isolation-culture-cells-from-nephrogenic-zone-embryonic-mouse

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.

The MHC class I-like IgG receptor controls perinatal IgG transport, IgG homeostasis, and fate of IgG-Fc-coupled drugs.

Die MHC-Klasse-I-&#xE4;hnliche IgG-Rezeptor-IgG steuert perinatale Verkehr, IgG Hom&#xF6;ostase, und das Schicksal von IgG-Fc-gekoppelten Medikamente.

Minor histocompatibility antigens (minor H antigen) elicit strong T-cell-mediated responses during both graft rejection and graft versus leukemia (GvL) among MHC-matched individuals (where MHC is major histocompatibility complex). Employing expression-cloning methodology, we have identified a cDNA clone, MI-35, encoding the immunodominant H4b minor H antigen within the classical mouse H4 complex. The minimal antigenic epitope derived from H4b presented on Kb class I MHC is SGIVYIHL (SYL8) and the polymorphism is due to C-->T nucleotide modification in p3 resulting in the change of threonine (ACT) to isoleucine (ATT). The results presented here demonstrate that amino acid variation in the allelic epitopes results in the low abundance of H4a peptide. The differential peptide copy number resulted in an immunodominant cytotoxic T cells (CTL) response directed against H4b while the anti-B6 response directed against H4a was easily dominated. These results provide a molecular mechanism for the H4 minor H antigen and suggest a novel mechanism by which alloantigenic disparity caused by conservative amino acid changes can be augmented by posttranslational antigen processing events.

A single nucleotide polymorphism in the Emp3 gene defines the H4 minor histocompatibility antigen.

Ein Einzel-Nukleotid-Polymorphismus im Gen EMP3 definiert die H4 Nebenhistokompatibilit&#xE4;tsantigene Antigen.

There is a pressing need for interactive bioinformatics tools that empower investigators with the means to extract information and organize it in a simplified but meaningful format. A wealth of mammalian gene expression data is readily accessible, much of which is based on expressed sequence tags (ESTs). Many mammalian ESTs are derived from tissue-specific cDNA libraries in which the number of ESTs representing a specific gene approximates the transcriptional expression level in the source tissue. Our program ExQuest (Expressional Quantification of ESTs) organizes the public EST database (dbEST) into hierarchical tissue classes and reports tissue or developmental gene expression patterns for both mRNA and genomic sequences. ExQuest also displays tissue expression patterns of genes in the context of assembled chromosomes. These interactive "transcriptome" maps provide a novel tool for investigating the genomic basis of gene expression as well as prioritizing candidate genes within genetically mapped mutant and quantitative trait loci.

ExQuest, a novel method for displaying quantitative gene expression from ESTs.

Recent developments in sequence databases provide the opportunity to relate the expression pattern of genes to their genomic position, thus creating a transcriptome map. Quantitative trait loci (QTL) are phenotypically-defined chromosomal regions that contribute to allelically variant biological traits, and by overlaying QTL on the transcriptome, the search for candidate genes becomes extremely focused.

Searching QTL by gene expression: analysis of diabesity.

Suche nach QTL Genexpression: Analyse der Diabesity.

