isolation-culture-neural-crest-cells-from-embryonic-murine-neural

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 ...

Klf4 (formerly GKLF) is a zinc-finger transcription factor expressed in the epithelia of the skin, lungs, gastrointestinal tract and several other organs. In vitro studies have suggested that Klf4 plays an important role in cell proliferation and/or differentiation. Mice homozygous for a null mutation in Klf4 die within 15 hours of birth and show selective perturbation of late-stage differentiation structures in the epidermis, but the function of Klf4 in the gastrointestinal tract has not been investigated. To address this issue, we have generated Klf4(-/-) mice by homologous recombination in embryonic stem cells. In this study, we provide the first in vivo evidence that Klf4 is a goblet cell-specific differentiation factor in the colon. Klf4(-/-) mice exhibit normal cell proliferation and cell death rates in the colon on postnatal day 1. However, Klf4(-/-) mice demonstrate a 90% decrease in the number of goblet cells in the colon, show abnormal expression of the goblet cell-specific marker Muc2 by in situ hybridization, have abnormal staining of the colonic epithelium with Alcian Blue for acidic mucins, and lack normal goblet cell morphology by ultrastructural analysis. All other epithelial cell types are present in the colon of Klf4(-/-) mice. In summary, Klf4 plays a crucial role in colonic epithelial cell differentiation in vivo.

The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon.

A novel population of tissue-resident endothelial precursors (TEPs) was isolated from small blood vessels in dermal, adipose, and skeletal muscle of mouse based on their ability to be grown as spheres. Cellular and molecular analyses of these cells revealed that they were highly related regardless of the tissue of origin and distinct from embryonic neural stem cells. Notably, TEPs did not express hematopoietic markers, but they expressed numerous characteristics of angiogenic precursors and their differentiated progeny, such as CD34, Flk-1, Tie-1, CD31, and vascular endothelial cadherin (VE-cadherin). TEPs readily differentiated into endothelial cells in newly formed vascular networks following transplantation into regenerating skeletal muscle. Taken together, these experiments suggest that TEPs represent a novel class of endothelial precursors that are closely associated with small blood vessels in muscle, adipose, and dermal tissue. This finding is of particular interest since it could bring new insight in cancer angiogenesis and collateral blood vessels developed following ischemia. Disclosure of potential conflicts of interest is found at the end of this article.

Resident endothelial precursors in muscle, adipose, and dermis contribute to postnatal vasculogenesis.

Conditional gene targeting has been extensively used for in vivo analysis of gene function in Î²-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate Î²-cell- or pancreas-specific recombination, also drive Cre expression in the brain.

Conditional gene targeting in mouse pancreatic ÃŸ-Cells: analysis of ectopic Cre transgene expression in the brain.

Bedingte Gene targeting wurde ausgiebig f&uuml;r in-vivo-Analyse der Genfunktion in der &beta;-Zelle Biologie verwendet. Ziel dieser Studie war es zu pr&uuml;fen, ob die Maus transgene Cre Linien, verwendet, um die &beta;-Zelle oder Pankreas-spezifischen Rekombination, vermitteln auch Cre Ausdruck im Gehirn fahren.

Bedingte Gene targeting in Maus pankreatischen &szlig;-Zellen: Analyse der ektopische Cre Transgenexpression im Gehirn.

Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of insulin-expressing cells and insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic insulin granules and the presence of abnormal granules in insulin-expressing cells from mutant embryos. Together, these data demonstrate that the neural crest is a critical regulator of beta cell development on two levels: by negatively regulating beta cell proliferation and by promoting beta cell maturation.

Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation.

