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. Author manuscript; available in PMC: 2016 Mar 9.
Published in final edited form as: Cell Rep. 2016 Feb 18;14(8):1901–1915. doi: 10.1016/j.celrep.2016.01.067

Hox Proteins Coordinate Motor Neuron Differentiation and Connectivity Programs Through Ret/Gfrα Genes

Catarina Catela 1, Maggie M Shin 1, David H Lee 1, Jeh-Ping Liu 2, Jeremy S Dasen 1
PMCID: PMC4775310  NIHMSID: NIHMS756654  PMID: 26904955

Summary

The accuracy of neural circuit assembly relies on the precise spatial and temporal control of synaptic specificity determinants during development. Hox transcription factors govern key aspects of motor neuron (MN) differentiation; however, the terminal effectors of their actions are largely unknown. We show that Hox/Hox cofactor interactions coordinate MN subtype diversification and connectivity through Ret/Gfrα receptor genes. Hox and Meis proteins determine the levels of Ret in MNs and define the intrasegmental profiles of Gfrα1 and Gfrα3 expression. Loss of Ret or Gfrα3 leads to MN specification and innervation defects similar to those observed in Hox mutants, while expression of Ret and Gfrα1 can bypass the requirement for Hox genes during MN pool differentiation. These studies indicate that Hox proteins contribute to neuronal fate and muscle connectivity through controlling the levels and pattern of cell surface receptor expression, consequently gating the ability of MNs to respond to limb-derived instructive cues.

Graphical Abstract

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Introduction

Correct wiring of nervous systems involves both cell intrinsic factors that contribute to neuronal subtype identities and cell surface recognition systems that facilitate the connectivity of individual neurons. Although synaptic specificity determinants have been described in many systems (Dudanova and Klein, 2013; Kolodkin and Tessier-Lavigne, 2011; Sanes and Yamagata, 2009); the regulatory mechanisms governing the expression of guidance and adhesion molecules are poorly defined. Cell fate determinants likely play a key role in orchestrating synaptic specificity programs, but there are few examples where the relationships between transcription networks and cell surface receptors have been established (Polleux et al., 2007; Santiago and Bashaw, 2014; Zarin et al., 2014b).

Progress towards defining how neuronal identity directs connectivity has emerged through analysis of guidance decisions within the vertebrate spinal cord (Bonanomi and Pfaff, 2010; Klein and Kania, 2014). MNs targeting limb muscles are contained within the lateral motor columns (LMCs), which further differentiate into divisional and pool subtypes targeting specific limb regions (Landmesser, 2001). Neurons within the lateral division of the LMC innervate dorsal limb muscles, while medial LMC neurons project ventrally (Landmesser, 1978a; Tosney and Landmesser, 1985). MN pools positioned rostrally within the LMC typically pursue an anterior/proximal trajectory, while those residing more caudally innervate more posterior/distal limb muscles (Hollyday and Jacobson, 1990; Landmesser, 1978b). Experimental manipulations that alter MN or limb bud position indicate that motor axons can redirect their trajectories to find their appropriate targets (Ferguson, 1983; Lance-Jones and Landmesser, 1980, 1981; Stirling and Summerbell, 1988), suggesting that groups of MNs within the LMC are intrinsically programmed to select a specific pathway in response to limb-derived cues.

Depletion of factors involved in LMC subtype differentiation often disrupts the specificity of muscle target innervation, presumably due to changes in the expression of surface receptors on motor axons. For example, the Lim homeodomain (HD) transcription factors Lhx1 and Isl1 control expression of Eph guidance receptors and dictates the initial trajectories of motor axons at the base of the limb. Lhx1 induces expression of Epha4 in the lateral division of the LMC to direct motor axons dorsally within the limb, while Isl1 promotes Ephb1 expression in medial LMC neurons to direct axons ventrally (Helmbacher et al., 2000; Kania and Jessell, 2003; Luria et al., 2008). With the exception of this relatively simple binary decision, the mechanisms that restrict expression of surface receptors within specific MN subtypes are poorly understood.

A large family of transcription factors critical for MN subtype differentiation and connectivity are encoded by the Hox gene clusters, which comprise 39 genes organized in 4 chromosomal arrays (Philippidou and Dasen, 2013). Studies in chick have shown that Hox genes are differentially expressed by MNs along the rostrocaudal axis, and a network of ~20 Hox proteins defines the identities of MN pools targeting specific limb muscles (Dasen et al., 2003; Dasen et al., 2005; Liu et al., 2001). However, whether the pattern of Hox gene expression in MNs is predictive of muscle target specificity is largely untested, and the precise requirements for the majority of Hox genes are not well established. Moreover, with the exception of a handful of downstream transcription factors, the target effectors of Hox proteins in MNs are yet to be determined.

