Project Details
Projekt
The role of ER-resident glycosylation in vertebrate neural development
Applicants
Dr. Thomas Thumberger; Professor Dr. Joachim Wittbrodt
Subject Area
Developmental Biology
General Genetics and Functional Genome Biology
Cell Biology
General Genetics and Functional Genome Biology
Cell Biology
Term
since 2017
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 289991887
The perception of the extracellular environment is of key importance in particular for neural development. Crucial events in neural development e. g. the neural plate patterning, neuroblast differentiation and migration, or axonal outgrowth highly depend on cell-surface interactions to facilitate cell-cell contacts, the adhesion to the cellular matrix or perception of extracellular signals. Extracellular surface proteins are modified by N-glycosylation as well as C- and O-mannosylation or combinations of these. These modifications affect protein functionality. Consequently, impaired glycosylation results in severe defects of brain / neuronal development in human patients.Fish models offer alternative routes to study glycosylation during neural development. Extrauterine development on the one hand allows the observation of phenotypes from the earliest stages onwards. On the other hand, mutant cells can also be studied in the context of an otherwise wild type embryo by clonal analysis following experimental embryology or genetic approaches. This allows to overcome early embryonic lethality to study subsequent processes in neural development and growth. This fact, together with modern tools of genome editing (CRIPR/Cas9) facilitate the establishment and detailed molecular and developmental analyses of tractable animal models for that closely resemble human syndromes.We will focus on the clearly tractable effects in the developing retina as model for a complex neural structure. The life-long growth of the fish retina allows to continuously study the impact of glycosylation during the life-long neurogenesis in the retina. Established models in the lab take advantage of the stereotypic pattern of retinal differentiation and growth to instantly amplify even minute effects by clonal analysis. We build on our expertise in genome editing to establish GFP knock in alleles for the key enzymes worked on in the context of the Research Unit. Our study aims at addressing the role of the three major glycosylation pathways during neural development. This will be approached by interfering the with function of key enzymes for all three pathways by gene targeting. Targeting the loci of the Glyocosyl/Mannosyl transferases by knock in of GFP will instantly provide tools for the detailed expression analysis of the three major pathway components. We will employ heterozygous animals for long term imaging by light sheet microscopy to identify and highlight the potential domains of action during the patterning and differentiation of the forming nervous system with a particular focus on the eye. Depending on the severity of the phenotypes, mutant analysis will be performed in homozygous mutant embryos (if stages of neural development are reached), by clonal analysis (transplantation) or by conditional tools.The models established will provide the organismal context for the biochemical and molecular analyses performed by the other partners in the Research Unit.
DFG Programme
Research Units
