N-cadherin prevents the premature differentiation of anterior heart field progenitors in the pharyngeal mesodermal microenvironment

The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes. The mechanisms by which these progenitors are able to expand within the PM microenvironment without premature differentiation remain largely unknown. Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM. CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM. Notably, the phenotype of N-cadherin deficiency could be partially rescued by activating Wnt signaling, suggesting a delicate functional interaction between the adhesion role of N-cadherin and Wnt signaling in the early PM microenvironment. This study suggests a new mechanism for the early renewal of AHF progenitors where N-cadherin provides additional adhesion for progenitor cells in the PM, thereby allowing Wnt paracrine signals to expand the cells without premature differentiation

Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells

Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human–mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1+ vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo

A dual role for Sox10 in the maintenance of the postnatal melanocyte lineage and the differentiation of melanocyte stem cell progenitors

During embryogenesis, the transcription factor, Sox10, drives the survival and differentiation of the melanocyte lineage. However, the role that Sox10 plays in postnatal melanocytes is not established. We show in vivo that melanocyte stem cells (McSCs) and more differentiated melanocytes express SOX10 but that McSCs remain undifferentiated. Sox10 knockout (Sox10(fl); Tg(Tyr::CreER)) results in loss of both McSCs and differentiated melanocytes, while overexpression of Sox10 (Tg(DctSox10)) causes premature differentiation and loss of McSCs, leading to hair graying. This suggests that levels of SOX10 are key to normal McSC function and Sox10 must be downregulated for McSC establishment and maintenance. We examined whether the mechanism of Tg(DctSox10) hair graying is through increased expression of Mitf, a target of SOX10, by asking if haploinsufficiency for Mitf (Mitf(vga9) ) can rescue hair graying in Tg(DctSox10) animals. Surprisingly, Mitf(vga9) does not mitigate but exacerbates Tg(DctSox10) hair graying suggesting that MITF participates in the negative regulation of Sox10 in McSCs. These observations demonstrate that while SOX10 is necessary to maintain the postnatal melanocyte lineage it is simultaneously prevented from driving differentiation in the McSCs. This data illustrates how tissue-specific stem cells can arise from lineage-specified precursors through the regulation of the very transcription factors important in defining that lineage

A Sox10 Expression Screen Identifies an Amino Acid Essential for Erbb3 Function

The neural crest (NC) is a population of embryonic stem cells that gives rise to numerous cell types, including the glia and neurons of the peripheral and enteric nervous systems and the melanocytes of the skin and hair. Mutations in genes and genetic pathways regulating NC development lead to a wide spectrum of human developmental disorders collectively called neurocristopathies. To identify molecular pathways regulating NC development and to understand how alterations in these processes lead to disease, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen utilizing a mouse model sensitized for NC defects, Sox10 LacZ/+. Out of 71 pedigrees analyzed, we identified and mapped four heritable loci, called modifier of Sox10 expression pattern 1–4 (msp1–4), which show altered NC patterning. In homozygous msp1 embryos, Sox10 LacZ expression is absent in cranial ganglia, cranial nerves, and the sympathetic chain; however, the development of other Sox10-expressing cells appears unaffected by the mutation. Linkage analysis, sequencing, and complementation testing confirmed that msp1 is a new allele of the receptor tyrosine kinase Erbb3, Erbb3 msp1, that carries a single amino acid substitution in the extracellular region of the protein. The ENU-induced mutation does not alter protein expression, however, it is sufficient to impair ERBB3 signaling such that the embryonic defects observed in msp1 resemble those of Erbb3 null alleles. Biochemical analysis of the mutant protein showed that ERBB3 is expressed on the cell surface, but its ligand-induced phosphorylation is dramatically reduced by the msp1 mutation. These findings highlight the importance o

A Sox10 expression screen identifies an amino acid essential for Erbb3 function.

The neural crest (NC) is a population of embryonic stem cells that gives rise to numerous cell types, including the glia and neurons of the peripheral and enteric nervous systems and the melanocytes of the skin and hair. Mutations in genes and genetic pathways regulating NC development lead to a wide spectrum of human developmental disorders collectively called neurocristopathies. To identify molecular pathways regulating NC development and to understand how alterations in these processes lead to disease, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen utilizing a mouse model sensitized for NC defects, Sox10(LacZ/+). Out of 71 pedigrees analyzed, we identified and mapped four heritable loci, called modifier of Sox10 expression pattern 1-4 (msp1-4), which show altered NC patterning. In homozygous msp1 embryos, Sox10(LacZ) expression is absent in cranial ganglia, cranial nerves, and the sympathetic chain; however, the development of other Sox10-expressing cells appears unaffected by the mutation. Linkage analysis, sequencing, and complementation testing confirmed that msp1 is a new allele of the receptor tyrosine kinase Erbb3, Erbb3(msp1), that carries a single amino acid substitution in the extracellular region of the protein. The ENU-induced mutation does not alter protein expression, however, it is sufficient to impair ERBB3 signaling such that the embryonic defects observed in msp1 resemble those of Erbb3 null alleles. Biochemical analysis of the mutant protein showed that ERBB3 is expressed on the cell surface, but its ligand-induced phosphorylation is dramatically reduced by the msp1 mutation. These findings highlight the importance of the mutated residue for ERBB3 receptor function and activation. This study underscores the utility of using an ENU mutagenesis to identify genetic pathways regulating NC development and to dissect the roles of discrete protein domains, both of which contribute to a better understanding of gene function in a cellular and developmental setting

<i>Sox10</i> is required by bulb melanocytes postnatally.

