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Transmembrane potential of GlyCl-expressing instructor cells induces a neoplastic-like conversion of melanocytes via a serotonergic pathway.

Blackiston D, Adams DS, Lemire JM, Lobikin M, Levin M - Dis Model Mech (2010)

Bottom Line: Molecular-genetic depolarization of a sparse, widely distributed set of GlyCl-expressing cells non-cell-autonomously induces a neoplastic-like phenotype in melanocytes: they overproliferate, acquire an arborized cell shape and migrate inappropriately, colonizing numerous tissues in a metalloprotease-dependent fashion.A similar effect was observed in human melanocytes in culture.Depolarization of GlyCl-expressing cells induces these drastic changes in melanocyte behavior via a serotonin-transporter-dependent increase of extracellular serotonin (5-HT).

View Article: PubMed Central - PubMed

Affiliation: Center for Regenerative and Developmental Biology, Biology Department, 200 Boston Avenue, Suite 4600, Tufts University, Medford, MA 02155, USA.

ABSTRACT
Understanding the mechanisms that coordinate stem cell behavior within the host is a high priority for developmental biology, regenerative medicine and oncology. Endogenous ion currents and voltage gradients function alongside biochemical cues during pattern formation and tumor suppression, but it is not known whether bioelectrical signals are involved in the control of stem cell progeny in vivo. We studied Xenopus laevis neural crest, an embryonic stem cell population that gives rise to many cell types, including melanocytes, and contributes to the morphogenesis of the face, heart and other complex structures. To investigate how depolarization of transmembrane potential of cells in the neural crest's environment influences its function in vivo, we manipulated the activity of the native glycine receptor chloride channel (GlyCl). Molecular-genetic depolarization of a sparse, widely distributed set of GlyCl-expressing cells non-cell-autonomously induces a neoplastic-like phenotype in melanocytes: they overproliferate, acquire an arborized cell shape and migrate inappropriately, colonizing numerous tissues in a metalloprotease-dependent fashion. A similar effect was observed in human melanocytes in culture. Depolarization of GlyCl-expressing cells induces these drastic changes in melanocyte behavior via a serotonin-transporter-dependent increase of extracellular serotonin (5-HT). These data reveal GlyCl as a molecular marker of a sparse and heretofore unknown cell population with the ability to specifically instruct neural crest derivatives, suggest transmembrane potential as a tractable signaling modality by which somatic cells can control stem cell behavior at considerable distance, identify a new biophysical aspect of the environment that confers a neoplastic-like phenotype upon stem cell progeny, reveal a pre-neural role for serotonin and its transporter, and suggest a novel strategy for manipulating stem cell behavior.

