only recently been elucidated and the extent that these events remain conserved throughout vertebrate evolution remains unclear. This chapter summarizes historical perspectives as well as recent molecular and genetic advances regarding: (1) the mechanisms that regulate symmetry-breaking in the vertebrate egg and early embryo, (2) the pathways that are activated by these events, in particular the Wnt pathway, and the role of these pathways in the formation and function of the organizer, and (3) how these pathways also mediate anteroposterior patterning and axial morphogenesis. Emphasis is placed on comparative aspects of the egg-to-embryo transition across vertebrates and their evolution. The future prospects for work regarding self-organization and gene regulatory networks in the context of early axis formation are also discussed.

Conserved and divergent expression patterns of markers of axial development in eutherian mammals.

BACKGROUND: Mouse embryos are cup shaped, but most nonrodent eutherian embryos are disk shaped. Extraembryonic ectoderm (ExEc), which may have essential roles in anterior-posterior (A-P) axis formation in mouse embryos, does not develop in many eutherian embryos. To assess A-P axis formation in eutherians, comparative analyses were made on rabbit, porcine, and Suncus embryos. RESULTS: All embryos examined expressed Nodal initially throughout epiblast and visceral endoderm; its expression became restricted to the posterior region before gastrulation. Anterior visceral endoderm (AVE) genes were expressed in Otx2-positive visceral endoderm, with Dkk1 expression being most anterior. The mouse pattern of AVE formation was conserved in rabbit embryos, but had diverged in porcine and Suncus embryos. No structure that was molecularly equivalent to Bmp-positive ExEc, existed in rabbit or pig embryos. In Suncus embryos, A-P axis was determined at prehatching stage, and these embryos attached to uterine wall at future posterior side. CONCLUSIONS: Nodal, but not Bmp, functions in epiblast and visceral endoderm development may be conserved in eutherians. AVE functions may also be conserved, but the pattern of its formation has diverged among eutherians. Roles of BMP and NODAL gradients in AVE formation seem to have been established in a subset of rodents.

Conditional restoration and inactivation of Rbm47 reveal its tissue-context requirement for viability and growth.

Rbm47 encodes a RNA binding protein that is necessary for Cytidine to Uridine RNA editing. Rbm47(gt/gt) mutant mice that harbor inactivated Rbm47 display poor viability. Here it was determined that the loss of Rbm47(gt/gt) offspring is due to embryonic lethality at mid-gestation. It was further showed that growth of the surviving Rbm47(gt/gt) mutants is impaired. Rbm47 is expressed in both the visceral endoderm and the definitive endoderm. Using the utility of the switchable FlEx gene-trap cassette and the activity of Cre and FLP recombinases to generate mice that conditionally inactivate and restore Rbm47 function in tissue-specific manner, it was demonstrated that Rbm47 function is required in the embryo proper, and not the visceral endoderm, for viability and growth. genesis 54:115-122, 2016. © 2016 Wiley Periodicals, Inc.

Nodal signaling from the visceral endoderm is required to maintain Nodal gene expression in the epiblast and drive DVE/AVE migration.

In the early mouse embryo, a specialized population of extraembryonic visceral endoderm (VE) cells called the distal VE (DVE) arises at the tip of the egg cylinder stage embryo and then asymmetrically migrates to the prospective anterior, recruiting additional distal cells. Upon migration these cells, called the anterior VE (AVE), establish the anterior posterior (AP) axis by restricting gastrulation-inducing signals to the opposite pole. The Nodal-signaling pathway has been shown to have a critical role in the generation and migration of the DVE/AVE. The Nodal gene is expressed in both the VE and in the pluripotent epiblast, which gives rise to the germ layers. Previous findings have provided conflicting evidence as to the relative importance of Nodal signaling from the epiblast vs. VE for AP patterning. Here we show that conditional mutagenesis of the Nodal gene specifically within the VE leads to reduced Nodal expression levels in the epiblast and incomplete or failed DVE/AVE migration. These results support a required role for VE Nodal to maintain normal levels of expression in the epiblast, and suggest signaling from both VE and epiblast is important for DVE/AVE migration.

AVE protein expression and visceral endoderm cell behavior during anterior-posterior axis formation in mouse embryos: Asymmetry in OTX2 and DKK1 expression.

The initial landmark of anterior-posterior (A-P) axis formation in mouse embryos is the distal visceral endoderm, DVE, which expresses a series of anterior genes at embryonic day 5.5 (E5.5). Subsequently, DVE cells move to the future anterior region, generating anterior visceral endoderm (AVE). Questions remain regarding how the DVE is formed and how the direction of the movement is determined. This study compares the detailed expression patterns of OTX2, HHEX, CER1, LEFTY1 and DKK1 by immunohistology and live imaging at E4.5-E6.5. At E6.5, the AVE is subdivided into four domains: most anterior (OTX2, HHEX, CER1-low/DKK1-high), anterior (OTX2, HHEX, CER1-high/DKK1-low), main (OTX2, HHEX, CER1, LEFTY1-high) and antero-lateral and posterior (OTX2, HHEX-low). The study demonstrates how this pattern is established. AVE protein expression in the DVE occurs de novo at E5.25-E5.5. Neither HHEX, LEFTY1 nor CER1 expression is asymmetric. In contrast, OTX2 expression is tilted on the future posterior side with the DKK1 expression at its proximal domain; the DVE cells move in the opposite direction of the tilt.

