Neurogenese in Zentralen Nervensystem der Wirbeltiere – Molekulare Mechanismen zur regionsspezifischen Expression von neurogenin1 im Embryo des Zebrafischs Danio rerio

Während der Embryonalentwicklung von Vertebraten kontrollieren drei hoch konservierte Enhancer die raumzeitliche Expression orthologer neurog1-Gene. In Anamniota wie dem Zebrafisch wird die Transkription des proneuralen Gens nach Bildung der Neuralplatte durch zwei dieser cis-regulatorischen Elemente gesteuert. Transgenanalysen zeigen, dass der distale LSE-Enhancer neurog1 im Telencephalon und der proximale LATE-Enhancer im Diencepha-lon der Gehirnanlage aktiviert (Blader et al., 2003). Pax6 aktiviert die diencephale neurog1-Expression durch direkte Interaktion mit LATE, und es wird vermutet, dass diese Interaktion zur Kooption von neurog1 im lateralen Telencephalon der Maus und damit zur Evolution des Neocortex in Amniota entscheidend beigetragen hat. Jedoch müssen andere mit Pax6 kooper-ierende Faktoren an diesem Prozess beteiligt sein, einerseits, weil neurog1 in anderen Regio-nen auch unabhängig von Pax6 exprimiert wird, andererseits, weil Pax6 allein zur neurog1-Aktivierung nicht ausreicht, etwa im Telencephalon des Zebrafischs (Blader et al., 2004). Im Rahmen dieser Arbeit wurde untersucht, ob und wie weitere Transkriptionsfaktoren (TFs) in die Regulation der regionsspezifischen neurog1-Expression im Diencephalon des Zebra-fischs durch Interaktion mit dem LATE-Enhancer involviert sind. Eine Kombination aus Sequenzanalyse, Mutationsanalyse mit Reportertransgenen und Koexpressionsanalyse poten-tieller TF-Kandidaten weist auf eine Beteiligung von Homeodomänen-TFs der POU-Klasse (POU-TFs) an der LATE-vermittelten Regulation von neurog1 hin: Bioinformatische Analysen identifizieren POU-Bindestellen im stromabwärts der Pax6-Bindestelle benachbarten und in Vertebraten vollkommen konservierten LATE-Fragment, die selektiv mit rekombinantem Pou50-Protein physisch interagieren. Deletion dieses Fragments im LATE-Reporter induziert ektopische Transgenaktivität spezifisch im Telencephalon, was beweist, dass die anzestrale LATE-Funktion nicht auf diencephale Aspekte der neurog1-Expression beschränkt ist. LATE kooperiert hierbei mit dem LSE-Enhancer. Eine eigens für die Koexpressionsanalyse entwickelte Methode zur virtuellen 3D-Visualisierung von Genaktivitäten konnte nachweisen, dass in der ektopischen Transgendomäne neurog1, Pax6 und POU-TFs koexprimiert sind. Überdies kann die ektopische Transgen- und endogene neurog1-Aktivität spezifisch im Telencephalon durch HDAC-Inhibition reproduziert bzw. verstärkt werden. Die präsentierten Daten zeigen eindeutig, dass LATE ein direktes Ziel nicht nur positiv, sondern auch negativ regulierender Signale darstellt, und weisen darauf hin, dass neben transkriptioneller Aktivierung durch Kooption auch die Aufhebung transkriptioneller Repression für die Evolution des Neocortex entscheidend war

Fli+ etsrp+ hemato-vascular progenitor cells proliferate at the lateral plate mesoderm during vasculogenesis in zebrafish.

BACKGROUND:Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we use ets-related protein-1 (etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp(+) cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli(+)etsrp(+) hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp(+) cell numbers at the LPM. CONCLUSIONS/SIGNIFICANCE:This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish

Characterization of the astacin family of metalloproteases in C. elegans

Background: Astacins are a large family of zinc metalloproteases found in bacteria and animals. They have diverse roles ranging from digestion of food to processing of extracellular matrix components. The C. elegans genome contains an unusually large number of astacins, of which the majority have not been functionally characterized yet. Results: We analyzed the expression pattern of previously uncharacterized members of the astacin family to try and obtain clues to potential functions. Prominent sites of expression for many members of this family are the hypodermis, the alimentary system and several specialized cells including sensory sheath and sockets cells, which are located at openings in the body wall. We isolated mutants affecting representative members of the various subfamilies. Mutants in nas-5, nas-21 and nas-39 (the BMP-1/Tolloid homologue) are viable and have no apparent phenotypic defects. Mutants in nas-6 and nas-6; nas-7 double mutants are slow growing and have defects in the grinder of the pharynx, a cuticular structure important for food processing. Conclusions: Expression data and phenotypic characterization of selected family members suggest a diversity of functions for members of the astacin family in nematodes. In part this might be due to extracellular structures unique to nematodes.Science, Faculty ofZoology, Department ofNon UBCReviewedFacult

Spatial and temporal characterization of <i>mmp17b</i> expression.