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Related in: MedlinePlus

Early ivermectin exposure induces an increase in pigment cell proliferation. Embryos exposed to ivermectin from stages 10–24 (early) or 28–46 (late) both show darkening due to expansion of melanocytes. To determine whether there was also a corresponding increase in melanocyte number, photographs were taken of controls (A) and ivermectin-exposed (B) embryos after tricaine anesthetization, which contracts the pigment cells. The number of melanocytes in the eye field (red boxes) were then counted. Early exposed embryos showed a 1.5-fold increase in melanocyte number relative to controls (C), whereas no detectable difference was observed between late exposed embryos and controls. Error bars indicate one standard deviation; n=24 embryos for each treatment. Control embryos processed in in situ hybridization for the melanocyte marker Trp2 at stage 28 show the normal pattern of expression prior to the migration of melanocytes away from the dorsal neural tube (D). Ivermectin-treated embryos show precisely the same pattern (E) and exhibit no evidence of ectopic locations being converted into a melanocyte fate by the ivermectin treatment. Sectioning reveals that control (F) and ivermectin-treated (G) embryos have the same number of melanocytes at the neural tube, also ruling out local shifts of neural crest cells into the melanocyte lineage as the explanation for later hyperpigmentation. Red arrows indicate positive signal (melanocytes indicated by Trp2 expression), whereas white arrows indicate lack of signal. (H-I′) To directly analyze proliferation in melanocytes, embryos were stained for the melanocyte marker Trp2 using in situ hybridization to identify pigment cells, and were then sectioned and processed for immunohistochemistry with anti-H3B-P antibody. (H) Trp2 section in control embryos; (H′) corresponding signal of H3B-P stain in the same section. (I) Trp2 section in ivermectin-exposed embryos; (I′) corresponding signal of H3B-P stain in the same section. Overlays of the bright-field and fluorescent signals from the same section allowed quantification of the number of melanocytes that were in mitosis. At stage 28, there was no difference (P>0.2, n=6) between controls and ivermectin-treated embryos. By stage 35, there was a significant increase in the number of mitotic melanocytes in the ivermectin-treated embryos (P<0.009, n=6).
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f3-0040067: Early ivermectin exposure induces an increase in pigment cell proliferation. Embryos exposed to ivermectin from stages 10–24 (early) or 28–46 (late) both show darkening due to expansion of melanocytes. To determine whether there was also a corresponding increase in melanocyte number, photographs were taken of controls (A) and ivermectin-exposed (B) embryos after tricaine anesthetization, which contracts the pigment cells. The number of melanocytes in the eye field (red boxes) were then counted. Early exposed embryos showed a 1.5-fold increase in melanocyte number relative to controls (C), whereas no detectable difference was observed between late exposed embryos and controls. Error bars indicate one standard deviation; n=24 embryos for each treatment. Control embryos processed in in situ hybridization for the melanocyte marker Trp2 at stage 28 show the normal pattern of expression prior to the migration of melanocytes away from the dorsal neural tube (D). Ivermectin-treated embryos show precisely the same pattern (E) and exhibit no evidence of ectopic locations being converted into a melanocyte fate by the ivermectin treatment. Sectioning reveals that control (F) and ivermectin-treated (G) embryos have the same number of melanocytes at the neural tube, also ruling out local shifts of neural crest cells into the melanocyte lineage as the explanation for later hyperpigmentation. Red arrows indicate positive signal (melanocytes indicated by Trp2 expression), whereas white arrows indicate lack of signal. (H-I′) To directly analyze proliferation in melanocytes, embryos were stained for the melanocyte marker Trp2 using in situ hybridization to identify pigment cells, and were then sectioned and processed for immunohistochemistry with anti-H3B-P antibody. (H) Trp2 section in control embryos; (H′) corresponding signal of H3B-P stain in the same section. (I) Trp2 section in ivermectin-exposed embryos; (I′) corresponding signal of H3B-P stain in the same section. Overlays of the bright-field and fluorescent signals from the same section allowed quantification of the number of melanocytes that were in mitosis. At stage 28, there was no difference (P>0.2, n=6) between controls and ivermectin-treated embryos. By stage 35, there was a significant increase in the number of mitotic melanocytes in the ivermectin-treated embryos (P<0.009, n=6).

Mentions: We next investigated whether the hyperpigmentation effect involves increased numbers of melanocytes, in addition to ectopic migration and shape change. Exposed embryos were anesthetized and photographed; we then counted the number of melanocytes within a standard region defined by the eyes (Fig. 3A,B), and compared each treatment to age-matched control siblings. Embryos exposed to 10 μM ivermectin from stages 12–24 (gastrulation through to the completion of neurulation), washed three times and cultured in plain 0.1× MMR, show a 1.5-fold increase in melanocyte number compared with controls (Fig. 3C; ANOVA Tukey post-hoc, P<0.05). These results were not confined to the eye field: melanocyte counts in the tip of the tail also showed a significant 1.5-fold increase when exposed to ivermectin throughout development (Student’s t-test, t=6.069, P≤0.001). Importantly, we detected no increase in total melanin content above that explained by the increase in cell number (absorption at 414 nm: control=0.0534, ivermectin-treated=0.0656, n=5 embryos per treatment, repeated five times, 1.2-fold difference); because the increase in melanocyte number (1.5-fold) is bigger than the increase in melanin content, each melanocyte is actually less pigmented in ivermectin-exposed tadpoles, ruling out higher pigment synthesis as a contributing factor to the hyperpigmented appearance.