The MHC class I-like Fc receptor FcRn plays an essential role in extending the half-life (t(1/2)) of IgG antibodies and IgG-Fc-based therapeutics in the circulation. The goal of this study was to analyze the effect of human IgG1 (hIgG1) antibodies with enhanced in vitro binding to FcRn on their in vivo t(1/2) in mice expressing human FcRn (hFcRn). Mutants of the humanized monoclonal Herceptin antibody (Hu4D5-IgG1), directed against human epidermal growth factor receptor 2 (p185 (HER2)), show altered pH-dependent binding to hFcRn in vitro. Two engineered IgG1 mutants (N434A and T307A/E380A/N434A) showed a considerably extended t(1/2) in vivo compared with wild-type antibody in mice expressing an hFcRn transgene (Tg) but not in mice expressing the endogenous mouse FcRn. The efficiency of hFcRn-mediated protection was dependent on hFcRn Tg copy number. Moreover, when injected into FcRn-humanized mice at a concentration sufficient to partially saturate hFcRn, the engineered IgG1 mutants with an extended serum t(1/2) were most effective in reducing the t(1/2) of a tracer hIgG1 antibody. Finally, administration of mutant with high binding to hFcRn ameliorated arthritis induced by passive transfer with human pathogenic plasma. These results indicate that Fc regions modified for high binding affinity to hFcRn increases serum persistence of therapeutic antibodies, that the same approach can be exploited as an anti-autoimmune therapy to promote the clearance of endogenous pathogenic IgG and that FcRn-humanized mice are a promising surrogate for hIgG therapeutic development.

Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease.

Der MHC-Klasse-wie Fc-Rezeptor FcRn spielt eine wesentliche Rolle bei der Ausweitung der Half-Life (t(1/2)) von IgG-Antik&ouml;rpern und IgG-Fc-basierte Therapeutika in der Zirkulation. Das Ziel dieser Studie war es, die Auswirkungen von menschlichen IgG1 analysieren (hIgG1) Antik&ouml;rper mit verbesserter in-vitro Bindung an FcRn auf ihre in-vivo t(1/2) bei M&auml;usen menschliche FcRn (hFcRn) zum Ausdruck zu bringen. Mutanten von der humanisierte monoklonale Herceptin-Antik&ouml;rper (Hu4D5-IgG1), gegen den humanen epidermalen Wachstumsfaktor-Rezeptor 2 (p185 (HER2)), Karte ver&auml;ndert pH-Wert-abh&auml;ngige Bindung an in-vitro-hFcRn. Zwei entwickelt IgG1 Mutanten (N434A und T307A/E380A/N434A) zeigte eine deutlich erweiterte t(1/2) in-vivo im Vergleich zu Wildtyp Antik&ouml;rper in M&auml;usen Ausdruck einer hFcRn Transgen (Tg), aber nicht in den M&auml;usen zum Ausdruck der endogenen Maus FcRn. Die Effizienz der hFcRn-vermittelte Schutz war hFcRn Tg Kopienzahl abh&auml;ngig. Dar&uuml;ber hinaus waren, als in FcRn vermenschlicht M&auml;use in einer Konzentration, die ausreicht, um die teilweise hFcRn s&auml;ttigen injiziert, die manipulierte IgG1-Mutanten mit einer erweiterten Serum-t(1/2) am effektivsten bei der Verringerung der t(1/2) der eine Beobachter-hIgG1-Antik&ouml;rper. Verwaltung der Mutant mit hoher Bindung an hFcRn einged&auml;mmt und schlie&szlig;lich von passiven &Uuml;bertragung mit pathogenen Blutplasma induzierte Arthritis. Diese Ergebnisse zeigen, dass Fc Regionen ge&auml;ndert f&uuml;r hohe Bindungsaffinit&auml;t zu hFcRn erh&ouml;ht Serum Persistenz von therapeutischen Antik&ouml;rpern, dass der gleiche Ansatz ausgenutzt werden kann, als eine anti-autoimmune Therapie zur F&ouml;rderung des Rechnungsabschlusses endogene pathogenen IgG und FcRn vermenschlicht M&auml;usen vielversprechende Surrogat f&uuml;r hIgG therapeutische Entwicklung sind.

Verbesserte Halbwertszeit von gentechnisch IgG1-Antik&ouml;rper in einem humanisierten Mausmodell FcRn: m&ouml;gliche Anwendung in humorally vermittelte Autoimmunerkrankung.

Bone morphogenic proteins (BMPs) play diverse roles in embryonic kidney development, regulating essential aspects of both ureteric bud and nephron development. In this review, we provide an overview of reported expression patterns and functions of BMP signaling components within the nephrogenic zone or nephron progenitor niche of the developing kidney. Reported in situ hybridization results are relatively challenging to interpret and sometimes conflicting. Comparing these with high-resolution microarray gene expression data available in Gudmap, we propose a consensus gene expression pattern indicating that essential components of both the Smad-mediated pathway and the Smad-independent MAPK pathways are expressed in the nephron progenitor cell compartment and may be activated by BMPs, but that cortical interstitium may only be able to respond to BMPs through mitogen activated protein kinase (MAPK) signaling. Localization of phosphorylated Smad transcription factors and studies of a BMP reporter mouse strain however indicate limited transcriptional responsiveness to Smad-mediated signaling in cap mesenchyme. An overview of genetic inactivation, organ culture, and primary cell studies indicates that BMP signaling may elicit two important biological outcomes in the nephrogenic zone: survival of the cap mesenchyme, and the physical segregation of interstitial and progenitor cell compartments. Ongoing studies using a novel primary cell system that establishes the nephrogenic zone ex vivo are pursuing the concept that the balance between Smad-mediated and Smad-independent responses to BMP ligand may underlie these distinct outcomes.

BMP signaling in the nephron progenitor niche.

Knochen Morphogene Proteine (BMPs) spielen verschiedene Rollen in embryonale Nieren Entwicklung, wesentliche Aspekte der ureteric Knospe und Nephron-Entwicklung. In diesem Bericht bieten wir eine &Uuml;bersicht der gemeldeten Muster und Funktionen von BMP Signalling Komponenten innerhalb des nephrogenen Gebiets oder Nephron Stammvater Nische der entwickelnden Niere. Berichtet in situ-Hybridisierung Ergebnisse sind relativ schwierig zu interpretieren und manchmal widerspr&uuml;chlich. Vergleichen diese mit hochaufl&ouml;senden Microarray-Gen Ausdruck in Gudmap verf&uuml;gbaren Daten, schlagen wir eine Konsens Gen Ausdruck Muster, wonach wesentliche Bestandteile der beiden die Smad-vermittelter Signalweg und die Smad-unabh&auml;ngige MAPK-Wege in das Nephron Stammvater Zelle Fach ausgedr&uuml;ckt und durch BMPs aktiviert werden k&ouml;nnen, aber diese kortikale Interstitium m&ouml;glicherweise nur auf BMPs durch Mitogen-aktivierte Protein-Kinase (MAPK) Signale zu reagieren. Lokalisierung von phosphoryliert Smad Transkriptionsfaktoren und Studien eine BMP-Reporter-Maus-Stamm zeigen jedoch begrenzt transkriptionelle Reaktionsf&auml;higkeit auf Smad-vermittelte signalisieren in GAP Mesenchym. Ein &Uuml;berblick &uuml;ber genetische Inaktivierung, Orgel-Kultur und Prim&auml;rzellen Studien zufolge BMP Signalisierung zwei wichtige biologische Ergebnisse in der Nephrogene Zone hervorrufen k&ouml;nnten: &Uuml;berleben, die GAP-Mesenchym und die physische Trennung der interstitielle und Vorl&auml;uferzellen Zelle F&auml;chern. Laufende Studien mit einem neuartigen Prim&auml;rzellen-System, die die Nephrogene Zone ex Vivo herstellt verfolgen das Konzept, dass das Gleichgewicht zwischen Smad-vermittelte und Smad-unabh&auml;ngige Antworten auf BMP-Ligand diese unterschiedlichen Ergebnisse zugrunde liegen kann.

BMP signalisieren in der Nephron Stammvater Nische.