<p>(A–B) <i>Sox10^fl/fl</i> (<i>fl/fl; +/+</i>) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (<i>fl/fl; Cre/+</i>) pups treated with TAM by IP injection to the lactating mother on P0–3 display variegated hypopigmentation on the belly and back and exhibit a white head spot upon the emergence of the morphogenetic coat (P10 shown here, n>5). (C) Number of PAX3⁺ melanocytes per hair bulb in skins harvested from these mice at P10 are significantly decreased in <i>Sox10^fl/fl; Tyr::CreERT2</i> animals compared to similarly-treated <i>Sox10^fl/fl</i> animals (*p = 0.002). (D–E) Adult <i>Sox10^fl/fl; Tyr::CreERT2</i> mice treated with TAM by IP injection on 0–3dpp exhibit white hairs within the plucked region upon hair regrowth that is not visible in similarly treated <i>Sox10^fl/fl</i> mice (brackets indicate plucked region, lower image is a magnification of plucked region). (F) Number of PAX3⁺ melanocytes per hair bulb in skins harvested from similarly-treated mice at 7dpp are significantly decreased in <i>Sox10^fl/fl; Tyr::CreERT2</i> animals compared to <i>Sox10^fl/fl</i> animals (*p = 0.001). (G–H) Fluorescent and corresponding brightfield images of hair bulbs from mice described in D–E. Arrows and arrowheads indicate PAX3⁺/SOX10⁺ and PAX3⁺/SOX10⁻ melanocytes, respectively. (I) Distribution of melanocytes double-labeled for PAX3 and SOX10 within pigmented (gray) and non-pigmented (white) hair bulbs in skins from <i>Sox10^fl/fl</i> (n = 3) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (n = 4) harvested on 7dpp from mice treated with TAM on 0–3dpp (*p<0.006).</p

<i>Sox10</i> is required by LPP melanocytes postnatally.

<p>(A) Number of KIT⁺ LPP melanocytes within hairs from <i>Sox10^fl/fl</i> (<i>fl/fl; +/+</i>) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (<i>fl/fl; Cre/+</i>) mice. P0–3/P10 indicates skins harvested from pups on P10 that were maintained by lactating mothers that were IP injected with TAM on P0–3. 0–3dpp/7dpp indicates skins harvested from adult mice on 7dpp after IP injections of TAM on 0–3dpp. (B) White hairs remain visible in adult <i>Sox10^fl/fl; Tyr::CreERT2</i> mice that were treated with TAM by IP injection on 0–3dpp, allowed for complete hair regeneration, replucked and allowed for a second round of hair regrowth (brackets indicate plucked/replucked region, lower image is a magnification of plucked region; mouse in 2E and 3B are the same, imaged prior to and after replucking). (C) Number of PAX3⁺ bulb melanocytes within hairs from <i>Sox10^fl/fl</i> and <i>Sox10^fl/fl; Tyr::CreERT2</i> mice treated as described in B but harvested on 7dpp after replucking (0–3dpp/7dpp repluck). (D) Distribution of melanocytes double-labeled for PAX3 and SOX10 within pigmented (gray) and non-pigmented (white) hair bulbs in skins from <i>Sox10^fl/fl</i> (n = 3) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (n = 3) mice treated as described in B but harvested on 7dpp after replucking (*p<0.002). (E) Persistent hair graying is visible in <i>Sox10^fl/fl; Tyr::CreERT2</i> mice treated with IP TAM for pulse of five days beginning at five weeks old and imaged at one and two years old.</p

LPP melanocytes are reduced in <i>Tg(DctSox10)</i> homozygotes during hair morphogenesis.

<p>(A) Brightfield images of hairs in <i>Tg(DctSox10)</i> and <i>+/+</i> littermates at P2. (B) Number of DCT⁺ melanocytes within the LPP of hairs at P2 (stage 6 hairs) and P7/8. At both time points, LPP melanocytes per hair are reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> compared to <i>Tg(DctSox10)/+</i> and <i>+/+</i> mice (*p<0.017). (C, D) Quantitative immunohistochemical analysis of stage 6 hairs from P2 skins for DCT and TRP1, or DCT and KIT. The population of DCT⁺/TRP1⁺ cells is significantly reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> in comparison to <i>Tg(DctSox10)/+</i> and +/+ mice (*p<0.008). <i>Tg(DctSox10)</i> also causes a switch in KIT intensity from KIT^hi in wild type to KIT^low in <i>Tg(DctSox10)</i> animals (*KIT^lo and **KIT^hi comparisons made between +/+ and <i>Tg(DctSox10)/+</i> or <i>+/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i>; p<0.005).</p

<i>Tg(DctSox10)</i> results in congenital white spotting and premature hair graying.

<p>(A, B) Ventral and dorsal views demonstrating variable hypopigmentation in <i>Tg(DctSox10)/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i> mice during hair morphogenesis and adult hair cycling. (C) Frequency of pigmented (pig+) and non-pigmented (pig−) anagen III/IV (7dpp) hairs that contain (DCT+ LPP cells) or do not contain (no LPP cells) LPP melanocytes within <i>Tg(DctSox10)</i> or <i>+/+</i> mice. The ages of mice analyzed ranged between 9–22 weeks at harvest. Significance determined by chi-square analysis (p<<0.0001) and evaluation of standardized residuals (*, z = −8.84; **, z = 12.24).</p