Spinal motor neurons (MNs) innervating the limb are contained within the lateral motor columns (LMCs), which are produced specifically at brachial and lumbar levels of the spinal cord. LMC neurons are subsequently divided into two populations, lateral LMC (LMCl) neurons that innervate the dorsal part of the limb and medial LMC (LMCm) neurons that innervate the ventral part of the limb (Landmesser, 1978; Hollyday, 1980a; Hollyday, 1980b; Tosney and Landmesser, 1985b; Tosney and Landmesser, 1985a; Sockanathan et al., 2003; Ji et al., 2006). The molecular mechanism of how morphogenetic signaling molecules cooperate with transcription factors to define MN subtype specification has been extensively studied (Shirasaki and Pfaff, 2002; Lee and Pfaff, 2001). Retinoic acid (RA) is essential for the diversification of MN subtype and MN columnar organization. At brachial level, within the LMCs, RA is synthesized in subpopulations of MNs expressing the RA-synthesizing enzyme, RALDH2, and specifies migrating MN precursor cells into the LMCl neurons (Vermot et al., 2005; Sockanathan et al., 2003). However, it remains poorly understood whether other signaling molecules also contribute to the specification and/or maintenance of LMC and other MN columns.

Sonic hedgehog (Shh) signaling is a highly conserved pathway that is essential in directing cell proliferation and patterning during early embryogenesis (Ingham et al., 2011; Briscoe and Thérond, 2013). In spinal neuron development, Shh, released from the notochord and floor plate, leads to the generation of distinct classes of progenitor domains including MN progenitor (pMN) within the ventral side of spinal cord (Lee and Pfaff, 2001). In Shh-responsive cells, protein kinase A (PKA) plays crucial roles in fate specification and proliferation by modulating the activity of Shh signaling (Kotani, 2012; Asaoka, 2012). In the absence of Shh, PKA phosphorylates Gli transcription factors, promoting the production of repressor forms of Gli and thus repressing the Shh target gene expression, while Shh antagonizes this action of PKA (Pan et al., 2009). As both decreased and increased activity of PKA results in abnormal cell proliferation and cell fate specification, the basal level of PKA activity should be precisely controlled spatiotemporally to ensure its proper action (Kotani, 2012). PKA activation occurs upon binding of cyclic AMP (cAMP) to its regulatory subunits (PKAR), causing the release of its catalytic subunits (PKAC) (Taylor et al., 1990). Shh is able to induce phosphoinositide 3-kinase (PI3-kinase)-dependent AKT phosphorylation in cell lines such as LIGHT cells and HUVEC cells (Kanda et al., 2003; Riobó et al., 2006). Interestingly, PI3-kinase-dependent AKT activation further potentiates Shh signaling in the neuronal fate specification (Riobó et al., 2006). It was suggested that AKT might target proteins that modulate PKA kinase activity or the interaction between PKA and Gli (Riobó et al., 2006), but the identity of such proteins remain unknown.

During MN development, two LIM-homeodomain (LIM-HD) transcription factors, Islet-1 (Isl1) and LIM homeobox-3 (Lhx3) act as essential players in MN fate specification (Pfaff et al., 1996; Sharma et al., 1998; Tanabe et al., 1998; Thaler et al., 2002; Lee et al., 2013) by forming a hexameric complex, named the Isl1-Lhx3 complex, which consists of two Isl1:Lhx3 dimers and a nuclear LIM interactor (NLI) dimer (Lee et al., 2008; Lee et al., 2013). Genome-wide analyses of the Isl1-Lhx3 complex binding sites from chromatin immunoprecipitation-sequencing (ChIP-seq) datasets (Lee et al., 2013; Mazzoni et al., 2013) combined with MN transcriptome analyses (Lee et al., 2012; Mazzoni et al., 2013) identified many novel signaling pathways and regulators that are directly regulated by the Isl1-Lhx3 complex. Studies of individual target genes of the Isl1-Lhx3 complex in MN specification uncovered critical effector genes in MN specification such as genes involved in cholinergic neuronal identity determination (Cho et al., 2014), miR-218 as a downstream effector of Isl1-Lhx3 complex (Thiebes et al., 2015) and STAM1 as an endosomal sorting machinery necessary for ventral motor axon projection (Nam and Lee, 2016).

ARHGAP36, a putative Rho GTPase-activating protein, was identified from a genome-scale cDNA overexpression screen as a positive regulator of the Shh pathway (Rack et al., 2014). Overexpression of ARHGAP36 recapitulates Shh signaling transduction and these effects are independent of Smo and require kinesin family member 3a (Kif3a) and intraflagellar transport protein 88 (Ift88) (Rack et al., 2014). Recently, ARHGAP36 has emerged as a potent antagonist of PKA signaling. ARHGAP36 interacts with PKAC and inhibits PKAC catalytic activity as a PKA pseudosubstrate inhibitor (Eccles et al., 2016). It also targets PKAC for ubiquitin-dependent proteolysis by the endosomal sorting complex required for transport (ESCRT) pathway (Eccles et al., 2016). Moreover, ARHGAP36 interaction with Patched1 leads to the removal of centrosomal ARHGAP36 with ciliary Patched1 and the accumulation of centrosomal PKA that phosphorylates Inversin and this Patched1-ARHGAP36-PKA-Inversin axis determines the ciliary translocation of Smoothened and consequent hedgehog pathway activation (Zhang et al., 2019). Although recent studies in cultured cells suggest that ARHGAP36 regulates Shh activity through inhibiting PKA kinase activity, it remains unexplored whether ARHGAP36 functions as a key modulator of Shh signaling pathway in vivo.

Here we report that Shh expression is induced in postmitotic MNs at brachial and lumbar levels but not at thoracic level at later stages of development when motor columnar identities are established. Shh is required for proper LMC formation as the knock-down of Shh in the developing chick spinal cord and the deletion of Shh in the developing MNs of mouse embryos result in reduction of FoxP1⁺ LMC. We further show that ARHGAP36 is a critical MN-enriched Shh transduction component and Arhgap36 is a direct target gene of Isl1-Lhx3 complex during MN generation. The action of ARHGAP36 is to promote Gli-dependent transactivation partly through inhibition of PKA activity. Moreover, AKT, known as an inhibitor of PKA activity, interacts with ARHGAP36 and stabilizes ARHGAP36 protein, which further enforces suppression of PKA activity by ARHGAP36. Consistently, blocking AKT activity reduces the protein level of ARHGAP36 and hinders the efficiency of in vitro MN differentiation from mouse ESCs. Consistently, deletion of Arhgap36 gene in mouse results in defects in LMC formation at brachial level, which might be caused by dysregulation of Shh signaling pathway. Our results define a new regulatory axis of Shh-AKT-ARHGAP36-PKA in LMC specification, in which ARHGAP36 functions as an essential effector of Shh and AKT in repressing PKA activity.

While the role of Shh signaling pathway to induce ventral progenitor domains in the spinal cord has been relatively well characterized (Persson et al., 2002; Jessell, 2000), its role in LMC specification has not been studied. In this report, we discovered a novel role of Shh in inducing LMC specification, which involves coordination of multiple signaling pathways by ARHGAP36, a key modulator of Shh signaling pathway. First, we discovered that Shh is expressed in MNs at brachial and lumbar levels of the spinal cord where LMC neurons are specified and is required for proper LMC formation in developing chick and mouse spinal cord (Figures 1–3). Second, we identified ARHGAP36, along with Shh, as a protein highly expressed in the LMC regions of differentiating MNs (Figure 5C). Third, ARHGAP36 modulates the activity of PKA, an inhibitor of Shh pathway, thereby enhancing the activity of Gli-dependent transcription in the spinal cord (Figure 6—figure supplement 1). Fourth, ARHGAP36 levels seem to be tightly regulated by AKT during MN generation, as shown by the increase of ARHGAP36 protein levels by WT AKT and a constitutively active form of AKT and a decrease in ARHGAP36 levels by a dominant negative form of AKT and AKT inhibitor (Figure 7 and Figure 7—figure supplements 1–2). Fifth, deletion of Arhgap36 in mouse results in specific reduction of FoxP1⁺ LMC MNs in the developing mouse embryonic spinal cord (Figure 8), which is similar to what was observed in Shh knock-down chick spinal cord and Shh-cKO mouse spinal cord (Figures 2 and 3). Taken together, our results reveal a regulatory axis consisting of Shh-AKT-ARHGAP36-PKA, which plays crucial roles in modulating the activity of Shh signaling in a spatiotemporal manner for LMC specification.

Once MN progenitors, produced in the pMN progenitor domain of the ventral neural tube in response to the morphogen Shh, give birth to MNs (Jessell, 2000), MNs are further organized into distinct motor columns that are responsible for innervating each target muscle along the rostral-caudal neural tube (Dasen and Jessell, 2009; Stifani, 2014). MMC neurons innervate dorsal epaxial muscles, whereas HMC neurons project to the ventral hypaxial muscles. The LMC neurons innervate limb muscles and PGC neurons innervate sympathetic ganglia (Stifani, 2014; Dasen and Jessell, 2009). Motor column specific transcription factors and morphogenetic signaling molecules collaborate to define MN subtype specification (Shirasaki and Pfaff, 2002; Lee and Pfaff, 2001). RA is essential for the diversification of MN subtype and MN columnar organization. Also Hox genes, which encode a family of transcription factors, determine MN subtypes and there is a clear relationship between Hox protein expression and motor columnar specification. Moreover, FoxP1 has been shown to function as a critical Hox cofactor in regulating MN subtype diversity especially for specification of both the LMC and PGC neurons (Rousso et al., 2008; Pfaff, 2008; Dasen et al., 2008; Arber, 2008). It has been shown that abnormal expression of Hox proteins within postmitotic MNs result in subtype switching (Jung et al., 2010; Wu et al., 2008; Dasen et al., 2005; Lin and Carpenter, 2003). The spatiotemporal expression of these HD factors together with extrinsic signaling suggest that MN subtype identity remains plastic even after they exit the cell cycle and it should be tightly regulated to generate proper MN columnar subtypes. It is intriguing that two prominent extrinsic cues, RA and Shh, participate in LMC specification. Future studies should be directed at elucidating whether Shh pathway involving ARHGAP36 integrates with RA and Hox genes in LMC specification.

Because LMC and PGC neurons do not express Lhx3, it is not clear whether ARHGAP36 induced by the Isl1-Lhx3 complex at earlier stages of MN development (Figure 5) persists in LMC and PGC neurons or another mechanism independently induces the expression of ARHGAP36 in these specific motor columns at later stages. Because Shh stabilizes ARHGAP36 through AKT activation (Figure 7) and Shh is only expressed in LMC neurons at later developmental stages (Figure 1), Shh may be responsible for the continuous existence of ARHGAP36 at least in LMC neurons via its ability to stabilize ARHGAP36 protein. It is also possible that Hox and its cofactor Pbx may additionally upregulate the expression of ARHGAP36 at later stages of MN columnar specification (Hanley et al., 2016).

We found that there was no obvious defect in the general MN formation in Arhgap36 knockout mouse embryonic spinal cord at early stages, suggesting that its function is not necessary for the initial MN differentiation. This conclusion is supported by the lack of Shh expression in early born MNs and with our notion that Shh agonist is likely needed for the activity of ARHGAP36 (Figure 6—figure supplement 2) as well as our finding that overexpression of AKT shows specific effect only on FoxP1⁺ LMC neurons (Figure 7). Because ARHGAP36 is also expressed in early born MNs, however, ARHGAP36 may also have a critical role in initial MN generation. This action of ARHGAP36 may have a redundant functional homologue, given no deficits were observed in early MN generation in Arhgap36 knockout mouse embryonic spinal cord. Furthermore, there was no obvious defect even in the LMC formation in male mutant spinal cord at later stages. Based on the amino acid sequence of ARHGAP36, it is predicted to be a Rho GAP family member, but it lacks the ‘arginine finger’ motif for Rho GTPase activity (Rittinger et al., 1997), suggesting that the catalytic GAP domain is not required for Gli activation. Arhgap6 is the closest homolog of Arhgap36, which does not affect Shh activation (Rack et al., 2014). It will be interesting to further investigate whether another functional homologue to ARHGAP36 controls initial MN generation as well as LMC formation particularly in male embryos. At E12.5, we observed that LMCm (Isl1⁺/FoxP1⁺) neurons are increased, whereas LMCl (Lhx1⁺/FoxP1⁺) neurons are decreased in Arhgap36 mutant spinal cord (Figure 8—figure supplement 1). But later on, the number of FoxP1⁺ LMC neurons decreased significantly in Arhgap36 deficient mouse embryonic spinal cord. These results suggest that the increased early born LMCm neurons in the Arhgap36 mutant spinal cord somehow adopt other cell fates or degenerate. These possibilities need to be further investigated in the future. Later born LMCl neurons have to migrate through earlier born LMCs (Sockanathan et al., 2003; Maden, 2006) but it is not known whether early born LMCm neurons can change their fate into LMCl neurons by receiving signals such as RA and Shh from the neighboring cells. It will be interesting to further investigate whether early born LMCm neurons affect LMCl neuron specification through Shh signaling pathway and its modulator ARHGAP36.