Interaktionen zwischen Zellen aus dem Ektoderm und Mesoderm beeinflussen die Entwicklung der Bauchspeicheldr&uuml;se endodermally abgeleitet. W&auml;hrend viel bekannt ist, &uuml;ber wie Mesoderm Pankreas Entwicklung reguliert, ist relativ wenig verstanden, &uuml;ber wie und wann der Neuralleiste ectodermally abgeleiteten Pankreas Entwicklung und insbesondere Beta-Zelle Reifung regelt. Eine fr&uuml;here Studie gezeigt, dass die Signale aus der Neuralleiste regulieren Beta Zellproliferation und letztlich Beta cell Masse. Hier erweitern wir diese Arbeit zu beschreiben Timing der Neuralleiste-Ankunft der entwickelnden Pankreas Knospe und erweitern unser Wissen &uuml;ber die nicht-Zelle autonome Rolle f&uuml;r Neuralleiste Derivate in den Prozess der Reifung der Beta-Zelle. Wir bewiesen, dass murinen Neuralleiste der Pankreas Mesenchym zwischen 26 und 27 Somiten Stadien (ca. 10,0 Dpc) eingegeben und mit Pankreas Vorl&auml;uferzellen vermischt wurde, wie das Epithel in den umliegenden Mesenchym verzweigt. Verwenden eine L&ouml;schung der Neuralleiste-spezifische des Transkriptionsfaktors Forkhead Foxd3, ablated wir Neuralleistenzellen, die in der Bauchspeicheldr&uuml;se Urzeit migrieren. Einklang mit fr&uuml;heren Daten in das Fehlen von Foxd3, und daher das Fehlen der Neuralleistenzellen, Proliferation von Zellen Insulin-Ausdr&uuml;cken und Insulin-positiven Bereich werden erh&ouml;ht. Analyse der Genexpression von endokrinen Zellen in Ermangelung der Neuralleiste gezeigt, dass, obwohl die Anzahl der Insulin-Ausdruck Zellen erh&ouml;ht wurde, Beta-Zelle Reifung erheblich beeintr&auml;chtigt wurde. Verminderte MafA und Pdx1 Ausdruck illustriert den Mangel in der Beta-Zelle Reifung; Wir entdeckten, dass ohne Neuralleiste, gab es eine Verringerung des Anteils der Insulin-positiven Zellen, die Glut2 co-expressed und Pdx1 im Vergleich zu Kontrollen. Dar&uuml;ber hinaus verringert &Uuml;bertragung Elektronenmikroskopie Analysen ergab Zahlen von charakteristischen Insulin-Granulaten und das Vorhandensein von anormale K&ouml;rnchen in Insulin-Ausdruck Zellen von mutant Embryonen. Zusammen, diese Daten zeigen, dass der Neuralleiste ein wichtiger Regulator der Beta-Zell-Entwicklung auf zwei Ebenen: durch die Regelung negativ Beta Zellproliferation und durch F&ouml;rderung der Beta-Zelle Reifung.

Einfluss und den Zeitpunkt der Ankunft des murinen Neuralleiste Betazellen Entwicklung und Reifung.

The spinal cord contains a diverse array of physiologically distinct interneuron cell types that subserve specialized roles in somatosensory perception and motor control. The mechanisms that generate these specialized interneuronal cell types from multipotential spinal progenitors are not known. In this study, we describe a temporally regulated transcriptional program that controls the differentiation of Renshaw cells (RCs), an anatomically and functionally discrete spinal interneuron subtype. We show that the selective activation of the Onecut transcription factors Oc1 and Oc2 during the first wave of V1 interneuron neurogenesis is a key step in the RC differentiation program. The development of RCs is additionally dependent on the forkhead transcription factor Foxd3, which is more broadly expressed in postmitotic V1 interneurons. Our demonstration that RCs are born, and activate Oc1 and Oc2 expression, in a narrow temporal window leads us to posit that neuronal diversity in the developing spinal cord is established by the composite actions of early spatial and temporal determinants.

Renshaw cell interneuron specialization is controlled by a temporally restricted transcription factor program.

Das R&uuml;ckenmark enth&auml;lt eine Vielzahl von physiologisch unterschiedlichen Interneuron Zelltypen, die spezielle Rollen in somatosensorische Wahrnehmung und Motorsteuerung subserve. Die Mechanismen, die diesen speziellen interneuronales Zelltypen aus multipotenzielle spinal Vorl&auml;uferzellen zu generieren sind nicht bekannt. In dieser Studie beschreiben wir ein zeitlich geregelten transkriptionelle Programm, das die Differenzierung der Renshaw Zellen (RCs), eine anatomisch und funktional diskreter spinal Interneuron Untertyp steuert. Wir zeigen, dass die selektive Aktivierung der Transkriptionsfaktoren Onecut Oc1 und Oc2 bei der ersten Welle der V1 Interneuron Neurogenese ein wichtiger Schritt in der RC Differenzierung-Programm ist. Die Entwicklung der RCs ist zus&auml;tzlich abh&auml;ngig von den Transkriptionsfaktor Forkhead Foxd3, der allgemein in postmitotischen V1 Interneuronen angegeben wird. Unsere Demonstration, dass RCs sind geboren, und aktivieren Sie Oc1 und Oc2 Ausdruck, in einem engen zeitlichen Fenster uns f&uuml;hrt zu postulieren, dass neuronale Vielfalt im entwickelnden R&uuml;ckenmark wird durch die zusammengesetzten Aktionen der fr&uuml;hen r&auml;umliche und zeitliche Faktoren festgelegt.

