zebrafish-whole-mount-high-resolution-double-fluorescent-situ

Zebrafish Whole Mount High-Resolution Double Fluorescent <em>In Situ</em> Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. We have performed a detailed analysis of the mouse Hoxb4 intron enhancer to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. As these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site.

Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY.

R&#xE4;umlich spezifische Expression von HOXB4 ist abh&#xE4;ngig von der ubiquit&#xE4;ren Transkriptionsfaktor NFY.

Hox genes are key determinants of anteroposterior patterning of animal embryos, and spatially restricted expression of these genes is crucial to this function. In this study, we demonstrate that expression of Hoxb4 in the paraxial mesoderm of the mouse embryo is transcriptionally regulated in several distinct phases, and that multiple regulatory elements interact to maintain the complete expression domain throughout embryonic development. An enhancer located within the intron of the gene (region C) is sufficient for appropriate temporal activation of expression and the establishment of the correct anterior boundary in the paraxial mesoderm (somite 6/7). However, the Hoxb4 promoter is required to maintain this expression beyond 8.5 dpc. In addition, sequences within the 3' untranslated region (region B) are necessary specifically to maintain expression in somite 7 from 9.0 dpc onwards. Neither the promoter nor region B can direct somitic expression independently, indicating that the interaction of regulatory elements is crucial for the maintenance of the paraxial mesoderm domain of Hoxb4 expression. We further report that the domain of Hoxb4 expression is restricted by regulating transcript stability in the paraxial mesoderm and by selective translation and/or degradation of protein in the neural tube. Moreover, the absence of Hoxb4 3'-untranslated sequences from transgene transcripts leads to inappropriate expression of some Hoxb4 transgenes in posterior somites, indicating that there are sequences within region B that are important for both transcriptional and post-transcriptional regulation.

Multiple levels of transcriptional and post-transcriptional regulation are required to define the domain of Hoxb4 expression.

Mehrere Ebenen der Transkriptions und posttranskriptionalen Regulation erforderlich sind, um die Dom&#xE4;ne von HOXB4 Ausdruck definieren.

Somites are the mesodermal segments of vertebrate embryos that become the vertebral column, skeletal muscle and dermis. Somites arise within the paraxial mesoderm by the periodic, bilaterally symmetric process of somitogenesis. However, specification of left-right asymmetry occurs in close spatial and temporal proximity to somitogenesis and involves some of the same cell signaling pathways that govern segmentation. Here, we review recent evidence that identifies cross-talk between these processes and that demonstrates a role for retinoic acid in maintaining symmetrical somitogenesis by preventing impingement of left-right patterning signals upon the paraxial mesoderm.

Balancing segmentation and laterality during vertebrate development.

Beginnen werden die mesodermale Segmente des vertebrate Embryos, die der Wirbels&auml;ule, der Skelettmuskulatur und der Lederhaut geworden. Beginnen entstehen innerhalb der Paraxial Mesoderm durch den regelm&auml;&szlig;igen, bilateral symmetrischen Prozess des Somitogenesis. Jedoch Spezifikation der Links-rechts-Asymmetrie tritt in r&auml;umlicher und zeitlicher N&auml;he zum Somitogenesis und beinhaltet einige der gleichen Zelle Signalisierung Bahnen, die Segmentierung zu regieren. Hier schauen wir uns den letzten Beweis, kennzeichnet Nebensprechen zwischen diesen Prozessen und zeigt, dass eine Rolle f&uuml;r Retinoic S&auml;ure in die Aufrechterhaltung der symmetrische Somitogenesis durch Zusammenstoss der Links-rechts-Musterung-Signale auf das paraxiale Mesoderm zu verhindern.

Ausgleichende Segmentierung und Lateralit&auml;ts w&auml;hrend der vertebrate Entwicklung.

Somites are segmental units of the mesoderm in vertebrate embryos that give rise to the axial skeleton, muscle, and dermis. Somitogenesis occurs in a periodic manner and is governed by a segmentation clock that causes cells to undergo repeated oscillations of gene expression. Here, we present a detailed analysis of cis-regulatory elements that control oscillating expression of the zebrafish her1 gene in the anterior presomitic mesoderm. We identify binding sites for Her proteins and demonstrate that they are necessary for transcriptional repression. This result confirms that direct negative autoregulation of her gene expression constitutes part of the oscillator mechanism. We also characterize binding sites for fused somites/Tbx24 and Suppressor of Hairless proteins and show that they are required for activation of her1 expression. These data provide the foundation for a precise description of the regulatory grammar that defines oscillating gene expression in the zebrafish segmentation clock.

Expression of the oscillating gene her1 is directly regulated by Hairy/Enhancer of Split, T-box, and Suppressor of Hairless proteins in the zebrafish segmentation clock.

