Melanoma cells influence the differentiation pattern of human epidermal keratinocytes.

BACKGROUND: Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC). METHODS: Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK. RESULTS: Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes. CONCLUSION: We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

TVT-S in the U position--anatomical study.

INTRODUCTION AND HYPOTHESIS: The objective is to describe the anatomical position of tension-free vaginal tape Secur (TVT-S) in the U position regarding possible injury and fixation site. METHODS: We placed TVT-S inserters bilaterally in 13 embalmed and five fresh frozen female bodies. After dissection, we measured distances from the obturator bundle. RESULTS: In embalmed bodies, the mean distance of TVT-S from the obturator bundle was 2.83 cm (standard deviation (SD) 0.87 cm) on the left, 2.92 cm (SD 1.24 cm) on the right. Perforation of the fascia of obturator internus muscle occurred in 38.5%. In fresh frozen bodies, results were fundamentally similar (p > 0.05). CONCLUSIONS: There is a risk of injury to the obturator bundle and urinary bladder during TVT-S; however, there is a significant risk of inserting the TVT-S inserter outside the obturator internus muscle (into the lesser pelvis). The position of TVT-S does not change significantly after legs mal-positioning.

Somitic origin of the medial border of the mammalian scapula and its homology to the avian scapula blade.

The scapula is the main skeletal element of the pectoral girdle allowing muscular fixation of the forelimb to the axial skeleton. The vertebrate limb skeleton has traditionally been considered to develop from the lateral plate mesoderm, whereas the musculature originates from the axial somites. However, in birds, the scapular blade has been shown to develop from the somites. We investigated whether a somitic contribution was also present in the mammalian scapula. Using genetic lineage-tracing techniques, we show that the medial border of the mammalian scapula develops from somitic cells. The medial scapula border serves as the attachment site of girdle muscles (serratus anterior, rhomboidei and levator scapulae). We show that the development of these muscles is independent of the mechanism that controls the formation of all other limb muscles. We suggest that these muscles be specifically referred to as medial girdle muscles. Our results establish the avian scapular blade and medial border of the mammalian scapula as homologous structures as they share the same developmental origin.

[HOX genes and the limb development in the clinical praxis and in the experiment]. / HOX geny a vývoj koncetin v klinické medicíne i v experimentu.

In human, congenital malformations of the limbs are ranked among the most prevalent of all congenital birth defects. Substantial portion of these defects has genetic origin. Increasing knowledge about the particular mutations responsible for limb malformations in human results in the increasing availability of DNA diagnostic procedures for confirmation of clinical diagnosis and family counselling. Increasing understanding of the underlying developmental processes revealed by studying limb defects both in human and animal models may offer better therapeutic options in the future. This review concentrates on the role of Hox genes in limb development. Man, as well as other mammals, has 39 HOX genes, divided into 4 complexes (clusters). HOX genes play a major role in body plan layout and development of many organ systems. Experimental data show that during the limb development, HOX genes influence patterning along the proximodistal and anteroposterior (thumb-little finger) axis of the limb bud. In human, limb malformation was described in patients with mutations in HOXA11, HOXA13, HOXD10, and HOXD13 genes. Most frequent among these malformations are hand-foot-genital syndrome caused by HOXA13 gene mutation, and synpolydactyly caused by HOXD13 mutation. Severity of the phenotype manifestation of these diseases is variable, and depends on the particular mutation type, where point mutations, polyalanine expansions and deletions can take part.

[Cancer microenvironment affects biological properties of tumor cells--tumor as an embryologic problem?]. / Nádorové mikroprostredí ovlivnuje biologické vlastnosti nádorové bunky--nádor jako embryologický problém?

Mutual epithelial-mesenchymal interaction represents a fundamental control mechanism during the development of organs and tissues. This review article demonstrates the importance of such interaction for tumor formation where it influences the biological properties of cancer stem cell and tumor itself. The teratoma and melanoma are employed as examples to demonstrate the influence of embryonic microenvironment on the biological properties of tumor, mainly on its potential to metastasize. The manipulation of cancer microenvironment represents the perspective therapeutic tool for cancer treatment in future.

Analysis of dermal fibroblasts isolated from neonatal and child cleft lip and adult skin: Developmental implications on reconstructive surgery.