Renshaw Zelle Interneuron Spezialisierung wird von einem zeitlich beschr&auml;nkten Transkription Faktor-Programm gesteuert.

The enteric nervous system (ENS) arises from the coordinated migration, expansion and differentiation of vagal and sacral neural crest progenitor cells. During development, vagal neural crest cells enter the foregut and migrate in a rostro-to-caudal direction, colonizing the entire gastrointestinal tract and generating the majority of the ENS. Sacral neural crest contributes to a subset of enteric ganglia in the hindgut, colonizing the colon in a caudal-to-rostral wave. During this process, enteric neural crest-derived progenitors (ENPs) self-renew and begin expressing markers of neural and glial lineages as they populate the intestine. Our earlier work demonstrated that the transcription factor Foxd3 is required early in neural crest-derived progenitors for self-renewal, multipotency and establishment of multiple neural crest-derived cells and structures including the ENS. Here, we describe Foxd3 expression within the fetal and postnatal intestine: Foxd3 was strongly expressed in ENPs as they colonize the gastrointestinal tract and was progressively restricted to enteric glial cells. Using a novel Ednrb-iCre transgene to delete Foxd3 after vagal neural crest cells migrate into the midgut, we demonstrated a late temporal requirement for Foxd3 during ENS development. Lineage labeling of Ednrb-iCre expressing cells in Foxd3 mutant embryos revealed a reduction of ENPs throughout the gut and loss of Ednrb-iCre lineage cells in the distal colon. Although mutant mice were viable, defects in patterning and distribution of ENPs were associated with reduced proliferation and severe reduction of glial cells derived from the Ednrb-iCre lineage. Analyses of ENS-lineage and differentiation in mutant embryos suggested activation of a compensatory population of Foxd3-positive ENPs that did not express the Ednrb-iCre transgene. Our findings highlight the crucial roles played by Foxd3 during ENS development including progenitor proliferation, neural patterning, and glial differentiation and may help delineate distinct molecular programs controlling vagal versus sacral neural crest development.

Enteric nervous system specific deletion of Foxd3 disrupts glial cell differentiation and activates compensatory enteric progenitors.