Beginnen sind segmentale Einheiten das Mesoderm in vertebrate Embryos, die der Axial-Skelett, Muskeln und Haut hervorrufen. Somitogenesis tritt in einer regelm&auml;&szlig;igen Weise und unterliegt einer Segmentierung-Uhr, die bewirkt, dass die Zellen wiederholte Oszillationen der Genexpression zu unterziehen. Hier pr&auml;sentieren wir eine detaillierte Analyse der Cis-regulatorischen Elementen, die oszillierende Ausdruck im Zebrafisch-her1-gen in der vorderen presomitic Mesoderm zu steuern. Wir identifizieren Bindungsstellen f&uuml;r ihre Proteine und zeigen, dass sie f&uuml;r transcriptional Repression erforderlich sind. Dieses Ergebnis best&auml;tigt, dass direkte Negative &uuml;berl&auml;sst ihre Gen-Ausdrucks ist Bestandteil der Oszillator-Mechanismus. Wir auch Bindungsstellen f&uuml;r gegossenes beginnen/Tbx24 und Suppressor of Hairless Proteine zu charakterisieren und zeigen, dass sie f&uuml;r die Aktivierung der her1 Ausdruck ben&ouml;tigt werden. Diese Daten bilden die Grundlage f&uuml;r eine genaue Beschreibung der regulatorischen Grammatik, die oszillierende Genexpression in der Zebrafisch-Segmentierung-Uhr definiert.

Ausdruck der oszillierenden gen her1 wird direkt von haarigen/Enhancer von Split, T-Box, reguliert und Suppressor of Hairless Proteine in der Zebrafisch-Segmentierung Uhr.

Using in vitro and in vivo assays, we define a network of Her/Hes dimers underlying transcriptional negative feedback within the zebrafish segmentation clock. Some of the dimers do not appear to be DNA-binding, whereas those dimers that do interact with DNA have distinct preferences for cis regulatory sequences. Dimerization is specific, with Hes6 serving as the hub of the network. Her1 binds DNA only as a homodimer but will also dimerize with Hes6. Her12 and Her15 bind DNA both as homodimers and as heterodimers with Hes6. Her7 dimerizes strongly with Hes6 and weakly with Her15. This network structure engenders specific network dynamics and imparts greater influence to the Her7 node. Computational analysis supports the hypothesis that Her7 disproportionately influences the availability of Hes6 to heterodimerize with other Her proteins. Genetic experiments suggest that this regulation is important for operation of the network. Her7 therefore has two functions within the zebrafish segmentation clock. Her7 acts directly within the delayed negative feedback as a DNA-binding heterodimer with Hes6. Her7 also has an emergent function, independent of DNA binding, in which it modulates network topology via sequestration of the network hub.

The Her7 node modulates the network topology of the zebrafish segmentation clock via sequestration of the Hes6 hub.

Mit in-vitro- und in-vivo-Assays, definieren wir ein Netzwerk von ihr / Hes-Dimer transkriptionelle negatives Feedback innerhalb der Zebrafisch-Segmentierung zugrundeliegenden clock. Einige der Dimere erscheinen nicht zu DNA-Bindung, w&auml;hrend diese Dimere, die mit der DNA interagieren unterschiedliche Pr&auml;ferenzen f&uuml;r Cis-regulatorische Sequenzen haben. Dimerisierung ist spezifisch, mit Hes6 als die Nabe des Netzes. HER1 bindet DNA nur als ein Homodimer, sondern wird auch mit Hes6 Mitgliedern bilden. Her12 und Her15 binden DNA Homodimers sowohl als Heterodimere mit Hes6. Her7 dimerizes stark mit Hes6 und schwach Her15. Diese Netzwerkstruktur erzeugt spezifische Netzwerk-Dynamik und vermittelt mehr Einfluss auf den Her7-Knoten. Computational Analysis unterst&uuml;tzt die Hypothese, dass Her7 unverh&auml;ltnism&auml;&szlig;ig der Verf&uuml;gbarkeit von Hes6, um Heterodimere mit anderen ihre Proteine beeinflusst. Genetische Experimente deuten darauf hin, dass diese Verordnung wichtig f&uuml;r den Betrieb des Netzes. Her7 hat also zwei Funktionen innerhalb der Zebrafisch-Segmentierung-Uhr. Her7 fungiert als ein DNA-bindenen Heterodimer mit Hes6 direkt in die verz&ouml;gerte negatives Feedback. Her7 hat auch eine emergente Funktion, unabh&auml;ngig von der DNA-Bindung, in dem es Netzwerktopologie &uuml;ber Beschlagnahme von Netzwerkhub moduliert.

Article	Year
Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY. Development (Cambridge, England)\| PubMed ID: 12135926	2002
Multiple levels of transcriptional and post-transcriptional regulation are required to define the domain of Hoxb4 expression. Development (Cambridge, England)\| PubMed ID: 12736215	2003
Balancing segmentation and laterality during vertebrate development. Seminars in cell & developmental biology\| PubMed ID: 19084074	2009
Expression of the oscillating gene her1 is directly regulated by Hairy/Enhancer of Split, T-box, and Suppressor of Hairless proteins in the zebrafish segmentation clock. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 19795510	2009
The Her7 node modulates the network topology of the zebrafish segmentation clock via sequestration of the Hes6 hub. Development (Cambridge, England)\| PubMed ID: 22278920	2012

Tim Brend

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