Functional analysis of IRF6 and SPECC1L in zebrafish models of orofacial cleft

Background Cleft lip and/or palate (CL/P) is the most frequent craniofacial malformation affecting 1 in 700 births. Children born with CL/P have to undergo multiple staged procedures early in their lifetime and may suffer from severe functional sequelae. Surgery remains their only therapeutic option. CL/P can be caused by perturbation of cranial neural crest cell proliferation, migration, and apoptosis in early embryogenesis. Environmental and genetic factors can cause CL/P. We investigated the genetic and developmental basis of craniofacial morphogenesis with the goal to find new therapeutic and potentially preventive measures to mitigate cleft phenotypes. This thesis focused on the functional analysis of IRF6 and SPECC1L, two genes involved in syndromic and nonsyndromic CL/P and oblique facial cleft, respectively. We used the zebrafish to study gene function in vivo, as this versatile vertebrate animal model allows us to easily manipulate and follow craniofacial developmental processes in real time. Methods and Results In manuscript 1, we showed that zebrafish and amniote lip/primary palate formation is analogous by fusion of the paired maxillary prominences (MXPs) and the frontonasal prominence (FNP). We suggested that the V-shaped fusion seam between FNP and MXP is analogous to the Y-shaped fusion seam found in humans. Further, we showed that cleft malformation is conserved in zebrafish by creating a dominant negative transgenic line harboring a known human mutation of IRF6 (R84C). Moreover, we described palate extension in detail as a process of morphological change, intercalation, proliferation and integration of cells and showed that wnt9a and irf6 are required for extension and integration, respectively. In manuscript 2, we described the lineage-tracing method that was used to determine analogy of palate formation between amniotes and zebrafish. We described selection and mounting of embryos, as well as imaging software settings. The three aspects of the protocol (mounting, imaging, software) have wide application, as every part can be used as a guide to confocal microscopy of any tissue. Manuscript 3 took advantage of knowledge gained in manuscripts 1 and 2. We investigated SPECC1L, the first gene implicated in ObFC. We showed that specc1lb is the zebrafish ortholog (conserved function) of human SPECC1L and showed expression in epithelia surrounding chondrocytes. We proposed that specc1lb is required for convergence of facial prominences and integration of the FNP and MXPs. In our analysis, migration to pharyngeal arches was not perturbed, although CNCCs contributing to the mandibular prominence failed to form the mandible, and anterior CNCCs did not converge to contribute to their respective facial prominences (FNP, MXP). Discussion and Conclusions IRF6 has been studied extensively. However, gene expression in different cell types, function of regulatory elements and the irf6 signaling pathways remain to be elucidated. In order to assess irf6 expression and activity in detail, we are currently working on identifying the irf6 promoter region, as well as regulatory elements upstream of irf6. In addition to investigating the gain of function dominant negative irf6 phenotype described in manuscript 1, we have created CRISPR knockout lines to assess the loss of function genotype. We hope that a combination of stable transgenic lines with irf6 gene expression pattern, as well as irf6 CRISPR lines, will allow us to study gene function and perform genetic and chemical screens to find substrates that can mitigate cleft phenotypes. Although SPECC1L MO knockdown phenotypes in manuscript 3 did not show perturbation of CNCC migration, CRISPR lines that are currently growing up may show NCC migration and delamination defects that were not uncovered by MO. Recent evidence from Co-Immunoprecipitation with a custom made anti-specc1l antibody followed by mass spectrometry provide evidence in support of our hypothesis that SPECC1L plays a role in directional migration during NCC movement. It is possible that SPECC1L is part of cell-cell and cell-ECM junctions, as well as actin and microtubulemembrane junctions to transduce and maybe potentiate mechano-sensitive signals. Further, it may be able to change the conformation of cell-cell and cell-ECM contacts as well