The nonsyndromic cleft is one of the most frequent congenital defects in humans. Clinical data demonstrated improved and almost scarless neonatal healing of reparative surgery. Based on our previous results on crosstalk between neonatal fibroblasts and adult keratinocytes, the present study focused on characterization of fibroblasts prepared from cleft lip tissue samples of neonates and older children, and compared them with samples isolated from normal adult skin (face and breast) and scars. Although subtle variances in expression profiles of children and neonates were observed, the two groups differed significantly from adult cells. Compared with adult cells, differences were observed in nestin and smooth muscle actin (SMA) expression at the protein and transcript level. Furthermore, fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine, transforming growth factor-ß1 (TGF-ß1). Dysregulation of the TGF-ß signalling pathway, including low expression of the TGF-ß receptor II, may contribute to reducing scarring in neonates. Fibroblasts of facial origin also exhibited age independent differences from the cells prepared from the breast, reflecting the origin of the facial cells from neural crest-based ectomesenchyme.

Functional differences between neonatal and adult fibroblasts and keratinocytes: Donor age affects epithelial-mesenchymal crosstalk in vitro.

Clinical evidence suggests that healing is faster and almost scarless at an early neonatal age in comparison with that in adults. In this study, the phenotypes of neonatal and adult dermal fibroblasts and keratinocytes (nestin, smooth muscle actin, keratin types 8, 14 and 19, and fibronectin) were compared. Furthermore, functional assays (proliferation, migration, scratch wound closure) including mutual epithelial­mesenchymal interactions were also performed to complete the series of experiments. Positivity for nestin and α smooth muscle actin was higher in neonatal fibroblasts (NFs) when compared with their adult counterparts (adult fibroblasts; AFs). Although the proliferation of NFs and AFs was similar, they significantly differed in their migration potential. The keratinocyte experiments revealed small, poorly differentiated cells (positive for keratins 8, 14 and 19) in primary cultures isolated from neonatal tissues. Moreover, the neonatal keratinocytes exhibited significantly faster rates of healing the experimentally induced in vitro defects in comparison with adult cells. Notably, the epithelial/mesenchymal interaction studies showed that NFs in co-culture with adult keratinocytes significantly stimulated the adult epithelial cells to acquire the phenotype of small, non-confluent cells expressing markers of poor differentiation. These results indicate the important differences between neonatal and adult cells that may be associated with improved wound healing during the early neonatal period.

Apoptosis of Merkel cells in neurotrophin-3 null mice.

Postnatal mice lacking neurotrophin-3 (NT3) are deficient in Merkel cells of touch domes and whisker follicles. We examined the mechanism of Merkel cell loss by immunocytochemistry and electron microscopy. Merkel cell of whisker follicles of NT3 null newborns exhibited decreased immunoreactivity for cytokeratin 8 and contained apoptotic bodies that were positive for cleaved caspase-3, a marker of active apoptosis. By electron microscopy, the Merkel cells displayed aggregation of chromatin along the nuclear membrane, with the marginated chromatin forming caps at the periphery of the nucleus. Ribosomes aggregated in the cytoplasm, while dense core granules characteristic of Merkel cells were still discernible. Finally, the Merkel cells and their nuclei fragmented into apoptotic bodies. None of the apoptotic Merkel cells were contacted by nerve fibers, and their desmosomal contacts with surrounding keratinocytes disappeared. After postnatal day 6 apoptotic Merkel cells were no longer observed, and the number of surviving Merkel cells was severely reduced. They were flat and contained few osmiophilic granules. We conclude that perinatal apoptosis is responsible for the loss of Merkel cells lacking innervation in NT3 null mice.

The role of NT-3 signaling in Merkel cell development.

