Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Vanderbilt University Medical Center

Vanderbilt University

Skin-derived precursors (SKPs) are an attractive stem cell model for cell-based therapies. SKPs can be readily generated from embryonic and adult mice and adult humans, exhibit a high degree of multipotency, and have the potential to serve as a patient autologous stem cell. The advancement of these cells toward therapeutic use depends on the ability to control precisely the self-renewal and differentiation of SKPs. Here we show that two well-known stem cell factors, Foxd3 and Sox2, are critical regulators of the stem cell properties of SKPs. Deletion of Foxd3 completely abolishes the sphere-forming potential of these cells. In the absence of Sox2, SKP spheres can be formed, but with reduced size and frequency. Our results provide entry points into the gene regulatory networks dictating SKP behavior, and pave the way for future studies on a therapeutically relevant stem cell.

Ground-state transcriptional requirements for skin-derived precursors.

Understanding when and how multipotent progenitors segregate into diverse fates is a key question during embryonic development. The neural crest (NC) is an exemplary model system with which to investigate the dynamics of progenitor cell specification, as it generates a multitude of derivatives. Based on 'in ovo' lineage analysis, we previously suggested an early fate restriction of premigratory trunk NC to generate neural versus melanogenic fates, yet the timing of fate segregation and the underlying mechanisms remained unknown. Analysis of progenitors expressing a Foxd3 reporter reveals that prospective melanoblasts downregulate Foxd3 and have already segregated from neural lineages before emigration. When this downregulation is prevented, late-emigrating avian precursors fail to upregulate the melanogenic markers Mitf and MC/1 and the guidance receptor Ednrb2, generating instead glial cells that express P0 and Fabp. In this context, Foxd3 lies downstream of Snail2 and Sox9, constituting a minimal network upstream of Mitf and Ednrb2 to link melanogenic specification with migration. Consistent with the gain-of-function data in avians, loss of Foxd3 function in mouse NC results in ectopic melanogenesis in the dorsal tube and sensory ganglia. Altogether, Foxd3 is part of a dynamically expressed gene network that is necessary and sufficient to regulate fate decisions in premigratory NC. Their timely downregulation in the dorsal neural tube is thus necessary for the switch between neural and melanocytic phases of NC development.

A dynamic code of dorsal neural tube genes regulates the segregation between neurogenic and melanogenic neural crest cells.

Comprendre quand et comment s&eacute;parer les cellules souches multipotentes dans divers destins est une question essentielle au cours du d&eacute;veloppement embryonnaire. La cr&ecirc;te neurale (NC) est un syst&egrave;me de mod&egrave;le exemplaire permettant d&#39;&eacute;tudier la dynamique de la sp&eacute;cification des cellules prog&eacute;nitrices, car il g&eacute;n&egrave;re une multitude de produits d&eacute;riv&eacute;s. Bas&eacute; sur &laquo; in ovo &raquo; analyse de la lign&eacute;e, nous avons pr&eacute;c&eacute;demment sugg&eacute;r&eacute; une restriction sort d&eacute;but du tronc pr&eacute;migratoire NC pour g&eacute;n&eacute;rer neurale par rapport aux destins de la m&eacute;lanogen&egrave;se, mais le timing de la s&eacute;gr&eacute;gation de sort et les m&eacute;canismes sous-jacents sont rest&eacute;s inconnu. L&#39;analyse des cellules souches exprimant un journaliste Foxd3 r&eacute;v&egrave;le que prospective m&eacute;lanoblastes downregulate Foxd3 et ont d&eacute;j&agrave; s&eacute;par&eacute;s des lign&eacute;es neuronales avant l&#39;&eacute;migration. Lorsque cette diminution de l&#39;expression est emp&ecirc;ch&eacute;e, MC/1 et le r&eacute;cepteur d&#39;orientation Ednrb2, g&eacute;n&eacute;rant plut&ocirc;t gliales cellules que P0 expresse et Fabp et fin-&eacute;migrer aviaires pr&eacute;curseurs ne parviennent pas &agrave; r&eacute;guler positivement les marqueurs de la m&eacute;lanogen&egrave;se Mitf. Dans ce contexte, Foxd3 se trouve en aval de Snail2 et Sox9, constituant un r&eacute;seau minimum en amont de Mitf et Ednrb2 de lier la sp&eacute;cification de la m&eacute;lanogen&egrave;se et migration. Compatible avec les donn&eacute;es de gain de fonction dans avians, perte de fonction de Foxd3 &agrave; souris NC entra&icirc;ne m&eacute;lanog&eacute;n&egrave;se ectopique dans le goulot de la dorsale et les ganglions sensitifs. Au total, Foxd3 fait partie d&#39;un r&eacute;seau de g&egrave;ne exprim&eacute; de mani&egrave;re dynamique qui est n&eacute;cessaire et suffisant pour r&eacute;glementer les d&eacute;cisions sort en Caroline du Nord pr&eacute;-migratoires. Il faut donc leur r&eacute;gulation en temps opportun dans le tube neural dorsal pour le commutateur entre neurones et m&eacute;lanocytaires phases du d&eacute;veloppement de la NC.