In the developing central nervous system, a number of differentiated cells degenerate during development (Buss et al., 2006; Oppenheim, 1991). Initially overproduced MNs undergo cell death by competing for a limited amount of neurotropic factors when they arrive at the target muscles (Yamamoto and Henderson, 1999). LMC neuronal axons navigate through a very long distance to their final destinations by integrating intrinsic factors and extrinsic pro- and anti-apoptotic signals. Based on the well-known function of AKT in cell survival, we speculate that ARHGAP36 proteins stabilized by AKT may also function to maintain the proper number of differentiated LMC neurons by blocking their apoptosis. Indeed, we observed an increase in cell death with a reduction of FoxP1⁺ LMC neurons in Arhgap36 KO mouse (Figure 8—figure supplement 1D), suggesting that ARHGAP36 may function as an essential downstream effector of AKT-directed cell survival.

It remains to be further investigated how ARHGAP36 affects the FoxP1⁺ LMC cell fate specification. It has been shown that FoxP1 expression is regulated by PI3K/AKT/p70S6K signaling cascade in breast cancer (Halacli and Dogan, 2015). These results further support our proposal that the expression of FoxP1 in LMC neurons is likely regulated by activated AKT in collaboration with ARHGAP36. Furthermore, ARHGAP36 is up-regulated in medulloblastoma where Shh pathway is aberrantly activated (Rack et al., 2014), suggesting that similar mechanisms may be applied to tumorigenesis. Therefore, it will be interesting to further study the function of ARHGAP36 in Shh-dependent tumorigenesis, for instance using the cerebellar granule neuron progenitors (GNPs) from Arhgap36^-/- mice.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Heejin Nam

   College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea

   Contribution

   Data curation, Formal analysis, Validation, Writing—original draft

   Competing interests

   No competing interests declared

2. Shin Jeon

   1. College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea
   2. Neuroscience Section, Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science Uiversity, Portland, United States

   Contribution

   Resources, Validation, Methodology

   Competing interests

   No competing interests declared

3. Hyejin An

   College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea

   Contribution

   Data curation, Formal analysis, Writing—review and editing, Generated new images with Shh KD experiment for the revision and counted cell numbers for quantification

   Competing interests

   No competing interests declared

4. Jaeyoung Yoo

   College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea

   Contribution

   Data curation, Formal analysis, Writing—review and editing, Generated new images for early stage Arhgap36 KO embryos for the revision and counted cell numbers for quantification

   Competing interests

   No competing interests declared

5. Hyo-Jong Lee

   College of Pharmacy and Inje Institute of Pharmaceutical Sciences and Research, Inje University, Gyungnam, Republic of Korea

   Contribution

   Data curation, Formal analysis, Writing—review and editing, Generated new images of Shh-cKO embryos with BrdU incorporation, Ki67, MN columnar marker stainings for the revision and counted cell numbers for quantification

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4826-7954

6. Soo-Kyung Lee

   1. Neuroscience Section, Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science Uiversity, Portland, United States
   2. Vollum Institute, Oregon Health and Science University, Portland, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1745-256X

7. Seunghee Lee

   College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   leeseung@snu.ac.kr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5483-0016

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