Our study describes the expression of Gαi2 in the spinal cord, and identifies a function for Gαi2 in the control of spinal motor neuron differentiation. We show that GDP-bound forms of Gαi2 preferentially interact with GDE2, a known regulator of motor neuron differentiation and that this interaction is necessary for generating the normal complement of spinal motor neurons. Our data suggest that Gαi2 interactions are unlikely to be mediated by upstream GPCR signals and invoke instead, a non-canonical function for Gαi2 in regulating motor neuron differentiation.
The progression of motor neuron differentiation can be monitored by the cell-body position of prospective motor neurons within the spinal cord. Cycling progenitors are located medially within the VZ, while cells in transition to a differentiated state are situated in the IZ and terminally differentiated motor neurons are located laterally in the MZ (Jessell, 2000
; Hollyday, 2001
; Price and Briscoe, 2004
). We show that different members of the Gαi family of proteins are expressed in overlapping patterns along the medial-lateral axis of the spinal cord. This expression pattern suggests that different Gαi proteins might function at different stages of motor neuron development, where medially expressed Gαi proteins such as Gαi2, play roles in motor neuron differentiation while more laterally expressed subunits might function in specifying postmitotic motor neuron fate or in regulating their function. Consistent with this prediction, knockdown of Gαi2
by siRNAs results in a reduction of postmitotic motor neurons, but no changes in the patterning of motor neuron progenitors, the number of Olig2+
progenitors or the rate of progenitor proliferation. We find that Gαi3 expression overlaps with Gαi2 in IZ cells of the spinal cord, suggesting potential redundant roles for Gαi3 in regulating motor neuron differentiation. Indeed, reports indicate that there is functional redundancy between Gαi2 and Gαi3, for instance, single Gαi2
null mutants are viable but Gαi2/Gαi3
double knockouts are lethal (Wettschureck et al., 2004
). In support of this possibility, we find that Gαi3 is capable of binding GDE2. Moreover, Gαi3 interacts with GDE2 when bound to GDP rather than GTP, suggestive of shared mechanisms between Gαi2 and Gαi3 in regulating GDE2-dependent motor neuron differentiation (G.P. and S.S., unpublished observations).
Our results suggest that Gαi2 mediates motor neuron differentiation in part through its interaction with GDE2. Interestingly, Gαi2 binds preferentially to GDE2 when bound to GDP rather than GTP. We infer from this observation that GDE2 is not an effector of Gαi2 mediated signaling from upstream GPCRs. If so, how might Gαi2 interactions with GDE2 operate to regulate motor neuron differentiation? One possibility is that GDE2 regulates downstream pathways through mechanisms utilizing Gαi2 in a manner similar to the mode of GPCR function. Some studies have described GDE2 as adopting a seven transmembrane structure, but these conclusions were based on predictions generated from computer based structural algorithms (Zheng et al., 2000
; Nogusa et al., 2004
; Yanaka, 2007
). In contrast to these predictions, epitope tagging experiments indicate that the N and C-termini of GDE2 are intracellular while the GDPD domain is extracellular, thus providing strong biochemical evidence that GDE2 is a six transmembrane protein (Rao and Sockanathan, 2005
). Since GDE2 does not conform to the typical seven transmembrane GPCR structure, it is highly unlikely that it functions as a classical GPCR. However, it is possible that the GDE family of proteins utilizes similar components of the GPCR signal transduction machinery to perform its functions. In support of this idea, the related two pass transmembrane GDPD protein GDE1 is known to bind RGS16, a protein that regulates G-protein signaling through its GTPase activity (Zheng et al., 2000
Alternative possibilities for how Gαi2-GDP might function in concert with GDE2 to regulate motor neuron differentiation arises from known GPCR-independent roles for Gαi2-GDP in asymmetric cell division and in receptor trafficking (Hampoelz and Knoblich, 2004
; Sans et al., 2005
). While the possibility that Gαi2-GDP interactions with GDE2 are involved in asymmetric cell division, which is a known prerequisite for triggering neuronal differentiation, is exciting, this function is unlikely, as GDE2 and Gαi2 expression overlap in non-dividing cells of the ventral spinal cord. In terms of receptor trafficking, it remains possible that Gαi2-GDP is required for optimal transport of GDE2 to or from, the cell surface. By analogy with the NR2B subunit of the NMDA receptor, Gαi2-GDP would increase levels of GDE2 to the membrane, thereby acting as a positive regulator of GDE2 signaling and promoting motor neuron differentiation (Sans et al., 2005
). This model is consistent with our results where ablation of Gαi2 causes reductions in postmitotic motor neuron numbers and a broadening of the domain of NeuroM expression in the ventral spinal cord. The sustained expression of NeuroM may result from slowing of the rate of GDE2 trafficking to the cell surface in the absence of Gαi2, and is suggestive of a delay rather than an absence of differentiation. Nevertheless, the mechanisms by which Gαi2-GDP interactions mediate GDE2-dependent motor neuron differentiation remain open and await further investigation.
In conclusion, our study identifies a role for GDP bound forms of Gαi2 in regulating neuronal differentiation that is likely to be distinct from its roles in mediating GPCR signaling. These observations raise the possibility that GDP forms of Gαi proteins may have new cellular functions in regulating multiple aspects of neuronal development and function.