Merkel cells originate from the neural crest. They are located in hairy and glabrous skin and have neuroendocrine characteristics. Together with A beta afferents, Merkel cells form a slowly adapting mechanoreceptor, the Merkel nerve ending, which transduces steady skin indentation. Neurotphin-3 (NT-3) plays important roles in neural crest cell development. We thus sought to determine whether neurotrophin signaling is essential for Merkel cell development in the whisker pad of the mouse. Our data indicate that at embryonic day 16.5 (E 16.5), NT-3 and its receptors, p75 neurotrophin receptor (p75NTR) and tyrosine kinase receptor, TrkC are not expressed at detectable levels in Merkel cells. After a perinatal switch, however, Merkel cells in whiskers of newborn mice are immunoreactive for p75NTR, TrkC and NT-3. Immunoreactivity of all three markers persists into adulthood. By contrast, innervating fibers are intensely p75NTR-immunoreactive in E16.5 whiskers, but no TrkC immunoreactivity is detected. At birth, and at 6 weeks of age, afferent fibers are intensely immunoreactive for both p75NTR and TrkC. In TrkC null whiskers, numerous Merkel cells are present at E16.5, and they are innervated. We draw three major conclusions from these observations: (i) NT-3 signaling through p75NTR or TrkC is not required for the development and prenatal survival of either a major subset or of all Merkel cells, (ii) the postnatal survival of Merkel cells is supported by autocrine or paracrine NT-3, rather than by neuron-derived NT-3, and (iii) Merkel cell-derived NT-3 is not a chemoattractant for innervating A beta fibers, but is likely to be involved in maintaining Merkel cell innervation postnatally.

Friedrich Sigmund Merkel and his "Merkel cell", morphology, development, and physiology: review and new results.

Merkel nerve endings are mechanoreceptors in the mammalian skin. They consist of large, pale cells with lobulated nuclei forming synapse-like contacts with enlarged terminal endings of myelinated nerve fibers. They were first described by F.S. Merkel in 1875. They are found in the skin and in those parts of the mucosa derived from the ectoderm. In mammals (apart from man), the largest accumulation of Merkel nerve endings is found in whiskers. In all vertebrates, Merkel nerve endings are located in the basal layer of the epidermis, apart from birds, where they are located in the dermis. Cytoskeletal filaments consisting of cytokeratins and osmiophilic granules containing a variety of neuropeptides are found in Merkel cells. In anseriform birds, groups of cells resembling Merkel cells, with discoid nerve terminals between cells, form Grandry corpuscles. There has been controversy over the origin of Merkel cells. Results from chick/quail chimeras show that, in birds, Merkel cells are a subpopulation of cells derived from the neural crest, which thus excludes their development from the epidermis. Most recently, also in mammals, conclusive evidence for a neural crest origin of Merkel cells has been obtained. Merkel cells and nerve terminals form mechanoreceptors. Calcium ions enter Merkel cells in response to mechanical stimuli, a process which triggers the release of calcium from intracellular stores resulting in exocytosis of neurotransmitter or neuromodulator. Recent results suggest that there may be glutamatergic transmission between Merkel cell and nerve terminal, which appears to be essential for the characteristic slowly adapting response of these receptors during maintained mechanical stimuli. Thus, we are convinced that Merkel cells with associated nerve terminals function as mechanoreceptor cells. Cells in the skin with a similar appearance as Merkel cells, but without contact to nerve terminals, are probably part of a diffuse neuroendocrine system and do not function as mechanoreceptors. Probably these cells, rather than those acting as mechanoreceptors, are the origin of a highly malignant skin cancer called Merkel cell carcinoma.

Anatomical relationship and fixation of tension-free vaginal tape Secur.

OBJECTIVE: The objective is to describe the anatomical localisation of tension-free vaginal tape Secur (TVT-S) in the H-position regarding possible injury of vessels and fixation site. METHODS: We placed TVT-S inserters bilaterally in 14 embalmed and five fresh frozen female bodies. After dissection, we measured distances from the obturator bundle (obturator nerve and obturator vessels). RESULTS: In embalmed bodies, the mean distance of TVT-S from the obturator bundle was 3.05 cm (standard deviation (SD) 1.18 cm) on the left, 3.07 cm (SD 1.17 cm) on the right. Perforation of the fascia of obturator internus muscle occurred in 46.4%. In fresh frozen bodies, results were fundamentally similar. Injury of variable vessels can occur. CONCLUSION: There is a minimal risk of injury to the obturator bundle during TVT-S; however, there is a significant risk of inserting the TVT-S inserter into the obturator fossa. The position of TVT-S does not change significantly after legs mal-positioning.

Deletion of a conserved noncoding sequence in Plzf intron leads to Plzf down-regulation in limb bud and polydactyly in the rat.

