Milos Grim

Pluripotent Neural Crest Stem Cells in the Adult Hair Follicle

We report the presence of pluripotent neural crest stem cells in the adult mammalian hair follicle. Numerous neural crest cells reside in the outer root sheath from the bulge to the matrix at the base of the follicle. Bulge explants from adult mouse whisker follicles yield migratory neural crest cells, which in clonal culture form colonies consisting of over a thousand cells. Clones contain neurons, smooth muscle cells, rare Schwann cells and melanocytes, demonstrating pluripotency of the clone‐forming cell. Targeted differentiation into Schwann cells and chondrocytes was achieved with neuregulin‐1 and bone morphogenetic protein‐2, respectively. Serial cloning in vitro demonstrated self‐renewal capability. Together, the data show that the adult mouse whisker follicle contains pluripotent neural crest stem cells, termed epidermal neural crest cells (eNCSC). eNCSC are promising candidates for diverse cell therapy paradigms because of their high degree of inherent plasticity and due to their easy accessibility in the skin. Developmental Dynamics 231:258–269, 2004.

The Splotch mutation interferes with muscle development in the limbs

SummaryHomozygosity for the Splotch mutation causes neural tube and neural crest defects in mice. It has been demonstrated that Splotch mutant mice carry mutations in the homeodomain of the Pax-3 gene. Pax-3 is expressed in the neural tube, some neural crest derivatives, the mesenchyme of the limb bud and the somites. We have examined the development of the somite-derived skeletal muscles in homozygotes carrying the Splotch (Sp1H) mutation. Our results suggest that the Splotch mutation affects the development of skeletal muscles in a region-specific way: 1. The expression of the CMZ transgene in homozygotes reveals a disorganisation of the dermomyotome in whole stained embryos. 2. The axial musculature is reduced in size along a rostro-caudal gradient. 3. The muscle anlagen in the limbs develop much more slowly. Muscles of the head and the ventral body wall are normally developed in the mutant on day 13.5 of gestation. Recently, it has been shown that the myogenic precursors of the limbs are derived from the lateral half of the somite. The specific disturbance of muscle development in the limbs of Splotch mutants thus suggests a role for Pax-3 in the organisation of the somite, the production of trophic factors in the limb mesenchyme or an alteration of myogenic and mesenchymal cells.

Melanoma cells influence the differentiation pattern of human epidermal keratinocytes

BackgroundNodular 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).MethodsComparative 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.ResultsEpidermis 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.ConclusionWe 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.

Local signalling in dermomyotomal cell type specification

SummaryThe development and differentiation of the avian myotome was studied after removal of the neural tube, including neural crest, and after replacement of dorsal half-somites by ventral half-somites. Results show that in the absence of neural tissue myoblast differentiation within the somites does not take place. Ventral halfsomites are able to undergo muscle differentiation if they were grafted in place of dorsal half somites. It is suggested that local signals must be responsible for the dorsalisation of the newly formed somite including myoblast differentiation. Neural crest cells are discussed as possible sources of these signals.

Neural crest origin of mammalian Merkel cells

Here, we provide evidence for the neural crest origin of mammalian Merkel cells. Together with nerve terminals, Merkel cells form slowly adapting cutaneous mechanoreceptors that transduce steady indentation in hairy and glabrous skin. We have determined the ontogenetic origin of Merkel cells in Wnt1-cre/R26R compound transgenic mice, in which neural crest cells are marked indelibly. Merkel cells in whiskers and interfollicular locations express the transgene, beta-galactosidase, identifying them as neural crest descendants. We thus conclude that murine Merkel cells originate from the neural crest.

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.

Experimental hypoxia and embryonic angiogenesis

We examined the role of hypoxia and HIF factors in embryonic angiogenesis and correlated the degree of hypoxia with the level of HIF and VEGF expression and blood vessel formation. Quail eggs were incubated in normoxic and hypoxic (16% O2) conditions. Tissue hypoxia marker, pimonidazol hydrochloride, was applied in vivo for 1 hr and detected in sections with Hypoxyprobe‐1 Ab. VEGF and HIF expression was detected by in situ hybridization. HIF‐1α protein was detected in sections and by Western blot. Endothelial cells were visualized with QH‐1 antibody. Hypoxic regions were detected even in normoxic control embryos, mainly in brain, neural tube, branchial arches, limb primordia, and mesonephros. The expression patterns of HIF‐1α and HIF‐1β factors followed, in general, the Hypoxyprobe‐1 marked regions. HIF‐2α was predominantly expressed in endothelial cells. Diffuse VEGF expression was detected in hypoxic areas of neural tube, myocardium, digestive tube, and most prominently in mesonephros. Growing capillaries were directed to areas of VEGF positivity. Hypoxic regions in hypoxic embryos were larger and stained more intensely. VEGF and HIF‐1 factors were proportionately elevated in Hypoxyprobe‐1 marked regions without being expressed at new sites and were followed by increased angiogenesis. Our results demonstrate that normal embryonic vascular development involves the HIF‐VEGF regulatory cascade. Experimentally increasing the level of hypoxia to a moderate level resulted in over‐expression of HIF‐1 factors and VEGF followed by an increase in the density of developing vessels. These data indicate that embryonic angiogenesis is responsive to environmental oxygen tension and, therefore, is not entirely genetically controlled. Developmental Dynamics 235:723–733, 2006.

Origin of spinal cord meninges, sheaths of peripheral nerves, and cutaneous receptors including Merkel cells

SummaryThe origin of cells covering the nervous system and the cutaneous receptors was studied using the quail-chick marking technique and light and electron microscopy. In the first experimental series the brachial neural tube of the quail was grafted in place of a corresponding neural tube segment of the chick embryo at HH-stages 10 to 14. In the second series the leg bud of quail embryos at HH-stages 18–20 was grafted in place of the leg bud of the chick embryos of the same stages and vice versa. It was found that all meningeal layers of the spinal cord, the perineurium and the endoneurium of peripheral nerves, as well as the capsular and inner space cells of Herbst sensory corpuscles, develop from the local mesenchymal cells. Schwann cells and cells of the inner core of sensory corpuscles are of neural crest origin. The precursors of Merkel cells migrate similarly to the Schwann cells into the limb bud where they later differentiate. This means that in addition to the Schwann cells and the melanocytes a further neural crest-derived subpopulation of cells enters the limb.

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.

Transcription factor c-Myb is involved in the regulation of the epithelial-mesenchymal transition in the avian neural crest

Abstract.Multipotential neural crest cells (NCCs) originate by an epithelial-mesenchymal transition (EMT) during vertebrate embryogenesis. We show for the first time that the key hematopoietic factor c-Myb is synthesized in early chick embryos including the neural tissue and participates in the regulation of the trunk NCCs. A reduction of endogenous c-Myb protein both in tissue explants in vitro and in embryos in ovo, prevented the formation of migratory NCCs. A moderate over-expression of c-myb in naive intermediate neural plates triggered the EMT and NCC migration probably through cooperation with BMP4 signaling because (i) BMP4 activated c-myb expression, (ii) elevated c-Myb caused accumulation of transcripts of the BMP4 target genes msx1 and slug, and (iii) the reduction of c-Myb prevented the BMP4-induced formation of NCCs. The data show that in chicken embryos, the c-myb gene is expressed prior to the onset of hematopoiesis and participates in the formation and migration of the trunk neural crest.