The Early Isoform of Disabled-1 Functions Independently of Reelin-Mediated Tyrosine Phosphorylation in Chick Retina

Abstract

The Reelin-Disabled-1 (Dab1) signaling pathway plays a key role in the positioning of neurons during brain development. Two alternatively spliced Dab1 isoforms have been identified in chick retina and brain: Dab1-E, expressed at early stages of development, and Dab1-L (commonly referred to as Dab1), expressed at later developmental stages. The well-studied Dab1-L serves as an adaptor protein linking Reelin signal to its downstream effectors; however, nothing is known regarding the role of Dab1-E. Here we show that Dab1-E is primarily expressed in proliferating retinal progenitor cells whereas Dab1-L is found exclusively in differentiated neuronal cells. In contrast to Dab1-L, which is tyrosine phosphorylated upon Reelin stimulation, Dab1-E is not tyrosine phosphorylated and may function independently of Reelin. Knockdown of Dab1-E in chick retina results in a significant reduction in the number of proliferating cells and promotes ganglion cell differentiation. Our results demonstrate a role for Dab1-E in the maintenance of the retinal progenitor pool and determination of cell fate.

Signaling by the Extracellular Matrix Protein Reelin Promotes Granulosa Cell Proliferation in the Chicken Follicle

“…Disruption of the pathway leads to a reduction in the number of rod bipolar cells. Interestingly, in the chicken retina, a splice variant of Dab1 is also expressed that functions independently of the Reelin signaling pathway (25).…”

“…chicken ApoER2 was detected with ␣19 (24) and ␣187 (23). The following antibodies were purchased from the indicated sources: rabbit polyclonal anti mouse Dab1 (400 -555, Rockland; this antibody has been shown to cross-react with chicken Dab1 (25) and has already been used to evaluate Dab1 levels in chicken brain (26)); mouse monoclonal anti-Reelin, clone G10 (Millipore); rabbit polyclonal anti-p-Dab1 (Tyr-220) (Santa Cruz Biotechnology); rabbit polyclonal anti-Akt and rabbit polyclonal anti-p-Akt (Ser-473) (Cell Signaling Technology); mouse monoclonal anti-GFP, clone B-2 (Santa Cruz Biotechnology); mouse monoclonal anti-BrdU, clone 3D4 (BD Biosciences); goat monoclonal anti-mouse and anti-rabbit IgG HRP-conjugated antibodies (Jackson ImmunoResearch Laboratories); and Alexa Fluor 488-coupled goat anti-mouse and anti-rabbit IgG (HϩL, Invitrogen). Rabbit polyclonal antichicken Dab1-E antibody (25) was a gift from Roseline Godbout (University of Alberta, Edmonton, Canada).…”

“…C, specificity of a newly developed mouse monoclonal anti-chicken Dab1 antibody (6H9-1A4) was tested using RIPA lysates of NIH 3T3 fibroblasts transiently transfected with pEGFP-C1 (lanes 1, 4, and 7), pEGFP-C1 containing ggDab1-E cDNA (lanes 2, 5, and 8), or pEGFP-C1 containing ggDab1-L cDNA (lanes 3, 6, and 9). GFP-tagged ggDab1-E and ggDab1-L were detected with 6H9-1A4, with an anti-chicken Dab1-E antibody (25), and with an anti-GFP antibody (clone B-2). GFP was detected with an anti-GFP antibody (clone B-2).…”

Serine phosphorylation regulates disabled-1 early isoform turnover independently of Reelin

“…We have identified two alternatively-spliced Dab1 isoforms in the developing chick retina: Dab1-E expressed in undifferentiated retinal progenitor cells, and Dab1-L expressed in differentiated ganglion, horizontal and amacrine cells [23,24]. Dab1-L, commonly referred to as Dab1, contains the five tyrosine residues described above, whereas Dab1-E is missing two SFK tyrosine phosphorylation sites, but retains two Abl tyrosine phosphorylation sites.…”

Reelin-Disabled-1 signaling in neuronal migration: splicing takes the stage

“…As different SH2 domains are connected to different downstream effectors, these results indicate that different Dab1 isoforms may direct Reelin signaling to distinct downstream cascades, leading to differential responses to Reelin stimulation. Dab1 isoforms are expressed in different subsets of cells at different developmental stages [69][70][71]. Differential interactions of Dab1 isoforms with distinct sets of SH2 domains may therefore have an important impact on the temporo-spatial coordination of neuronal movement in response to Reelin stimulation.…”