Recent studies indicate that nephron progenitor cells of the embryonic kidney are arranged in a series of compartments of an increasing state of differentiation. The earliest progenitor compartment, distinguished by expression of CITED1, possesses greater capacity for renewal and differentiation than later compartments. Signaling events governing progression of nephron progenitor cells through stages of increasing differentiation are poorly understood, and their elucidation will provide key insights into normal and dysregulated nephrogenesis, as well as into regenerative processes that follow kidney injury. In this study, we found that the mouse CITED1(+) progenitor compartment is maintained in response to receptor tyrosine kinase (RTK) ligands that activate both FGF and EGF receptors. This RTK signaling function is dependent on RAS and PI3K signaling but not ERK. In vivo, RAS inactivation by expression of sprouty 1 (Spry1) in CITED1(+) nephron progenitors results in loss of characteristic molecular marker expression and in increased death of progenitor cells. Lineage tracing shows that surviving Spry1-expressing progenitor cells are impaired in their subsequent epithelial differentiation, infrequently contributing to epithelial structures. These findings demonstrate that the survival and developmental potential of cells in the earliest embryonic nephron progenitor cell compartment are dependent on FGF/EGF signaling through RAS.

FGF/EGF signaling regulates the renewal of early nephron progenitors during embryonic development.

Neuere Studien zeigen, dass Nephron Vorl&auml;uferzellen der embryonalen Niere, in einer Reihe von F&auml;chern von einem zunehmenden Zustand der Differenzierung angeordnet sind. Die fr&uuml;heste Stammvater Fach, zeichnen sich durch Ausdruck der CITED1, besitzt gr&ouml;&szlig;eren Kapazit&auml;t f&uuml;r die Erneuerung und Differenzierung als sp&auml;tere F&auml;cher. Ereignisse, die EZB Fortschreiten des Nephron Vorl&auml;uferzellen durch Phasen zunehmender Differenzierung sind schlecht verstanden, und ihre Aufkl&auml;rung liefern wichtige Erkenntnisse, Normal und defekten Nephrogenesis, sowie regenerative Prozesse, die Folgen von Verletzungen der Niere. In dieser Studie fanden wir, dass die Maus CITED1(+) Vorl&auml;uferzellen Fach als Reaktion auf Rezeptor-Tyrosin-Kinase (RTK)-Liganden verwaltet wird, die FGF und EGF-Rezeptoren aktivieren. Diese Meldefunktion RTK ist abh&auml;ngig von RAS und PI3K aber nicht ERK signalisieren. In vivo f&uuml;hrt RAS Inaktivierung durch Ausdruck sprouty 1 (Spry1) in CITED1(+) Nephron Vorfahre, Verlust der charakteristische Molekulare Marker Ausdruck und erh&ouml;hte Tod von Progenitorzellen. Herkunfts-Ablaufverfolgung zeigt, dass die &Uuml;berlebende Spry1 auszudr&uuml;cken-Vorl&auml;uferzellen in ihre sp&auml;tere epitheliale Differenzierung, selten zur epithelialen Strukturen beeintr&auml;chtigt sind. Diese Ergebnisse zeigen, dass das &Uuml;berleben und die Entwicklungsbiologie Potential der Zellen im fr&uuml;hesten embryonalen Nephron Stammvater Zelle Fach FGF/EGF Signalisierung durch RAS abh&auml;ngig sind.

Article	Year
The MHC class I-like IgG receptor controls perinatal IgG transport, IgG homeostasis, and fate of IgG-Fc-coupled drugs. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 12646614	2003
A single nucleotide polymorphism in the Emp3 gene defines the H4 minor histocompatibility antigen. Immunogenetics\| PubMed ID: 12845499	2003
ExQuest, a novel method for displaying quantitative gene expression from ESTs. Genomics\| PubMed ID: 14962679	2004
Searching QTL by gene expression: analysis of diabesity. BMC genetics\| PubMed ID: 15760467	2005
Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease. International immunology\| PubMed ID: 17077181	2006
BMP signaling in the nephron progenitor niche. Pediatric nephrology (Berlin, Germany)\| PubMed ID: 21373777	2011
FGF/EGF signaling regulates the renewal of early nephron progenitors during embryonic development. Development (Cambridge, England)\| PubMed ID: 22031548	2011

Aaron C. Brown

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