as the actin/ myosin skeleton in response to signals from neighboring cells, the ECM, primary cilia and lamellipodia. SPECC1L high-throughput screens of potential SPECC1L interactors in the zebrafish animal model will elucidate which regulatory elements of cell movement associate with SPECC1L. Finally, transgenic irf6 and specc1l zebrafish lines will be used to analyze if the two genes act in the same regulatory gene network. Potentially, a connection between pathways leading to more or less severe forms of cleft can be made.Hintergrund Lippen-, Kiefer-,Gaumensegelspalten (LKGS) sind die häufigsten kraniofazialen Fehlbildungen und betreffen eines aus 700 bis 1000 Geburten. Chirurgie ist momentan die einzige therapeutische Option für diese Patienten. Unser Labor untersucht den genetischen Hintergrund kraniofazialer Entwicklung, mit dem Ziel, neue therapeutische und potenziell vorbeugende Maßnahmen zur Therapie und Prävention von LKGS zu finden. Dieses Dissertationsprojekt hat die zwei LKGS Gene IRF6 und SPECC1L untersucht. Wir haben die Gen-Funktion in vivo im Zebrafisch Tiermodell untersucht. Methoden und Ergebnisse In Manuskript 1 zeigten wir, dass die Bildung des Gaumens zwischen dem Zebrafisch und Menschen analog durch Fusion der Oberkieferwülste mit dem medialen Nasenwulst erfolgt. Wir schlugen vor, dass die V-förmige Fusionsnaht zwischen dem medialen Nasenwulst und dem Oberkieferwulst der Y-förmigen Fusionsnaht beim Menschen entspricht. Weiterhin beschrieben wir, dass Spaltfehlbildungen im Zebrafisch konserviert sind, indem wir eine bekannte LKGS Mutation im Menschen im Zebrafisch erzeugten (Tg: IRF6 R84C). Außerdem untersuchten wir die Entwicklung des Gaumens im Zebrafisch im Detail und zeigten, dass wnt9a für die Proliferation der Zellen und irf6 für die Fusion der Zellen erforderlich ist. In Manuskript 2 besprachen wir die Methode, welche verwendet wurde, um die Entwicklung des Gaumens im Zebrafisch in vivo zu beobachten. In Manuskript 3 analysierten wir SPECC1L, das erste Gen, welches nachweislich in komplexen schrägen Gesichtsspalten mutiert ist. Wir zeigten, dass specc1lb die Zebrafisch-Form des menschlichen SPECC1L ist und zeigten Expression im Epithel rund um Knorpelzellen des Gaumens. Wir schlugen vor, dass specc1lb für die Konvergenz der Gesichtswülste erforderlich ist. Diskussion und Schlussfolgerungen Um die Aktivität von IRF6 im Detail zu untersuchen, versuchen wir derzeit die Region des IRF6 Promoters zu identifizieren. Außerdem prüfen wir, ob regulatorische Elemente vor IRF6 im Zebrafisch konserviert sind. Zusätzlich haben wir CRISPR Knockout-Linien erstellt, um zu untersuchen was passiert, wenn die irf6 Gen Funktion komplett verloren geht. Wir hoffen, dass eine Kombination von transgenen Zebrafisch-Linien mit irf6 Gen-Expressionsmuster sowie IRF6 Knockout-Phänotypen uns erlauben wird, genetische und chemische Screenings durchzuführen, um Substanzen zu finden, die den LKGS Phänotypen verhindern oder verstärken. Obwohl SPECC1L MO Phänotypen in Manuskript 3 keine Störungen der Wanderung von Neuralleisten Zellen zeigt, könnten CRISPR Knockout-Linien, die derzeit heranwachsen, Migrationsstörungen zeigen. Neuere Erkenntnisse aus Co-Immunpräzipitation mit einem maßgeschneiderten anti-specc1l Antikörper, gefolgt von Massenspektrometrie bieten Hinweise, dass SPECC1L in gezielter Migration von Zellen eine Rolle spielt. Es ist möglich, dass SPECC1L Teil von Zell-Zell und Zell- Extrazellulärmatrix Übergängen ist. Ausserdem bindet SPECC1L Aktin und Mikrotubuli und könnte mechanosensitive Signale transduzieren. Zusätzlich könnte es die Konformation von Zell-Zell-und Zell-Extrazellulärmatrix Übergängen sowie das Aktin/ Myosin-Skelett in Reaktion auf Signale von benachbarten Zellen, Extrazellulärmatrix, primären Zilien und Lamellipodien ändern. Ein Screening von potenziellen Molekülen, die mit SPECC1L interagieren, wird zeigen, welche regulatorische Elemente in der Zelle von SPECC1L beeinflusst werden. Schlussendlich werden wir irf6- und specc1l transgene Zebrafischlinien verwenden, um zu analysieren, ob die beiden Gene in der Entwicklung des Gaumens und LKGS zusammenwirken. Potentiell kann eine Verbindung zwischen beiden Genen hergestellt werden, was Unterschiede im Schweregrad zwischen LKGS erklären könnte.submitted by Lisa GfrererAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, Med. Univ., Diss., 2015OeBB(VLID)171533

Technique Refinement in Prepectoral Implant Breast Reconstruction with Vicryl Mesh Pocket and Acellular Dermal Matrix Support

Summary: Direct-to-implant reconstruction after mastectomy is routinely performed with excellent oncologic and aesthetic outcomes. Several different techniques for placement of the prosthesis including total and partial subpectoral coverage have been described. Prepectoral implant placement is increasingly reported as a safe method, while patient selection and techniques are areas of further work. Most prepectoral prosthesis placement techniques describe complete acellular dermal matrix (ADM) coverage of the implant, often requiring multiple or larger ADM sheets than a comparable subpectoral implant procedure, resulting in high cost per reconstructed breast. This article describes the use of a vicryl mesh pocket and ADM support in prepectoral breast reconstruction achieving predicable and safe results at a lower cost

Ability to Cope with Pain Puts Migraine Surgery Patients in Perspective

Candidates for migraine surgery are chronic pain patients with significant disability. Currently, migraine-specific questionnaires are used to evaluate these patients. Analysis tools widely used in evaluation of better understood pain conditions are not typically applied. This is the first study to include a commonly used pain questionnaire, the Pain Self-Efficacy Questionnaire (PSEQ) that is used to determine patients' pain coping abilities and function. It is an important predictor of pain intensity/disability in patients with musculoskeletal pain, as low scores have been associated with poor outcome. Ninety patients were enrolled prospectively and completed the Migraine Headache Index and PSEQ preoperatively and at 12 months postoperatively. Scores were evaluated using paired t tests and Pearson correlation. Representative PSEQ scores for other pain conditions were chosen for score comparison. All scores improved significantly from baseline (p < 0.01). Mean preoperative pain coping score (PSEQ) was 18.2 ± 11.7, which is extremely poor compared with scores reported for other pain conditions. Improvement of PSEQ score after migraine surgery was higher than seen in other pain conditions after treatment (112 percent). Preoperative PSEQ scores did not influence postoperative outcome. The PSEQ successfully demonstrates the extent of debility in migraine surgery patients by putting migraine pain in perspective with other known pain conditions. It further evaluates functional status, rather than improvement in migraine characteristics, which significantly adds to our understanding of outcome. Poor preoperative PSEQ scores do not influence outcome and should not be used to determine eligibility for migraine surgery. Therapeutic, I

The Peripheral Nerve Surgeon's Role in the Management of Neuropathic Pain

Neuropathic pain (NP) underlies significant morbidity and disability worldwide. Although pharmacologic and functional therapies attempt to address this issue, they remain incompletely effective for many patients. Peripheral nerve surgeons have a range of techniques for intervening on NP. The aim of this review is to enable practitioners to identify patients with NP who might benefit from surgical intervention. The workup for NP includes patient history and specific physical examination maneuvers, as well as imaging and diagnostic nerve blocks. Once diagnosed, there is a range of options surgeons can utilize based on specific causes of NP. These techniques include nerve decompression, nerve reconstruction, nerve ablative techniques, and implantable nerve-modulating devices. In addition, there is an emerging role for preoperative involvement of peripheral nerve surgeons for cases known to carry a high risk of inducing postoperative NP. Lastly, we describe the ongoing work that will enable surgeons to expand their armamentarium to better serve patients with NP