Lx mutation in SHR.Lx rat manifests in homozygotes as hindlimb preaxial polydactyly. It was previously mapped to a chromosome 8 segment containing the Plzf gene. Plzf (promyelocytic leukemia zinc finger protein) influences limb development as a direct repressor of posterior HoxD genes. However, the Plzf coding sequence is intact in the Lx mutants. Using linkage mapping in F2 hybrids, we downsized the segment containing Lx to 155 kb and sequenced conserved noncoding elements (CNEs) inside. A 2,964-bp deletion in Plzf intron 2, never detected in control animals, is the only candidate for Lx. The deletion removes the most deeply conserved CNE in the 155-kb segment, suggesting a regulatory influence on Plzf expression. Correspondingly, using in situ hybridization and quantitative real-time polymerase chain reaction, we found a decrease of Plzf expression in Lx/Lx limb buds with concomitant anterior expansion of expression domains of its targets, Hoxd10-13 genes, in the absence of ectopic Sonic hedgehog expression. Upstream regulation of Plzf in limb buds is currently unknown. We present here the first candidate Plzf cis-regulatory sequence.

Sonic hedgehog is required for the assembly and remodeling of branchial arch blood vessels.

Sonic hedgehog (Shh) is a morphogen involved in many developmental processes. Injection of cells (5E1) that produce a Shh-blocking antibody causes an attenuation of the Shh response, and this causes vascular malformations and impaired remodeling characterized by hemorrhages and protrusions of the anterior cardinal vein and outflow tract, delayed fusion of the dorsal aortae, impaired branching of the internal carotid artery, and delayed remodeling of the aortic arches. Distribution of smooth muscle cells in the vessel wall is unchanged. In 5E1-injected embryos, we also observed impaired assembly of endothelial cells into vascular tubes, particularly in the sixth branchial arch, around the anterior cardinal vein and around the dorsal aorta. In 5E1-treated embryos, increased numbers of macrophage-like cells, apoptotic cells, and a decreased level of proliferation were observed in head mesenchyme. Together, these observations show that Shh signaling is required at multiple stages for proper vessel formation and remodeling.

Abnormal myocardial and coronary vasculature development in experimental hypoxia.

Oxygen availability is one of the necessary prerequisites for normal embryonic development. In our previous study we found that quail embryos incubated under hypoxic conditions (16% O(2)) die at embryonic day (ED) 9 with signs of heart failure. By ED4 and ED6 we found thinner ventricular wall and increased capillary density. We thus hypothesized that the cause of death would lie in severe myocardial and coronary maldevelopment. ED6 and 7 hypoxic hearts had thinner ventricular wall, especially left. There was a simultaneous increase in capillary density, most pronounced in the interventricular septum. This site corresponds to an area of tissue hypoxia and ensuing increased angiogenesis, and also formation of ventricular conduction system. Hypoxia had a positive effect on normal sequence of maturation of the conduction system evaluated by optical mapping at ED7. In sections from ED9 hypoxic hearts we found, in addition to thinner ventricular walls, irregularities in development of coronary tree (missing coronary ostia, absence of one coronary artery, and irregular arterial wall). This deficiency was due to decreased myocyte proliferation rather than to increased apoptosis. By Indian ink injection through the left ventricle we found in normoxic hearts regular coronary branching pattern, while in the hypoxic ones there was often only an irregular plexus. Embryonic hypoxia thus leads to increased capillarity and trabeculation to minimize diffusion distance. In the subsequent period there is a failure in organization of vascular plexus into normal vasculature, resulting in thin compact myocardium that likely leads to heart failure and embryonic death.

Characterization of epidermal neural crest stem cell (EPI-NCSC) grafts in the lesioned spinal cord.

We have characterized in the contusion-lesioned murine spinal cord the behavior of acutely implanted epidermal neural crest stem cells (EPI-NCSC, formerly eNCSC). EPI-NCSC, a novel type of multipotent adult stem cell, are remnants of the embryonic neural crest. They reside in the bulge of hair follicles and have the ability to differentiate into all major neural crest derivatives (Sieber-Blum, M., Grim, M., Hu, Y.F., Szeder, V., 2004. Pluripotent neural crest stem cells in the adult hair follicle. Dev. Dyn. 231, 258-269). Grafted EPI-NCSC survived, integrated, and intermingled with host neurites in the lesioned spinal cord. EPI-NCSC were non-migratory. They did not proliferate and did not form tumors. Significant subsets expressed neuron-specific beta-III tubulin, the GABAergic marker glutamate decarboxylase 67 (GAD67), the oligodendrocyte marker, RIP, or myelin basic protein (MBP). Close physical association of non-neuronal EPI-NCSC with host neurites was observed. Glial fibrillary acidic protein (GFAP) immunofluorescence was not detected. Collectively, our data indicate that intraspinal EPI-NCSC demonstrate several desirable characteristics that may include local neural replacement and re-myelination.

