Gab1, Gab2, and Gab3 null mice have profoundly different phenotypes ranging from embryonic lethality (Gab1) to apparent normalcy (Gab3; Itoh et al., 2000
; Gu et al., 2001
; Seiffert et al., 2003
), consistent with the Gab proteins having distinct functional roles in development. In this work, we used RNA interference to silence Gab2 and Gab3 in the P19 EC cell line and showed that these proteins have nonoverlapping functions in bFGF survival signaling. Gab2, but not Gab3 suppression, reduced bFGF-mediated AKT activity and abrogated bFGF's ability to protect P19 cells from RA-induced cell death. In addition, Gab2 relayed the bFGF signal to AKT and not ERK, and it required both its PH domain and p85/PI3K binding sites to signal downstream. These results support the conclusion that Gab2 is a critical positive regulator of bFGF-stimulated signaling and that it modulates bFGF signaling mainly through the AKT pathway. Using the EC and ES system, we also demonstrated for the first time that Gab2 expression increased dramatically during embryonic neural differentiation, and in EC cells, Gab2 silencing significantly affected this process. Gab2 was already highly expressed in NSCs, suggesting that up-regulation of Gab2 may occur at the neural commitment stage, before neuronal differentiation. Lastly, Gab2 silencing in NSCs markedly reduced the ability of bFGF to support neurosphere proliferation and inhibited neuronal survival when NSCs were induced to differentiate. As Gab2 has been reported to participate in mast cell and macrophage differentiation (Gu and Neel, 2003
), it may have a more general role in cellular differentiation.
During development, various extracellular signaling molecules modulate neuronal differentiation. RA is one such molecule. RA is teratogenic when administrated in excess to pregnant animals and affects multiple systems including the central nervous system (Maden and Holder, 1992
). In Xenopus laevis
, RA regulates pattern formation of the vertebrate neural plate, by up-regulating prepattern and neurogenic genes, and by down-regulating genes that inhibit neurogenesis (Franco et al., 1999
). FGFs are also important factors of neuronal differentiation. bFGF participates in the development of the cerebral cortex and supports the proliferation of NSCs from the SVZ (Reynolds and Weiss, 1992
; Dono et al., 1998
). The FGF and RA pathways have been shown to have opposing effects in vivo (Appel and Eisen, 2003
). During spinal cord development in the chick embryo, FGF blocks expression of class I HD/bHLH transcription factors and Sonic hedgehog. This inhibits neurogenesis and maintains the caudal region as a stem zone. On the other hand, RA, arising from somites, antagonizes the FGF signal and promotes neurogenesis by up-regulating class I genes. Thus, FGF and RA coordinate the initiation of neurogenesis.
Gab2 is expressed in the brain but little is known about its function there. Our results firmly establish that Gab2 has a role in neural function and provides a link between bFGF and RA in embryonic neural differentiation. bFGF plays a complex role during neural development. It supports the proliferation and survival of ES cells and is needed for the expansion and maintenance of neuroectodermal and neural precursor cells (Reynolds and Weiss, 1992
). When neural precursor cells are induced to differentiate, the ratio of neurons to other cell types in culture appears to depend on the levels of bFGF present, low doses (0.1 ng/ml) favoring neuronal differentiation and high doses (10 ng/ml) glial generation (Qian et al., 1997
). In EC cells, Gab2 expression is markedly increased during RA-induced neural differentiation and is further enhanced by the addition of bFGF. Depletion of endogenous bFGF gives rise to a phenotype which is similar to that induced by Gab2 silencing. This leads to the interesting possibility that Gab2 may play a role in bFGF-dependent maintenance/expansion of neural precursors. Consistently, NSCs that are already committed to the neural fate but still retain the potential to differentiate, show high levels of Gab2 expression and Gab2 silencing significantly reduced NSC proliferation. bFGF may further modulate neurogenesis when NSCs are induced to differentiate. In the presence of exogenous bFGF, more cells survive but there is a concomitant reduction in the ratio of neurons to other cells, consistent with previous observations that high dose bFGF promotes glial formation. In the absence of exogenous bFGF, low levels of bFGF may still be present in the conditioned medium because neurons and astrocytes are reported to secrete bFGF (Reuss and von Bohlen und Halbach, 2003
). Thus, the effect on differentiation due to Gab2 suppression could still reflect a bFGF effect. However, neurotrophins are also secreted by NSCs and their receptors are up-regulated during differentiation (Barnabe-Heider and Miller, 2003
). Therefore we cannot exclude the possibility that Gab2 also functions downstream of Trk receptors to modulate the fate specification of multipotential progenitors. Irrespective of which upstream signal Gab2 is responding to during NSC differentiation, our data clearly support an essential role for Gab2 in mediating neuronal survival. Interestingly, because Gab2 expression was prominent in neurons but not astrocytes after NSC differentiation, our findings also argue for a possible role of Gab2 in lineage selection.
A key finding of our studies is that Gab2 regulates the bFGF survival pathway mainly through AKT. Both the p85 binding sites and the PH domain of Gab2 are crucial for bFGF-stimulated AKT activation. Suppression of Gab2 does not completely eliminate AKT activation, indicating that Gab2-independent pathways also exist. Gab3 is not a major contributor because Gab3 silencing does not affect bFGF-induced AKT activation. Deletion of the PH domain significantly reduces bFGF-mediated Gab2 tyrosine phosphorylation and AKT activation, suggesting that Gab2 needs to be recruited to the plasma membrane via PIP3 before it can become tyrosine phosphorylated. Altogether, these results support a model in which bFGF stimulates a Gab2-independent pathway to induce early PI3K–AKT activation. PIP3 generated during this early phase then binds to the PH domain of Gab2, recruiting Gab2 to the plasma membrane, whereby it becomes tyrosine phosphorylated at sites including those that dock p85. In this fashion, an amplification loop is created resulting in further AKT activation. This model is consistent with the finding that 3YF-Gab2 when overexpressed, functions in dominant-negative fashion, possibly by sequestering PIP3 and blocking signaling to AKT.
As we did not observe any defect in bFGF-mediated ERK activation in Gab2-silenced P19 cells, FGFR probably uses other proteins such as FRS2 to activate the ERK pathway. This is supported by the report that FRS2 is required for FGF1-induced ERK activation. Moreover, similar to our observations on Gab2, Gab1 is essential for FGF1-induced PI3K–AKT, but not ERK, activation (Lamothe et al., 2004
). Although Gab2 does not appear to play a major role in bFGF-induced ERK activation, it clearly contributes to ERK activation in response to other stimuli. For example, while IgE-provoked ERK activation in Gab2 knockout mice is not affected (Gu et al., 2001
), Erk activation induced by BCR/ABL is clearly decreased (Sattler et al., 2002
). Thus, depending on the stimulus, Gab2 may have differential effects on ERK activation.
Until now, Gab2 was mainly considered to be a regulator of hematopoiesis. Data shown in this study provide the first demonstration that Gab2 also functions in the nervous system by acting as a critical link between bFGF and the PI3K–AKT pathway. No gross brain defect was reported in Gab2 null mice; however, bFGF−/− mice were also initially reported to have normal brain morphogenesis. It was only recently that these mice were found to have a significant decrease in the number of neurons in the cerebral cortex (Dono et al., 1998
). It will be interesting to see if an in-depth investigation of Gab2 knockout mice will reveal defects in neural development and neurological function.