In this study we assessed the mechanisms through which Hox genes control expression of cell surface receptors during MN subtype differentiation. We show that MNs extending along the major nerves within the forelimb are defined by specific profiles of Hox expression, and that Hox genes are essential in establishing the pattern and specificity of limb muscle innervation. Hox genes determine MN pool fates and connectivity patterns through controlling the profile of Ret and Gfrα expression, and by constraining the ability of MNs to respond to limb-derived signals. We suggest that Hox genes govern neuronal fate and target connectivity by defining both the level and pattern of cell surface receptor gene expression.

Results

Hox Profiles Define MN Groups Targeting Forelimb Muscles

To explore the relationship between profiles of Hox protein expression in MNs and peripheral target specificity, we first sought to establish a detailed molecular map of MN groups targeting forelimb and shoulder muscles in mice. We determined the position of MNs projecting along the nine major nerve pathways within the forelimb at embryonic stage (e) 12.5 (Figure 1A–B). Consistent with studies in postnatal rat and embryonic chick (Bacskai et al., 2013; Hollyday and Jacobson, 1990; Tosolini and Morris, 2012), MNs targeting proximal forelimb muscles were located in rostral spinal segments, while MNs projecting distally were positioned caudally. Furthermore, ventrally located MNs adopted a proximal trajectory whereas distal limb muscles were innervated by dorsally located MNs (Figure 1B, S1A–I).

Figure 1. Combinatorial Hox Profiles Define Forelimb Innervating MNs.

Figure 1

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(A) Schematic of the primary motor nerves supplying the mouse forelimb at e12.5. Dorso-ventral (D–V), medio-lateral (M–L) and proximo-distal (Pr-Di) axes are indicated. (B) Topographical organization of forelimb MNs. MN positions are based on retrograde labeling from nerves at e12.5 (see Figure S1A–I). Dorso-ventral (D–V) and medio-lateral (M–L) coordinates are indicated. Segmental position of labeled LMC neurons are indicated, and extend from cervical segment 4 (C4) to thoracic segment 1 (T1). Non-LMC MN populations present in these segments are also outlined. (C–G) Hoxc4, Hoxa5, Hoxc5, Hoxc6 and Hoxc8 expression in retrogradely labeled MNs projecting along the suprascapular, lateral pectoral, musculocutaneous, axillary, thoracodorsal, medial anterior lateral, radial, median and ulnar nerves. (H) Summary of Hox expression profiles in MNs projecting along the main forelimb motor nerves. See also Figure S1.

We next determined the profile of Hox protein expression in traced MN populations. We assessed the expression of Hoxc4, Hoxa5, Hoxc5, Hoxc6 and Hoxc8 as well as Hox co-factors belonging to the Meis and Pbx families. We found that Hox5, Meis1, Meis2, Pbx1 and Pbx3 proteins were restricted to rostral brachial LMC neurons, while Hoxc8 was expressed caudally (Figure 1D, E, G and Figure S1J–L). Neurons within the Hox5+ and Hoxc8+ populations could be further distinguished by differential expression of additional Hox proteins. While all rostral LMC neurons express Hoxc5, Hoxa5 was excluded from axillary MNs (Figure 1D, E). Hoxc4 was present in a subset of rostral Hox5+ LMC neurons (Figure 1C); while Hoxc6 was excluded from MNs targeting specific proximal and distal nerves (Figure 1F). Some MNs projecting along motor nerves shared identical or similar Hox codes, but could be discriminated by differential expression of the Lim HD proteins Lhx1 and Isl1 (Figure S1M,N). These results indicate that the expression of Hox proteins, Hox cofactors, and Lim HD proteins mark subsets of MNs supplying forelimb and shoulder muscles (Figure 1H).

Hox Genes are Essential for MN Topographical Organization and Innervation Pattern

To assess the function of Hox genes in the organization and connectivity of MNs we analyzed mice lacking Hox5 (Hoxa5 and Hoxc5), Hoxc6, and Hoxc8, as well as the HoxC gene cluster. We determined how loss of Hox function affects the differentiation of LMC neurons, the overall pattern of limb innervation, and the topographic relationship between MN position and target muscle specificity. Each Hox mutant line was crossed with Hb9::GFP mice to visualize motor axon projection patterns, and to assist in the identification of nerves for tracing assays.

Analysis of mice lacking Hox5 genes in MNs (Hox5MNΔ;Hb9::GFP mice) (Philippidou et al., 2012) revealed grossly normal patterns of limb innervation (Figure S2A,B), and expression of Hoxc4, Hoxc6, Meis and Foxp1 proteins was preserved (Figure S2I and data not shown). Because assessment of axonal trajectories alone does not provide information on whether MNs make appropriate decisions to target a specific region, we performed retrograde labeling assays in Hox5MNΔ;Hb9::GFP mice. Retrograde tracing from the suprascapular nerve labeled MNs that were scattered within the LMC and expressed high levels of Hoxc4 (Figure S2J), a profile typically observed for lateral pectoral MNs. Because the profile of Hoxc4 expression is unchanged in Hox5MNΔ mice (Figure S2I), these results indicate that some MNs lacking Hox5 genes adopt aberrant routes and target inappropriate muscles.