<p>A is RT-PCR showing temporal expression of <i>mmp17b</i> and <i>mmp17a</i> compared to β-actin. <i>Mmp17b</i> expression commences at around 18 hpf while <i>mmp17a</i> is expressed as early as 5 hpf. B-E are whole mount in situ hybridization (WISH) panels of <i>mmp17b</i> at different embryonic stages (B and B’, 17 hpf; C and C’, 20 hpf; D and D’, 26 hpf). Earlier in development <i>mmp17b</i> is expressed more dorsally and then moves more posterior and ventral as development continues. Panel E shows <i>mmp17b</i> fluorescent in situ hybridization (FISH) in red and immunofluorescence (IF) for Flk-GFP in a 26 hpf embryo, which illustrates lack of <i>mmp17b</i> expression in the vasculature. Panels F, G-H, I-J and K are three color WISH staining performed as described in materials and methods with different probes as indicated in each panel. In panel F, <i>mmp17b</i> (purple), lower left is 4D9 staining (green) the muscle pioneer cells, lower right is Flk-GFP marking (red) the endothelial cells, and upper right is a merge. In this panel you can observe that 4D9 staining is located in the same region as <i>mmp17b</i>. G-H panel shows three color ISH image of 26 hpf zebrafish trunk and plexus with <i>mmp17b</i> (blue), Flk-GFP (red), and <i>crestin</i> (green). In the trunk image, <i>crestin</i> and <i>mmp17b</i> are overlapping in expression suggesting co-expression of these two genes in the same cell. The expression of both <i>crestin</i> and <i>mmp17b</i> are more dorsal in posterior regions of the embryo. Panels I-J represent high-powered image of the panel G. J is an optical section of panel G. Panel K is a three color image of 26 hpf zebrafish trunk with <i>mmp17a</i> (green), <i>mmp17b</i> (blue), and Flk-GFP (red). This image illustrates that <i>mmp17a</i> and <i>mmp17b</i> are expressed in different regions of the developing embryo. B-D are lateral views, B’-D’ are dorsal views. D” is a close-up of the panel D. Panels L-N are single plane confocal images of a 26 hpf embryo stained for <i>mmp17b</i> using FISH (panel L, green), and immunostaining for sox10 cells labelled with RFP (panel M, red). Panel N is the merged images showing co-localization of <i>mmp17b</i> and sox10 as indicated by yellow color (arrowhead).</p

Mmp17b Is Essential for Proper Neural Crest Cell Migration <i>In Vivo</i>

<div><p>The extracellular matrix plays a critical role in neural crest (NC) cell migration. In this study, we characterize the contribution of the novel GPI-linked matrix metalloproteinase (MMP) zebrafish <i>mmp17b. Mmp17b</i> is expressed post-gastrulation in the developing NC. Morpholino inactivation of <i>mmp17b</i> function, or chemical inhibition of MMP activity results in aberrant NC cell migration with minimal change in NC proliferation or apoptosis. Intriguingly, a GPI anchored protein with metalloproteinase inhibitor properties, <u>Re</u>version-inducing-<u>C</u>ysteine-rich protein with <u>K</u>azal motifs (RECK), which has previously been implicated in NC development, is expressed in close apposition to NC cells expressing <i>mmp17b</i>, raising the possibility that these two gene products interact. Consistent with this possibility, embryos silenced for <i>mmp17b</i> show defective development of the dorsal root ganglia (DRG), a crest-derived structure affected in RECK mutant fish <i>sensory deprived</i> (<i>sdp</i>). Taken together, this study has identified the first pair of MMP, and their putative MMP inhibitor RECK that functions together in NC cell migration.</p> </div

Bioinformatic and biochemical analysis of Mmp17b.

<p>Panel A depicts amino acid alignment of human and mouse MMP17 and zebrafish Mmp17a and Mmp17b. Red color indicates conserved amino acids and blue color indicates less conserved regions. B is a cartoon of Mmp17b protein. The predicted domains include a zinc catalytic domain, hemopexin-like domains, and a GPI-anchor. C and D are myc and MMP17 western blots of HEK293T cell lysates respectively. Supernatant (S) and pellet (P) fractions were generated as described in Methods S1. Mmp17b protein is observed only in the P fraction. MMP17 and MMP25 proteins are more robustly expressed, and were observed in both S and P fractions. D depicts bands at the proper size for Mmp17b and MMP17 with some cross reactivity to MMP25. Bands of higher molecular weight are also observed. UT = Untransfected, S = supernatant, P = pellet, + = positive control, E = empty vector control. E-J are myc tagged <i>MMP17</i> and myc-HIS tagged <i>MMP17b</i> cDNAs transiently expressed in HEK293T cells. The enriched metalloproteinase fusion proteins were detected using human specific MMP17 antibody (E & H; shown in green) and a caveolin mAB antibody (F & I; shown in red). The overlay images (G & J) show co-localization of caveolin with MMPs in positively transfected cells. Image micrograph depicting nucleus stained with DAPI not shown. Scale bars are 10 micron.</p

RECK blocks MMP17 activity <i>in vitro</i>.

<p>Elavl1 staining of control MO (cMO) (A) or <i>mmp17b</i> MO1 (B) injected 72 hpf embryos. Asterisks show the location of DRGs (dorsal root ganglia). <i>Mmp17b</i> knockdown embryos lack proper DRG development compared to controls. Quantitation of number of DRGs and migrated DRGs are indicated in panel C. Panel D is immunoprecipitation of myc-Mmp17b and RFP-Reck in Cos7 cells. Left panels indicate input of the two proteins in the sample, and right panels are RFP antibody immunoprecipitated samples followed by western blot. Arrow shows the pull down Mmp17b protein. Panel E is immunoprecipitation of myc-MMP17 and RFP-Reck in Cos7 cells. Left panel indicates input of MMP17 proteins in the lysate and the right panels indicate RFP antibody immunoprecipitated samples followed by western for myc epitope. Panel F is western blot of myc-MMP17 unactivated and activated with 4-aminophenylmercuric acetate (APMA) using MMP17 antibody. Activated MMP lanes show multiple bands, which are absent in samples co-incubated with RECK protein. Panel G is FRET-based fluorescence readout for MMP activity using the samples run on western blots in panel F.</p

Mmp17b is involved in neural crest patterning.

<p>A-C and D-F are WISH staining for <i>crestin</i> in control MO (cMO) (A), MO1 (B), and MO2 (C) injected 26 hpf or DMSO (D), Marimastat (E) and ONO-4817 (F) treated 26 hpf embryos. A’-C’ are high powered images of the trunk regions of A-C. Arrowheads indicate <i>crestin</i>^<i>+</i> cells misplaced in the trunk. There is a mis-patterning of <i>crestin</i> in the trunk of the MO1 and MO2 injected embryos compared to control. There is also an accumulation of <i>crestin</i>⁺ cells in the posterior of the embryo (white bracket) compared to control. This is quantitated in panel G. N=25 for cMO; n=19 for MO1; n=18 for MO2. In panels D-F, WISH staining for <i>crestin</i> in MMP inhibitor treated 26 hpf embryos shows mis-patterning similar to <i>mmp17b</i> KD embryos (A-C). An accumulation of <i>crestin</i>⁺ cells in the posterior of the MMP inhibitor treated embryos is also observed (red brackets). This is quantitated in panel H. N=12 for DMSO; n=8 for ONO 4817; n=10 for Marimastat. I-K shows melanocyte quantitation done on 72 hpf fish. Dorsal images were taken of 72 hpf fish injected with either control MO (J) or <i>mmp17b</i> MO1 (I). The number of medial (M, red arrow) and lateral (L, red arrow) melanocytes is counted between the two vertical bars illustrated in panels I and J for 10 fish in each category. The results were quantitated in panel K. The medial cells were not statistically different but the lateral cells were at a p-value of less than 0.05.</p

Current concepts in cleft care: A multicenter analysis

The current surgical techniques used in cleft repair are well established, but different centers use different approaches. To determine the best treatment for patients, a multi-center comparative study is required. In this study, we surveyed all craniofacial departments registered with the German Society of Maxillofacial Surgery to determine which cleft repair techniques are currently in use. Our findings revealed much variation in cleft repair between different centers. Although most centers did use a two-stage approach, the operative techniques and timing of lip and palate closure were different in every center. This shows that a retrospective comparative analysis of patient outcome between the participating centers is not possible and illustrates the need for prospective comparative studies to establish the optimal technique for reconstructive cleft surgery. (c) 2018 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved