Project 1: Retinal development and retinoblastoma

Background: The central nervous system (CNS) consists of the brain, spinal cord and retina. Of these three tissues, the retina is the easiest to work with because it is well-characterized and relatively simple in structure. The retina originates from precursor cells that have the potential of differentiating into six classes of neuronal cells and one class of glial cell.

Dorsal expression of ALDH in retina

Goal: To identify and characterize genes involved in the differentiation of retinal precursor cells. We are particularly interested in genes that function as transcriptional regulators or as signaling molecules as they have the potential of affecting the expression of numerous genes during retinal development and in retinoblastoma.

Experimental approach: We have used a variety of screening procedures to identify genes that are differentially expressed during retinal development and differentiation. One of the genes that was identified using these screens is the transcription factor AP-2. Based on expression analyses and in situ hybridizations, AP-2 appears to be critical for the differentiation of retinal precursor cells into two neuronal cell lineages called amacrine and horizontal. We are using chromatin immunoprecipitation (ChIP) to identify the target genes of AP-2. The role of AP-2 in retinoblastoma is also under investigation. In a separate study, we have found that the signaling molecule Disabled-1 (Dab1) exists in two forms in the developing retina and brain: an early form restricted to precursor cells and a late form restricted to differentiated cells. We are studying the role of these two forms of Dab1 in the developing retina and in retinoblastoma using mutagenesis assays, DNA transfection experiments and expression analysis.   

       

 Retina tissue section immunostained with AP-2

Project 2: Role of DEAD box proteins in retinoblastoma and retinal development

Background: DEAD box proteins are putative RNA unwinding proteins that have been implicated in all aspects of RNA metabolism (transcription, splicing, processing, translation). We identified DEAD box 1 (DDX1) in a differential screen of mRNAs expressed in retinoblastoma compared to normal tissue. Subsequent experiments demonstrated that DDX1 was amplified and over-expressed in a subset of retinoblastomas as well as neuroblastomas, another type of childhood tumour.

  DEAD box proteins conserved motifs

Goal: To determine the function of DDX1 in retinoblastoma and in normal retinal cells.                 
Experimental approach: Using confocal microscopy and a biochemical approach, we have demonstrated that DDX1 is primarily located in the nucleus where it exists in close association with proteins implicated in RNA transcription and processing. To further address the role of DDX1 in normal and cancer cells, we are using a specialized immunoprecipitation technique to identify the RNAs associated with DDX1. We are also using the yeast two-hybrid system to identify proteins that interact with DDX1. Transgenic mice carrying multiple copies of the DDX1 gene have been generated and attempts are being made to produce DDX1 gene knock-out mice.

 

Immunofluorescence analysis of DDX1 protein

 

Project 3: Brain tumours and expression of glial cell differentiation markers

Background: Malignant gliomas are brain tumours that are very difficult to treat. Patients diagnosed with these cancers usually die within two years of diagnosis. We have found that a marker of glial cell differentiation called “brain fatty acid-binding protein” (B-FABP) is expressed in a subset of malignant glioma tumour cell lines. Interestingly, B-FABP is coordinately expressed with a second marker of glial cell differentiation, called glial fibrillary acidic protein (GFAP).
 

Gel shift assay

B-FABP and GFAP expression in differentiating  glial cells

Goal: To understand the role that glial cell differentiation markers play in the biology of malignant glioma tumours.

 

Experimental approach: We have identified malignant glioma cell lines that express B-FABP and GFAP, and malignant glioma cell lines that don’t express these glial differentiation markers. B-FABP has been introduced into the “negative” cell lines, while an RNA interference approach was used to reduce B-FABP levels in a “positive” cell line. We are using these lines to address the role that B-FABP plays in cellular growth properties such as proliferation rate, growth in soft agar, invasiveness, motility, etc. We are also using microchip cDNA arrays to find differences between populations of cells that express B-FABP compared to those that don’t.