Enterische Nervensystem (ENS) ergibt sich aus der koordinierten Migration, Erweiterung und Differenzierung der Progenitorzellen der Neuralleiste Vagusnerven und sakral. W&auml;hrend der Entwicklung Vagusnerven Neuralleistenzellen geben Sie den Vorderdarm und Migrieren in Rostro-kaudaler Richtung kolonisieren den gesamten Magen-Darm-Trakt und die Generierung des Gro&szlig;teil der ENS Sakrale Neuralleiste tr&auml;gt zu einer Teilmenge der enterischen Ganglien in der Hinterdarm, kolonisieren den Doppelpunkt in einer kaudalen zu K&ouml;rperabschnitt Welle. W&auml;hrend dieses Vorgangs Enterische Neuralleiste abgeleitete Vorl&auml;uferzellen (ENPs) erneuern und Marker der neuronalen und Gliazellen &Uuml;bertragungslinien auszudr&uuml;cken, wie sie den Darm Auff&uuml;llen beginnen. Unsere fr&uuml;heren Arbeit gezeigt, dass der Transkriptionsfaktor Foxd3 fr&uuml;h in der Neuralleiste abgeleitete Vorl&auml;uferzellen f&uuml;r Selbsterneuerung, die Multipotenz und die Einrichtung mehrerer Neuralleiste gewonnenen Zellen und Strukturen einschlie&szlig;lich der ens erforderlich ist Hier beschreiben wir in den fetalen und postnatale Darm Foxd3 Ausdruck: Foxd3 war stark in ENPs ausgedr&uuml;ckt, da sie den Magen-Darm-Trakt kolonisieren und beschr&auml;nkte sich zunehmend auf enterischen Gliazellen. Mit einer neuartigen Ednrb-iCre Transgene Foxd3 l&ouml;schen, nach dem Migrieren von Vagusnerven Neuralleistenzellen in den Mitteldarm, demonstrierten wir sp&auml;te zeitliche Voraussetzung f&uuml;r Foxd3 w&auml;hrend der ENS-Entwicklung. Linie Kennzeichnung von Ednrb-iCre auszudr&uuml;cken Zellen in Foxd3 mutant Embryonen ergab eine Reduktion der ENPs im gesamten Darm und Verlust von Ednrb-iCre Linie Zellen im distalen Dickdarm. Obwohl Mutante M&auml;use lebensf&auml;hig waren, waren M&auml;ngel in Musterung und Verteilung der ENPs reduzierte Proliferation und starker Reduktion von Gliazellen, abgeleitet von der Linie Ednrb-iCre zugeordnet. Analysen von ENS-&Uuml;bertragungslinie und Differenzierung in mutant Embryonen vorgeschlagen Aktivierung der kompensatorischen Einwohner Foxd3-positiven ENPs, die nicht die Transgene Ednrb-iCre Ausdr&uuml;cken. Unsere Ergebnisse markieren die entscheidende Rolle, die Foxd3 w&auml;hrend der ENS Entwicklung einschlie&szlig;lich Vorl&auml;uferzellen Proliferation, neuronale Musterbildung und Gliazellen Differenzierung und k&ouml;nnen helfen, unterschiedliche Molekulare Programme Steuern Vagusnerven gegen&uuml;ber sakralen Neuralleiste Entwicklung voneinander abzugrenzen.

Enterischen Nervensystem bestimmte Streichung des Foxd3 st&ouml;rt Gliazelle Differenzierung und kompensatorische Enterische Vorl&auml;uferzellen aktiviert.

Article	Year
The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. Development (Cambridge, England)\| PubMed ID: 12015290	2002
Genes & development\| PubMed ID: 12381664	2002
Developmental cell\| PubMed ID: 14667405	2003
Stem cells (Dayton, Ohio)\| PubMed ID: 15153600	2004
Nature cell biology\| PubMed ID: 15517002	2004
Developmental biology\| PubMed ID: 16039639	2005
Genes & development\| PubMed ID: 16510872	2006
Gene expression patterns : GEP\| PubMed ID: 16750430	2006
Advances in experimental medicine and biology\| PubMed ID: 17076284	2006
Development (Cambridge, England)\| PubMed ID: 17092955	2006
Development (Cambridge, England)\| PubMed ID: 17720696	2007
Resident endothelial precursors in muscle, adipose, and dermis contribute to postnatal vasculogenesis. Stem cells (Dayton, Ohio)\| PubMed ID: 17823241	2007
Development (Cambridge, England)\| PubMed ID: 18367558	2008
Stem cells (Dayton, Ohio)\| PubMed ID: 18653770	2008
Human molecular genetics\| PubMed ID: 18799476	2008
Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 19030797	2008
Developmental biology\| PubMed ID: 19576203	2009
Cell stem cell\| PubMed ID: 20621055	2010
Conditional gene targeting in mouse pancreatic ÃŸ-Cells: analysis of ectopic Cre transgene expression in the brain. Diabetes\| PubMed ID: 20802254	2010
Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation. Developmental biology\| PubMed ID: 21081123	2011
Development (Cambridge, England)\| PubMed ID: 21228004	2011
Genesis (New York, N.Y. : 2000)\| PubMed ID: 21254333	2011
Endocrinology\| PubMed ID: 21952247	2011
Renshaw cell interneuron specialization is controlled by a temporally restricted transcription factor program. Development (Cambridge, England)\| PubMed ID: 22115757	2012
Enteric nervous system specific deletion of Foxd3 disrupts glial cell differentiation and activates compensatory enteric progenitors. Developmental biology\| PubMed ID: 22266424	2012
Scientific reports\| PubMed ID: 22355623	2011

Patricia A. Labosky

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