Transmembrane potential of GlyCl-expressing instructor cells induces a neoplastic-like conversion of melanocytes via a serotonergic pathway.

Blackiston D, Adams DS, Lemire JM, Lobikin M, Levin M - Dis Model Mech (2010)

Early ivermectin exposure induces an increase in pigment cell proliferation. Embryos exposed to ivermectin from stages 10–24 (early) or 28–46 (late) both show darkening due to expansion of melanocytes. To determine whether there was also a corresponding increase in melanocyte number, photographs were taken of controls (A) and ivermectin-exposed (B) embryos after tricaine anesthetization, which contracts the pigment cells. The number of melanocytes in the eye field (red boxes) were then counted. Early exposed embryos showed a 1.5-fold increase in melanocyte number relative to controls (C), whereas no detectable difference was observed between late exposed embryos and controls. Error bars indicate one standard deviation; n=24 embryos for each treatment. Control embryos processed in in situ hybridization for the melanocyte marker Trp2 at stage 28 show the normal pattern of expression prior to the migration of melanocytes away from the dorsal neural tube (D). Ivermectin-treated embryos show precisely the same pattern (E) and exhibit no evidence of ectopic locations being converted into a melanocyte fate by the ivermectin treatment. Sectioning reveals that control (F) and ivermectin-treated (G) embryos have the same number of melanocytes at the neural tube, also ruling out local shifts of neural crest cells into the melanocyte lineage as the explanation for later hyperpigmentation. Red arrows indicate positive signal (melanocytes indicated by Trp2 expression), whereas white arrows indicate lack of signal. (H-I′) To directly analyze proliferation in melanocytes, embryos were stained for the melanocyte marker Trp2 using in situ hybridization to identify pigment cells, and were then sectioned and processed for immunohistochemistry with anti-H3B-P antibody. (H) Trp2 section in control embryos; (H′) corresponding signal of H3B-P stain in the same section. (I) Trp2 section in ivermectin-exposed embryos; (I′) corresponding signal of H3B-P stain in the same section. Overlays of the bright-field and fluorescent signals from the same section allowed quantification of the number of melanocytes that were in mitosis. At stage 28, there was no difference (P>0.2, n=6) between controls and ivermectin-treated embryos. By stage 35, there was a significant increase in the number of mitotic melanocytes in the ivermectin-treated embryos (P<0.009, n=6).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3008964&req=5

f3-0040067: Early ivermectin exposure induces an increase in pigment cell proliferation. Embryos exposed to ivermectin from stages 10–24 (early) or 28–46 (late) both show darkening due to expansion of melanocytes. To determine whether there was also a corresponding increase in melanocyte number, photographs were taken of controls (A) and ivermectin-exposed (B) embryos after tricaine anesthetization, which contracts the pigment cells. The number of melanocytes in the eye field (red boxes) were then counted. Early exposed embryos showed a 1.5-fold increase in melanocyte number relative to controls (C), whereas no detectable difference was observed between late exposed embryos and controls. Error bars indicate one standard deviation; n=24 embryos for each treatment. Control embryos processed in in situ hybridization for the melanocyte marker Trp2 at stage 28 show the normal pattern of expression prior to the migration of melanocytes away from the dorsal neural tube (D). Ivermectin-treated embryos show precisely the same pattern (E) and exhibit no evidence of ectopic locations being converted into a melanocyte fate by the ivermectin treatment. Sectioning reveals that control (F) and ivermectin-treated (G) embryos have the same number of melanocytes at the neural tube, also ruling out local shifts of neural crest cells into the melanocyte lineage as the explanation for later hyperpigmentation. Red arrows indicate positive signal (melanocytes indicated by Trp2 expression), whereas white arrows indicate lack of signal. (H-I′) To directly analyze proliferation in melanocytes, embryos were stained for the melanocyte marker Trp2 using in situ hybridization to identify pigment cells, and were then sectioned and processed for immunohistochemistry with anti-H3B-P antibody. (H) Trp2 section in control embryos; (H′) corresponding signal of H3B-P stain in the same section. (I) Trp2 section in ivermectin-exposed embryos; (I′) corresponding signal of H3B-P stain in the same section. Overlays of the bright-field and fluorescent signals from the same section allowed quantification of the number of melanocytes that were in mitosis. At stage 28, there was no difference (P>0.2, n=6) between controls and ivermectin-treated embryos. By stage 35, there was a significant increase in the number of mitotic melanocytes in the ivermectin-treated embryos (P<0.009, n=6).
Mentions: We next investigated whether the hyperpigmentation effect involves increased numbers of melanocytes, in addition to ectopic migration and shape change. Exposed embryos were anesthetized and photographed; we then counted the number of melanocytes within a standard region defined by the eyes (Fig. 3A,B), and compared each treatment to age-matched control siblings. Embryos exposed to 10 μM ivermectin from stages 12–24 (gastrulation through to the completion of neurulation), washed three times and cultured in plain 0.1× MMR, show a 1.5-fold increase in melanocyte number compared with controls (Fig. 3C; ANOVA Tukey post-hoc, P<0.05). These results were not confined to the eye field: melanocyte counts in the tip of the tail also showed a significant 1.5-fold increase when exposed to ivermectin throughout development (Student’s t-test, t=6.069, P≤0.001). Importantly, we detected no increase in total melanin content above that explained by the increase in cell number (absorption at 414 nm: control=0.0534, ivermectin-treated=0.0656, n=5 embryos per treatment, repeated five times, 1.2-fold difference); because the increase in melanocyte number (1.5-fold) is bigger than the increase in melanin content, each melanocyte is actually less pigmented in ivermectin-exposed tadpoles, ruling out higher pigment synthesis as a contributing factor to the hyperpigmented appearance.