Imprinted expression in cystic embryoid bodies shows an embryonic and not an extra-embryonic pattern.

A large subset of mammalian imprinted genes show extra-embryonic lineage (EXEL) specific imprinted expression that is restricted to placental trophectoderm lineages and to visceral yolk sac endoderm (ysE). Isolated ysE provides a homogenous in vivo model of a mid-gestation extra-embryonic tissue to examine the mechanism of EXEL-specific imprinted gene silencing, but an in vitro model of ysE to facilitate more rapid and cost-effective experiments is not available. Reports indicate that ES cells differentiated into cystic embryoid bodies (EBs) contain ysE, so here we investigate if cystic EBs model ysE imprinted expression. The imprinted expression pattern of cystic EBs is shown to resemble fetal liver and not ysE. To investigate the reason for this we characterized the methylome and transcriptome of cystic EBs in comparison to fetal liver and ysE, by whole genome bisulphite sequencing and RNA-seq. Cystic EBs show a fetal liver pattern of global hypermethylation and low expression of repeats, while ysE shows global hypomethylation and high expression of IAPEz retroviral repeats, as reported for placenta. Transcriptome analysis confirmed that cystic EBs are more similar to fetal liver than ysE and express markers of early embryonic endoderm. Genome-wide analysis shows that ysE shares epigenetic and repeat expression features with placenta. Contrary to previous reports, we show that cystic EBs do not contain ysE, but are more similar to the embryonic endoderm of fetal liver. This explains why cystic EBs reproduce the imprinted expression seen in the embryo but not that seen in the ysE.

Cyclin E1 plays a key role in balancing between totipotency and differentiation in human embryonic cells.

STUDY HYPOTHESIS: We aimed to investigate if Cyclin E1 (CCNE1) plays a role in human embryogenesis, in particular during the early developmental stages characterized by a short cell cycle. STUDY FINDING: CCNE1 is expressed in plenipotent human embryonic cells and plays a critical role during hESC derivation via the naïve state and, potentially, normal embryo development. WHAT IS KNOWN ALREADY: A short cell cycle due to a truncated G1 phase has been associated with the high developmental capacity of embryonic cells. CCNE1 is a critical G1/S transition regulator. CCNE1 overexpression can cause shortening of the cell cycle and it is constitutively expressed in mouse embryonic stem cells and cancer cells. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We investigated expression of CCNE1 in human preimplantation embryo development and embryonic stem cells (hESC). Functional studies included CCNE1 overexpression in hESC and CCNE1 downregulation in the outgrowths formed by plated human blastocysts. Analysis was performed by immunocytochemistry and quantitative real-time PCR. Mann-Whitney statistical test was applied. MAIN RESULTS AND THE ROLE OF CHANCE: The CCNE1 protein was ubiquitously and constitutively expressed in the plenipotent cells of the embryo from the 4-cell stage up to and including the full blastocyst. During blastocyst expansion, CCNE1 was downregulated in the trophectoderm (TE) cells. CCNE1 shortly co-localized with NANOG in the inner cell mass (ICM) of expanding blastocysts, mimicking the situation in naïve hESC. In the ICM of expanded blastocysts, which corresponds with primed hESC, CCNE1 defined a subpopulation of cells different from NANOG/POU5F1-expressing pluripotent epiblast (EPI) cells and GATA4/SOX17-expressing primitive endoderm (PrE) cells. This CCNE1-positive cell population was associated with visceral endoderm based on transthyretin expression and marked the third cell lineage within the ICM, besides EPI and PrE, which had never been described before. We also investigated the role of CCNE1 by plating expanded blastocysts for hESC derivation. As a result, all the cells including TE cells re-gained CCNE1 and, consequently, NANOG expression, resembling the phenotype of naïve hESC. The inhibition of CCNE1 expression with siRNA blocked proliferation and caused degeneration of those plated cells. LIMITATIONS, REASONS FOR CAUTION: The study is based on a limited number of good-quality human embryos donated to research. WIDER IMPLICATIONS OF THE FINDINGS: Our study sheds light on the processes underlying the high developmental potential of early human embryonic cells. The CCNE1-positive plenipotent cell type corresponds with a phenotype that enables early human embryos to recover after fragmentation, cryodamage or (single cell) biopsy on day 3 for preimplantation genetic diagnosis. Knowledge on the expression and function of genes responsible for this flexibility will help us to better understand the undifferentiated state in stem cell biology and might enable us to improve technologies in assisted reproduction. LARGE SCALE DATA: NA STUDY FUNDING AND COMPETING INTERESTS: This research is supported by grants from the Fund for Scientific Research - Flanders (FWO-Vlaanderen), the Methusalem (METH) of the VUB and Scientific Research Fond Willy Gepts of UZ Brussel. There are no competing interests.

An integrated approach identifies the molecular underpinnings of murine anterior visceral endoderm migration.

The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.

Actin dynamics switches two distinct modes of endosomal fusion in yolk sac visceral endoderm cells.

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using mouse yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells.