Transgene Mausmodelle zur Untersuchung von Nervenzellwanderungen

Im Rahmen dieser Arbeit wurden transgene Mausmodelle hergestellt, die eine weitere Aufklärung der Rolle des Transkriptionsfaktors Pax6 bei der Wanderung von Nervenzellen ermöglichen, sowie ein Kultursystem zur Darstellung embryonaler Wanderungen außerhalb des Mutterleibs entwickelt.Bei der YAC-transgenen Mäuselinie PhPax6-taulacZ wird das Reportergen taulacZ unter der Kontrolle des Pax6-Promotors exprimiert. Dadurch ist dort, wo Pax6 im Zellkern vorliegt, der Rest der Zelle über seine gesamte Ausdehnung mit der vom taulacZ-Transgen kodierten tau-b-Galactosidase markiert. Das räumlich-zeitliche Expressionsmuster von Pax6 und dem Transgen taulacZ wurde detailliert untersucht. Dabei wurde eine hohe Übereinstimmung festgestellt. Basierend auf der Darstellung der Zellen in ihrer gesamten Ausdehnung, die durch das taulacZ-Transgen erstmals möglich ist, wurde eine Klassifizierung Pax6-positiver Zelltypen vorgenommen. Zunächst wird Pax6 in Neuroepithelzellen, später in radialen Gliazellen exprimiert.Mit der zweiten transgenen Mäuselinie, PhPax6-tTA, wurde ein Werkzeug hergestellt, das die gezielte und hoch spezifische Expression von beliebigen Transgenen in Pax6-exprimierenden Zellen ermöglicht. In Pax6-positive Zellen der Medulla wurde das Grün Fluoreszierende Protein (GFP) eingeführt und das Wanderungsverhalten in vitro über mehrere Tage dargestellt. Erstmals können mit dieser Linie beliebige Expressionskonstrukte gezielt, hocheffizient und schnell in wandernde Neurone eingebracht werden, ohne störende Hinter-grundexpression in anderen Zellen.The transcription factor Pax6 has been found to be associated with neuronal migrations. To further elucidate the role of Pax6 in these migrations, transgenic mouse models were developed. Additionally, an organotypic culture system was established that allows to visualize embryonic migrations in vitro.In the YAC transgenic mouse line PhPax6-taulacZ, expression of the reporter gene taulacZ is controlled by the Pax6 promotor. In this line, cells expressing Pax6 in their nucleus will be marked by expression of tau-b-Galactosidase, the enzyme encoded by the gene taulacZ, in their whole periphery. The spatio-temporal expression pattern of Pax6 and the taulacZ transgene was analyzed in detail, revealing a high degree of parallel expression. Based on the whole cell staining performed here for the first time, a classification of Pax6 expressing cell types is proposed. Pax6 is primarily expressed in neuroepithelial cells, and is found in radial glia cells at a later stage.The second transgenic mouse line PhPax6-tTA serves as a new genetic tool, that allows any transgene to be expressed in a targeted and highly specific manner in Pax6 expressing cells. This was demonstrated by introducing the gene encoding the Green Fluorescent Protein (GFP) into Pax6 expressing cells of the medulla. The migration of these cells could be followed in vitro over the course of several days. With this new mouse line, expression constructs can be introduced in a targeted, highly efficient, and quick way into migrating neurons for the first time. Any background expression in other cells, and thus disturbances, is avoided

A novel approach to selectively target neuronal subpopulations reveals genetic pathways that regulate tangential migration in the vertebrate hindbrain.

International audienceVertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications

ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3

Item does not contain fulltextNephronophthisis is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. Here we identify ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVS (NPHP2) and NPHP3. We show that ANKS6 localizes to the proximal cilium and confirm its role in renal development through knockdown experiments in zebrafish and Xenopus laevis. We also identify six families with ANKS6 mutations affected by nephronophthisis, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN hydroxylates ANKS6 and INVS and alters the composition of the ANKS6-INVS-NPHP3 module. Knockdown of Hif1an in Xenopus results in a phenotype that resembles loss of other NPHP proteins. Network analyses uncovered additional putative NPHP proteins and placed ANKS6 at the center of this NPHP module, explaining the overlapping disease manifestation caused by mutation in ANKS6, NEK8, INVS or NPHP3.6 p

Risk Factors for Early Dialysis Dependency in Autosomal Recessive Polycystic Kidney Disease

© 2018 Elsevier Inc.Objective: To identify prenatal, perinatal, and postnatal risk factors for dialysis within the first year of life in children with autosomal recessive polycystic kidney disease (ARPKD) as a basis for parental counseling after prenatal and perinatal diagnosis. Study design: A dataset comprising 385 patients from the ARegPKD international registry study was analyzed for potential risk markers for dialysis during the first year of life. Results: Thirty-six out of 385 children (9.4%) commenced dialysis in the first year of life. According to multivariable Cox regression analysis, the presence of oligohydramnios or anhydramnios, prenatal kidney enlargement, a low Apgar score, and the need for postnatal breathing support were independently associated with an increased hazard ratio for requiring dialysis within the first year of life. The increased risk associated with Apgar score and perinatal assisted breathing was time-dependent and vanished after 5 and 8 months of life, respectively. The predicted probabilities for early dialysis varied from 1.5% (95% CI, 0.5%-4.1%) for patients with ARPKD with no prenatal sonographic abnormalities to 32.3% (95% CI, 22.2%-44.5%) in cases of documented oligohydramnios or anhydramnios, renal cysts, and enlarged kidneys. Conclusions: This study, which identified risk factors associated with onset of dialysis in ARPKD in the first year of life, may be helpful in prenatal parental counseling in cases of suspected ARPKD