Un code dynamique des g&egrave;nes du tube neural dorsal r&eacute;glemente la s&eacute;gr&eacute;gation entre neurog&egrave;ne et cellules neurales de cr&ecirc;te m&eacute;lanogen&egrave;se.

Capire quando e come progenitori multipotenti segregano in diversi destini &egrave; una questione chiave durante lo sviluppo embrionale. La cresta neurale (NC) &egrave; un sistema di modello esemplare con cui indagare le dinamiche della specifica delle cellule progenitrici, come esso genera una moltitudine di strumenti derivati. Basato su &#39;in ovo&#39; analisi di lineage, abbiamo precedentemente suggerito una restrizione destino precoce del tronco premigratory NC per generare neurale versus melanogenetico destini, ma i tempi della segregazione del destino e i meccanismi sottostanti &egrave; rimasto sconosciuto. Analisi dei progenitori esprimendo un reporter Foxd3 rivela che sottoregola prospettico melanoblasts Foxd3 e hanno gi&agrave; segregata da lignaggi neurale prima emigrazione. Quando &egrave; impedita questa downregulation, precursori aviario tardo-emigrare non riescono a potenziamento i marcatori melanogenetico Mitf e MC/1 e il recettore di orientamento Ednrb2, generando invece glial cellule quello espresso P0 e Fabp. In questo contesto, Foxd3 si trova a valle di Snail2 e Sox9, che costituiscono una rete minima a monte di Mitf ed Ednrb2 per collegare melanogenetico specifica con migrazione. Coerenti con i dati di guadagno di funzione in aviari, perdita della funzione Foxd3 in mouse NC risultati nella melanogenesi ectopica nel tubo dorsale e gangli sensoriali. Complessivamente, Foxd3 &egrave; parte di una rete di gene dinamicamente espressa che &egrave; necessario e sufficiente per regolamentare le decisioni del destino in premigratory NC. Loro tempestiva downregulation del tubo neurale dorsale &egrave; quindi necessaria per il passaggio tra fasi di sviluppo NC neurale e melanocitario.

Un codice dinamico del tubo neurale dorsale geni regola la segregazione tra neurogena e melanogenetico cellule della cresta neurale.

Skin melanocytes arise from two sources: either directly from neural crest progenitors or indirectly from neural crest-derived Schwann cell precursors after colonization of peripheral nerves. The relationship between these two melanocyte populations and the factors controlling their specification remains poorly understood. Direct lineage tracing reveals that neural crest and Schwann cell progenitor-derived melanocytes are differentially restricted to the epaxial and hypaxial body domains, respectively. Furthermore, although both populations are initially part of the Foxd3 lineage, hypaxial melanocytes lose Foxd3 at late stages upon separation from the nerve, whereas we recently found that epaxial melanocytes segregate earlier from Foxd3-positive neural progenitors while still residing in the dorsal neural tube. Gain- and loss-of-function experiments in avians and mice, respectively, reveal that Foxd3 is both sufficient and necessary for regulating the balance between melanocyte and Schwann cell development. In addition, Foxd3 is also sufficient to regulate the switch between neuronal and glial fates in sensory ganglia. Together, we propose that differential fate acquisition of neural crest-derived cells depends on their progressive segregation from the Foxd3-positive lineage.

Neural crest and Schwann cell progenitor-derived melanocytes are two spatially segregated populations similarly regulated by Foxd3.

Directional cell movement is universally required for tissue morphogenesis. Although it is known that cell/matrix interactions are essential for directional movement in heart development, the mechanisms governing these interactions require elucidation. Here we demonstrate that a novel protein/protein interaction between blood vessel epicardial substance (Bves) and N-myc downstream regulated gene 4 (NDRG4) is critical for regulation of epicardial cell directional movement, as disruption of this interaction randomizes migratory patterns. Our studies show that Bves/NDRG4 interaction is required for trafficking of internalized fibronectin through the "autocrine extracellular matrix (ECM) deposition" fibronectin recycling pathway. Of importance, we demonstrate that Bves/NDRG4-mediated fibronectin recycling is indeed essential for epicardial cell directional movement, thus linking these two cell processes. Finally, total internal reflectance fluorescence microscopy shows that Bves/NDRG4 interaction is required for fusion of recycling endosomes with the basal cell surface, providing a molecular mechanism of motility substrate delivery that regulates cell directional movement. This is the first evidence of a molecular function for Bves and NDRG4 proteins within broader subcellular trafficking paradigms. These data identify novel regulators of a critical vesicle-docking step required for autocrine ECM deposition and explain how Bves facilitates cell-microenvironment interactions in the regulation of epicardial cell-directed movement.

Bves and NDRG4 regulate directional epicardial cell migration through autocrine extracellular matrix deposition.

Vascular smooth muscle cells (VSMCs) are derived from distinct embryonic origins. Vessels originating from differing smooth muscle cell populations have distinct vascular and pathological properties involving calcification, atherosclerosis, and structural defects such as aneurysm and coarctation. We hypothesized that domains within a single vessel, such as the aorta, vary in phenotype based on embryonic origin. Gene profiling and myographic analyses demonstrated that embryonic ascending and descending aortic domains exhibited distinct phenotypes. In vitro analyses demonstrated that VSMCs from each region were dissimilar in terms of cytoskeletal and migratory properties, and retention of different gene expression patterns. Using the same analysis, we found that these same two domains are indistinguishable in the adult vessel. Our data demonstrate that VSMCs from different embryonic origins are functionally distinct in the embryonic mouse, but converge to assume a common phenotype in the aorta of healthy adults. These findings have fundamental implications for aortic development, function and disease progression.

Embryonic domains of the aorta derived from diverse origins exhibit distinct properties that converge into a common phenotype in the adult.

Failure of the ductus arteriosus (DA) to close at birth can lead to serious complications. Conversely, certain profound congenital cardiac malformations require the DA to be patent until corrective surgery can be performed. In each instance, clinicians have a very limited repertoire of therapeutic options at their disposal - indomethacin or ibuprofen to close a patent DA (PDA) and prostaglandin E1 to maintain patency of the DA. Neither treatment is specific to the DA and both may have deleterious off-target effects. Therefore, more therapeutic options specifically targeted to the DA should be considered. We hypothesized the DA possesses a unique genetic signature that would set it apart from other vessels. A microarray was used to compare the genetic profiles of the murine DA and ascending aorta (AO). Over 4,000 genes were differentially expressed between these vessels including a subset of ion channel-related genes. Specifically, the alpha and beta subunits of large-conductance calcium-activated potassium (BKCa) channels are enriched in the DA. Gain- and loss-of-function studies showed inhibition of BKCa channels caused the DA to constrict, while activation caused DA relaxation even in the presence of O2. This study identifies subsets of genes that are enriched in the DA that may be used to develop DA-specific drugs. Ion channels that regulate DA tone, including BKCa channels, are promising targets. Specifically, BKCa channel agonists like NS1619 maintain DA patency even in the presence of O2 and may be clinically useful.

Elise R. Pfaltzgraff

Department of Cell and Developmental Biology,
Center for Stem Cell Biology,
Department of Cell and Developmental Biology, Center for Stem Cell Biology

Vanderbilt University Medical Center

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Elise R. Pfaltzgraff

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell and Developmental Biology,Center for Stem Cell Biology,Department of Cell and Developmental Biology, Center for Stem Cell Biology

Vanderbilt University Medical Center

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Department of Cell and Developmental Biology,
Center for Stem Cell Biology,
Department of Cell and Developmental Biology, Center for Stem Cell Biology