Functional analysis – University of Copenhagen

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Functional analysis - Work package 3 : Functional analysis of candidate disease genes

This work package addresses the biological function of identified candidate disease genes, plus hitherto uncharacterized genes known to be associated with cilia and centrosomes using mouse and human cell cultures as well as zebrafish and mouse models with live cell and tissue imaging methods, transcriptomics and proteomic approaches.


The aim is to characterize how genetic defects lead to deficiencies in cellular processes that regulate embryonic and fetal developmental as well as tissue homeostasis in the adult. In this way, we wish to map how genetic defects cause severe developmental disorders and diseases, including congenital brain and heart defects. This work will form the basis for future diagnosis and treatment of patients with gene mutations.


Cilia and centrosomes play a critical role in coordinating cellular events associated with developmental processes and tissue homeostasis in the adult. Consequently, defects in these organelles lead to a variety of genetic diseases and developmental disorders, including early embryonic death, heterotaxy, congenital heart defects, craniofacial and skeletal patterning defects, renal and liver diseases, mental retardation, anosmia, obesity and tumourigenesis, also known as ciliopathies. A prominent example includes that of primary cilia, which are microtubules-based organelles that emanates from the centrosomal mother centriole as a single copy on the surface of almost every quiescent cell of the human body. Primary cilia function as cellular sites for mechano-, chemo- and osmosensation to regulate cell cycle control, differentiation, migration and other cellular processes during development and in tissue homeostasis. Multiple lines of evidence have indicated that the appropriate targeting of receptors and downstream signaling molecules into and out of the ciliary compartment is critical for their function, but much still remains to be learned about how primary cilia coordinate various signaling pathways in time and space and how this impacts on organogenesis and proper function of tissues and organs in the adult. Here we will address the biological function of candidate disease genes from patient material and biobanks in WP1 and WP2 as well as hitherto uncharacterized genes known to be associated with cilia and centrosomes. This biological/genetic focus was chosen since ciliopathies are pleiotropic causing dysfunction of a variety of organs, the ciliome constitutes 1/20 of the human proteome, but the number of known ciliopathies is still <100. The individual ciliopathy will likely be an orphan disorder, but will frequently have implications for phenotypically overlapping common complex disorders. Moreover, since the cilium proteome is known, targeted approaches can be used to identify candidate disease genes and study their function. Consequently, WP3 will give rise to new and important information on how gene mutations lead to life threatening diseases and disorders caused by breakdown in the function of cilia and centrosomes.


The biological function of candidate disease genes will be investigated by a multitude of approaches, including:

  • Cell cultures: We will use a variety of cell lines (mouse embryonic and human foreskin fibroblasts, human retinal pigment epithelial cells, mouse kidney epithelial cells, various stem cell lines). RNAi knockdown, expression of dominant-negative and/or fluorescent-tagged proteins, fluorescence microscopy (FM) and live cell imaging analyses will be used to study function and subcellular localization of gene products with particular focus on centrosomes, cilia, and cilia-related signalling pathways and cellular processes, including cell polarity, migration, differentiation and cell cycle control. The molecular signaling networks include the hedgehog (Hh), transforming growth factor beta/bone morphogenic protein (TGFβ/BMP), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and Wingless/int (Wnt) pathways.
  • Results obtained with cell cultures will be supplemented with transcriptomics, proteomics  and bioinformatics analyses.
  • The role of candidate genes in development and disease pathology will be addressed, with focus on heart (cardiomyogenesis) and brain (neurogenesis) development where centrosomes and cilia are of key importance. To this end, we will use mammalian embryonic and embryonic carcinoma stem cells (hESC, mESC, NT2, P19.CL6).
  • Using morpholino knockdown in zebrafish strains and live imaging we will study the effects from depletion of candidate disease genes on migration and polarization of progenitor cells during heart and brain morphogenesis. To investigate how primary cilia control signaling pathways in vivo during tissue and organ development we will use existing mutant fish strains with defective cilia and analyze the gene expression and protein localization in a spatial and temporal manner.
  • The role of selected disease genes in mammalian development will be further analyzed in mouse mutants, including, but not limited to, conditional knock outs. 
  • Mouse studies will be supplemented by immunohistochemical and in situ hybridization analysis of sections of human embryonic and fetal tissues to study the spatial and temporal expression of disease genes in tissues and organs during human development. This work will be carried out in both normal tissues as well as in tissues collected from spontaneous abortions from families with ciliopathies.

Visions for societal impact

The results of WP3 couple disease genes with relevant cellular/biochemical functions. This in turn will feed back on the genome mapping projects outlined in WP2, and will open a door to commercial exploitations related to diagnosis and treatment, which will be addressed in WP4. Ethical implications associated with patient information on identified disease genes will be dealt with in WP5.


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