A dual fate of the hindlimb muscle mass: cloacal/perineal musculature develops from leg muscle cells.

The cloaca serves as a common opening to the urinary and digestive systems. In most mammals, the cloaca is present only during embryogenesis, after which it undergoes a series of septation events leading to the formation of the anal canal and parts of the urogenital tract. During embryogenesis it is surrounded by skeletal muscle. The origin and the mechanisms regulating the development of these muscles have never been determined. Here, we show that the cloacal muscles of the chick originate from somites 30-34, which overlap the domain that gives rise to leg muscles (somites 26-33). Using molecular and cell labelling protocols, we have determined the aetiology of cloacal muscles. Surprisingly, we found that chick cloacal myoblasts first migrate into the developing leg bud and then extend out of the ventral muscle mass towards the cloacal tubercle. The development of homologous cloacal/perineal muscles was also examined in the mouse. Concordant with the results in birds, we found that perineal muscles in mammals also develop from the ventral muscle mass of the hindlimb. We provide genetic evidence that the perineal muscles are migratory, like limb muscles, by showing that they are absent in metd/d mutants. Using experimental embryological procedures (in chick) and genetic models (in chick and mouse), we show that the development of the cloacal musculature is dependent on proximal leg field formation. Thus, we have discovered a novel developmental mechanism in vertebrates whereby muscle cells first migrate from axially located somites to the pelvic limb, then extend towards the midline and only then differentiate into the single cloacal/perineal muscles.

The adult hair follicle: cradle for pluripotent neural crest stem cells.

This review focuses on the recent identification of two novel neural crest-derived cells in the adult mammalian hair follicle, pluripotent stem cells, and Merkel cells. Wnt1-cre/R26R compound transgenic mice, which in the periphery express beta-galactosidase in a neural crest-specific manner, were used to trace neural crest cells. Neural crest cells invade the facial epidermis as early as embryonic day 9.5. Neural crest-derived cells are present along the entire extent of the whisker follicle. This includes the bulge area, an epidermal niche for keratinocyte stem cells, as well as the matrix at the base of the hair follicle. We have determined by in vitro clonal analysis that the bulge area of the adult whisker follicle contains pluripotent neural crest stem cells. In culture, beta-galactosidase-positive cells emigrate from bulge explants, identifying them as neural crest-derived cells. When these cells are resuspended and grown in clonal culture, they give rise to colonies that contain multiple differentiated cell types, including neurons, Schwann cells, smooth muscle cells, pigment cells, chondrocytes, and possibly other types of cells. This result provides evidence for the pluripotentiality of the clone-forming cell. Serial cloning showed that bulge-derived neural crest cells undergo self-renewal, which identifies them as stem cells. Pluripotent neural crest cells are also localized in the back skin hair of adult mice. The bulge area of the whisker follicle is surrounded by numerous Merkel cells, which together with innervating nerve endings form slowly adapting mechanoreceptors that transduce steady skin indentation. Merkel cells express beta-galactosidase in double transgenic mice, which confirms their neural crest origin. Taken together, our data indicate that the epidermis of the adult hair follicle contains pluripotent neural crest stem cells, termed epidermal neural crest stem cells (eNCSCs), and one newly identified neural crest derivative, the Merkel cell. The intrinsic high degree of plasticity of eNCSCs and the fact that they are easily accessible in the skin make them attractive candidates for diverse autologous cell therapy strategies.

Neurotrophin-3 signaling in mammalian Merkel cell development.

Merkel cells are sensory cells of neural crest origin. Because little is known about the mechanisms that direct their differentiation, we have investigated the potential role of a candidate regulatory factor, neurotrophin-3 (NT-3). At embryonic day 16.5 (E 16.5), neither NT-3 nor its primary receptors, TrkC and p75NTR are expressed by Merkel cells in the murine whisker. At the time of birth, however, Merkel cells are immunoreactive for NT-3, TrkC and p75NTR. In TrkC null and NT-3 null mice, Merkel cells differentiate initially, but undergo apoptosis perinatally. These results show that NT-3 signaling is not required for the differentiation of Merkel cells, but that it is essential for their postnatal survival.