Current models suggest that newly born motor neurons are initially a blank slate in terms of subtype identity, and that motor columnar and pool fates are instructed in these generic new-born motor neurons by Hox transcriptional programs and extrinsically-derived signals (Dasen and Jessell, 2009
). Our analyses of GDE2 function prompt these concepts to be reexamined. We show here that GDE2 does not regulate the production of all motor neurons but that GDE2 is required for the timing and formation of motor neurons of defined columnar and pool-specific identities. Strikingly, postmitotic Hox protein expression and activities are not directly affected by GDE2. Instead, GDE2 downregulates Notch signaling pathways in neighboring progenitor cells through a non cell-autonomous mechanism that depends on extracellular GDE2 GDPD activity. This mechanism of GDE2 function is consistent with our observations that ablation of GDE2 decreases progenitor cell-cycle exit, prolongs the mitotic cell-cycle, delays the birth of prospective medially located LMC motor pools, and results in the failure of lateral motor pool formation. Thus, GDE2 regulates the generation of specific motor neuron subtypes through its role in triggering the differentiation of motor neuron progenitors into postmitotic motor neurons (Figure S6
These findings have several implications. First, they suggest that signals from postmitotic motor neurons are required for the formation of specific motor neuron subtypes at the level of motor neuron progenitor differentiation, a previously unrecognized concept in existing models of motor neuron diversification. In our model, MMC motor neurons, which are born prior to LMC neurons and do not require GDE2 for their formation, serve as an initial source of GDE2 that regulates the progressive generation of prospective LMC motor neurons from adjacent motor neuron progenitors. This function also applies to forelimb regions, as GDE2 is differentially required for the formation of C7–8 Pea3−
motor pools (P.S and S.S., unpublished observations). This strategy for building complexity within motor neuron populations is particularly compelling since the MMC is thought to be the ancestral motor column while the LMC is a more recent structure that evolved in accordance with limb development (Fetcho, 1992
; Dasen et al., 2008
). Feedback signaling mechanisms from postmitotic neurons to progenitor cells have been reported to control differentiation in other structures such as the cortex, where signals from cortical neurons can influence astrocyte generation during the neuronal to glial switch (Namahira et al, 2009
; Seuntjens et al., 2009
). Our finding that feedback signals also control subtype identity within a single class of neurons suggests that this strategy may form a general mechanism to control cell diversity in the developing nervous system.
A second implication from this study is that newly-born motor neurons are unlikely to be generic as previously believed given their differential requirements for GDE2 for their generation, but are inherently biased towards distinct postmitotic fates. The ability of Hox proteins to alter motor neuron identities in postmitotic motor neurons implies that such fates are not hardwired but are plastic to some degree. We suggest that hierarchical Hox transcriptional programs and additional signals act to consolidate and refine critical columnar and motor pool properties in newly born motor neurons, thus ensuring appropriate connectivity and function of motor circuits over time (Dasen et al., 2003
; Dasen et al., 2005
; Jung et al., 2010
). Conceptually, this model invokes that elements of postmitotic motor neuron identity are encoded in progenitor cells prior to their differentiation into postmitotic motor neurons, and implies that motor neuron progenitors are not uniform but are specified towards distinct postmitotic fates. While our data indicate that such specification includes columnar and pool identities, they also raise the possibility that alpha and gamma motor neuron identities might be encoded within motor neuron progenitors. This hypothesis stems from two observations: first, that the specific loss of LMC alpha motor neurons in postnatal Gde2−/−
animals correlates with the embryonic phenotype, where the formation of specific LMC motor pools is compromised while MMC motor neurons are unchanged; and second, that the reduction of LMC alpha motor neurons is highly unlikely to be a consequence of altered sensory neuron and interneuron formation in the absence of GDE2, as previous studies show that these neuronal subtypes are dispensable for alpha motor neuron formation and function (reviewed by Grillner and Jessell, 2009
). However, further study is required to test this hypothesis as our studies do not exclude alternative interpretations that are independent of progenitor specification, for instance that alpha motor neuron differentiation is predicated on the total number of motor neurons within a motor pool but that gamma motor neuron differentiation is not. Nevertheless, our data collectively suggest that similar to mechanisms that direct the diversification of different neuronal classes within the spinal cord, the acquisition of motor neuron subtype identity is a dynamic and progressive process that is initiated in motor neuron progenitors and continues in postmitotic motor neurons in accordance with their axial position relative to their final targets.
Our analysis of GDE2 function indicates that GDE2 triggers neighboring motor neuron progenitors to undergo differentiation by GDPD inhibition of Notch signaling. Notch signaling maintains the proliferative state of progenitor cells in part by inhibiting the expression of proneural genes such as Mash1 and Ngn2 (reviewed by Corbin et al., 2008
). Ngn2 in particular plays pivotal roles in synchronizing neurogenesis and motor neuron specification by decreasing Olig2:Ngn2 ratios to promote neuronal differentiation, and by directly interacting with Lhx3 and Isl1 to regulate the transcription of motor neuron-specific genes (Lee and Pfaff, 2003
). Overexpression of GDE2 in the chick spinal cord is sufficient to induce ectopic Ngn2 expression, supporting the model that GDE2 promotes motor neuron differentiation via the derepression of Notch-dependent Ngn2 inhibition (M.R and S.S, unpublished observations). It is widely accepted that Notch signaling plays important roles in generating diversity in neural progenitors. For example, differential Notch activity plays central roles in the sequential specification and binary fate choices of progenitors in the Drosophila peripheral nervous system, as well as in maintaining the heterogeneity of mammalian cortical progenitors (reviewed in Corbin et al., 2008
). Accordingly, it is possible that differential Notch signaling could similarly encode aspects of postmitotic motor neuron subtype identity in motor neuron progenitors, and that GDE2-dependent downregulation of Notch signaling could control the differentiation of pool-specific motor neurons. How GDE2 controls the temporal formation of medial LMC neurons via inhibition of Notch signals is less clear. The difference in GDE2 function in terms of regulating the timing of medially located LMC pool formation versus its requirement for the generation of laterally located motor neuron pools correlates with their birthdates, as medial motor pools are born earlier than lateral pools (Nornes and Carry, 1978
; Whitelaw and Hollyday, 1983
). We speculate that the levels of GDE2 targets might vary over time such that the precise modulation of Notch signaling could directly influence both motor neuron fates and birthdates.
Two major questions that emerge from this work are what are the direct targets of GDE2 GDPD activity, and how do they affect Notch signaling? Definitive identification of GDE2 GDPD substrates is currently underway; however, potential candidates are known from studies in non-neural cells, where GDE2 metabolizes glycerophosphocholine into glycerol-3-phosphate and choline (Gallazzani et al., 2008
). However, it is still unclear if glycerophosphocholine is indeed the physiological substrate for GDE2, and if so, how its metabolism could specifically inhibit Notch signaling. Further elucidation of the molecular mechanisms involved will provide key insight into how motor neuron diversity is generated, and may define general principles that underlie the regulation of neuronal differentiation in the developing nervous system.