Factors first identified as inductive signals that regulate cell fate and tissue organization have recently been shown to have crucial roles in acute activities such as growth cone guidance and axon path finding . This principle emerged from studies of the developmental actions of fibroblast growth factors and bone morphogenetic proteins (BMPs) [2–4], and has been shown more recently also to apply to Wnt [5, 6] and Hh  signaling. These observations pose the question of how distinctive developmental activities can be generated by the same ligand. In principle, a number of strategies might achieve such a dichotomy: different presentation of the ligand and/or mechanisms of selective receptor engagement could activate distinct intracellular pathways. The initiation of parallel or divergent signaling cascades presumably lies at the heart of distinct cellular events. But where and how such signaling pathways diverge remains unclear.
BMPs trigger long-term inductive signaling events that involve gene transcription and/or the acute cellular responses of chemotaxis and axon orientation, in both neurons and non-neuronal cells [3, 8]. Instances in which long-term and acute responses to the same BMP can occur concurrently in a single cell, illustrated in monocytes [9, 10], emphasize the requirement for divergent pathways and selective regulation of their activation. One cellular system that relies on sequential but distinct cellular responses to BMPs is the development of sensory projection neurons in the dorsal horn of the spinal cord. BMPs supplied by the roof plate initially specify the fates of several subsets of dorsal interneurons (dI neurons), directing expression of dI neuron class-specific transcription factors [11–14]. Subsequently, BMPs orient the axons of these post-mitotic dI neurons, directing their growth away from the dorsal midline [3, 4, 15] and also regulate the rate of growth of dI axons as they extend through the spinal cord . Both orientation and rate of growth appear to occur within minutes in vitro, suggesting they are regulated independently of the early inductive BMP pathways. Moreover, intriguingly, whereas the two highly related roof plate-derived BMPs, BMP7 and BMP6, both induce the differentiation of dI neurons [3, 4, 12, 13], BMP7, but not BMP6, is also able to orient dI axons in vitro and is required for appropriate dI axon projections in vivo [3, 4].
How BMPs signal the distinct activities in spinal neurons is unclear. The slow time course and molecular changes in dI neuronal specification in response to BMPs imply activation of a nuclear signaling pathway. The core pathway underlying the transduction of BMP signals from the surface of a cell to the nucleus typically involves ligand-induced recruitment and activation of a BMP receptor complex, which comprises one pair each of type I and type II receptor subunits. BMP binding promotes phosphorylation of type I by type II BMP receptors [17, 18]. Activated type I BMP receptors phosphorylate receptor-associated Smad1/5/8 proteins, resulting in nuclear translocation of Smad complexes and activation or repression of transcription of BMP target genes [18, 19]. In monocytes, BMP7 and BMP6 activate Smad1/5/8 phosphorylation and Smads are required for gene induction . However, a role for Smads as intracellular mediators in the induction of dI neuron-specific genes by BMPs has not been demonstrated and the question of how this pathway is transduced remains unsolved. In contrast to BMP-induced neural specification, the rapid time course of BMP-evoked growth cone orienting responses of dI neurons points to the recruitment of acute, transcription-independent pathways . Although there is a growing appreciation of the existence of transcription-independent responses to BMPs, much less is known about acute BMP signaling than its classical inductive counterpart. In monocytes, Smad4 appears not to be required for BMP7-evoked chemotaxis . Moreover, although in monocytes and other cell systems, effectors of cytoskeletal dynamics, such as PI3K, LIMK, and Rho family GTPases have been implicated as mediators of BMP-stimulated responses [10, 16, 20, 21], their role in BMP-evoked axon orientation in dI neurons remains to be determined. Indeed, recent studies suggest that the activation of LIMK by BMPs regulates the rate of extension of dI axons, but not their orienting response to BMP7 .
Elucidating signaling components is an important step towards understanding the differential selection of transduction pathways, but how might BMPs activate distinct intracellular signaling pathways? Experiments on BMP7-evoked gene induction and chemotaxis in monocytic cells suggest that recruitment of different canonical BMP receptor subunits may represent an early step in triggering divergent signaling paths. Most tellingly, although it seems likely that type II BMP receptors are required, the inductive pathway does not appear to depend on a specific type II receptor, whereas the selective involvement of two of the three known type II BMP receptor subunits, ActRIIA and BMPRII, is required for BMP7-evoked chemotaxis . The view that activation of particular type II BMP receptors is sufficient to initiate transcription-independent, acute cellular responses is supported by the observation that PI3K and LIMK can bind directly to the intracellular domains of type II BMP receptors [22–24]. Moreover, the BMPRII subunit has been implicated in eliciting LIMK-dependent responses to BMPs [16, 22]. However, the evidence that type II BMP receptors direct acute signaling that diverges from the classical inductive events does not resolve whether they act in the context of the canonical type I/type II BMP receptor complex. Type I BMP receptor activity has been linked previously with activation of transcriptional BMP responses [14, 25, 26]. Nevertheless, the loss of BMPRIB in dI neurons and in ventral retinal ganglion neurons results in aberrant axon guidance [27, 28]. From all these studies, a model is emerging in which canonical type I and type II BMP receptors support both the inductive specification and axon orienting activities of BMPs but the nature of the complex that drives orientation and the role of the individual receptor subunit activity remain unclear.
In the light of these findings, we have begun to resolve how BMPs exert their dual developmental effects on dI neurons by further evaluating the contributions of BMP receptor subunits and downstream signaling pathways to the inductive specification and axon orienting activities of BMP7. We have also examined how the selectivity of such responses is achieved. We have exploited the difference in axon orienting ability between BMP7 and BMP6, comparing requirements for their activities in neurons isolated in dissociated culture and in spinal explants. We demonstrate divergent BMP signaling pathways that operate concomitantly: a classical type I BMP receptor kinase-mediated path to BMP7-evoked Smad activation and neural specification, and a pathway dependent on PI3K activity, which independently mediates the orienting response of spinal axons to BMP7. Our results suggest a model in which BMP-evoked inductive specification in the dorsal spinal cord depends on type I BMP receptor activity and involves classical Smad signaling to the nucleus, whereas BMP7-elicited axon orientation depends on activation of PI3K signaling independent of type I BMP receptor activity and the Smad cascade, through differential engagement of type II BMP receptor subunits.