Bottom Line: Molecular-genetic depolarization of a sparse, widely distributed set of GlyCl-expressing cells non-cell-autonomously induces a neoplastic-like phenotype in melanocytes: they overproliferate, acquire an arborized cell shape and migrate inappropriately, colonizing numerous tissues in a metalloprotease-dependent fashion.A similar effect was observed in human melanocytes in culture.Depolarization of GlyCl-expressing cells induces these drastic changes in melanocyte behavior via a serotonin-transporter-dependent increase of extracellular serotonin (5-HT).

View Article: PubMed Central - PubMed

Affiliation: Center for Regenerative and Developmental Biology, Biology Department, 200 Boston Avenue, Suite 4600, Tufts University, Medford, MA 02155, USA.

ABSTRACT
Understanding the mechanisms that coordinate stem cell behavior within the host is a high priority for developmental biology, regenerative medicine and oncology. Endogenous ion currents and voltage gradients function alongside biochemical cues during pattern formation and tumor suppression, but it is not known whether bioelectrical signals are involved in the control of stem cell progeny in vivo. We studied Xenopus laevis neural crest, an embryonic stem cell population that gives rise to many cell types, including melanocytes, and contributes to the morphogenesis of the face, heart and other complex structures. To investigate how depolarization of transmembrane potential of cells in the neural crest's environment influences its function in vivo, we manipulated the activity of the native glycine receptor chloride channel (GlyCl). Molecular-genetic depolarization of a sparse, widely distributed set of GlyCl-expressing cells non-cell-autonomously induces a neoplastic-like phenotype in melanocytes: they overproliferate, acquire an arborized cell shape and migrate inappropriately, colonizing numerous tissues in a metalloprotease-dependent fashion. A similar effect was observed in human melanocytes in culture. Depolarization of GlyCl-expressing cells induces these drastic changes in melanocyte behavior via a serotonin-transporter-dependent increase of extracellular serotonin (5-HT). These data reveal GlyCl as a molecular marker of a sparse and heretofore unknown cell population with the ability to specifically instruct neural crest derivatives, suggest transmembrane potential as a tractable signaling modality by which somatic cells can control stem cell behavior at considerable distance, identify a new biophysical aspect of the environment that confers a neoplastic-like phenotype upon stem cell progeny, reveal a pre-neural role for serotonin and its transporter, and suggest a novel strategy for manipulating stem cell behavior.

Show MeSH
Related in: MedlinePlus