Hoxc8 is expressed by caudal brachial LMC neurons that innervate distal and posterior limb muscles. Within these segments MN pools targeting the cutaneous maximus (CM) and anterior latissimus dorsi (ALD) muscles are defined by expression of the transcription factor Pea3, while median and ulnar MNs express Scip (Jung et al., 2010; Livet et al., 2002). In mice lacking Hoxc8 in MNs (Hoxc8MNΔ mice) there was a marked reduction in expression of these markers (Figure S2M,N and O). Consistent with this defect, the median and ulnar nerves were thinner, the median nerve was truncated, and terminal arbors at the CM and ALD muscles were dramatically reduced (Figure 2A,B,E,F, S2A,B,E,F). Retrograde tracing from the ulnar, median, medial anterior thoracic and radial nerves revealed that MNs were scattered within the LMC in Hoxc8 mutants, in contrast to the stereotypic positioning and clustered organization in controls (Figure 2N–P, S2P).

Figure 2. Hox Genes Govern Limb Innervation Pattern and MN Pool Organization.

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(A–B) Wholemount GFP staining of control Hb9::GFP mice at e12.5 and e13.5. Schematic representations of innervation pattern are shown in right panels and motor nerves are color coded. Arrows indicate region where nerve diameters were measured. (C–H) Forelimb innervation patterns in Hoxc6, Hoxc8 and HoxC mutants at e12.5 and e13.5. Arrows and arrowheads indicate major nerve defects. Scale-bars represent 500 μm. Innervation patterns in control littermates are shown in Figure S2. (I) Quantification of muscle nerve diameters in indicated Hox mutant lines. Data are shown as mean +/− S.E.M. (J–P) Retrograde labeling of MNs in Hoxc8 mutants. Hoxc8 mutants display defects in the organization of suprascapular, lateral pectoral, medial anterior thoracic, median and ulnar MNs. (Q–S) Retrograde labeling of MNs projecting along the lateral pectoral, medial anterior thoracic and radial nerves in Hoxc6 mutants. The position and organization of MNs projecting along the suprascapular and musculocutaneous nerves are not affected in Hoxc6 mutants (Figure S2K, L). See also Figure S2.

Hoxc8 has been shown to be required for the exclusion of Hox5 genes from caudal LMC neurons in chick (Dasen et al., 2005). In Hoxc8MNΔ mice we observed a caudal extension of multiple rostrally-restricted factors, including Hoxc4, Hoxa5, Meis and Pbx proteins (Figure S2Q,R and data not shown). The diameters of nerves originating from rostral Hox5+ LMC neurons were increased in Hoxc8MNΔ embryos (Figure 2I), suggesting that some caudal LMC neurons are redirected to targets of Hox5+ MNs. To test this we performed retrograde tracing assays from the suprascapular and lateral pectoral nerves, revealing a caudal extension of Hox5+ MN pools (Figure 2J–M). To further assess whether a fate switch occurred in Hoxc8 mutants, we took advantage of a LacZ reporter inserted into the Hoxc8 locus. Staining for β Galactosidase (βGal) indicated that MNs within the caudal LMC coexpressed Hoxa5, Meis2, and βGal in Hoxc8 mutants (Figure S2S,T). Furthermore, MNs projecting along the suprascapular and lateral pectoral nerves expressed βGal in Hoxc8 mutants (Figure S2U,V). These studies suggest that in Hoxc8 mutants there is a partial transformation of caudal LMC neurons to a Hox5+ fate.

Hoxc6 is required for the expression of Pea3 and the innervation of the CM and ALD muscles (Figure 2C,D, S2C,D) (Lacombe et al., 2013). Further analysis of Hoxc6−/−;Hb9::GFP mice revealed additional defects not present in either Hox5 or Hoxc8 mutants. The diameters of the axillary, lateral pectoral and radial nerves were reduced by ~50% in Hoxc6 mutants while ulnar nerve diameters were increased by 31% (Figure 2C,D,I). Retrograde labeling assays revealed that MNs projecting along the lateral pectoral, radial and median anterior thoracic nerves were disorganized (Figure 2Q–S, S2K,L). Mice lacking the HoxC cluster displayed MN phenotypes that were a composite of defects in Hoxc6 and Hoxc8 mutants, including altered nerve diameters and a lack of intramuscular arborization at the CM and ALD muscles (Figure 2G,H,I, S2G,H). Collectively these analyses reveal essential roles for Hox genes in establishing the appropriate pattern of forelimb innervation and MN pool organization.

Ret and Gfrα Receptors are Selectively Expressed by Subsets of LMC Neurons

What are the effectors of Hox proteins in MNs, how are they regulated, and how do they contribute to LMC topographic organization and connectivity? To address these questions, we screened for surface receptors whose expression patterns correlated with Hox profiles along the rostrocaudal axis or within a single segment. One group of genes which met these criteria included the receptor tyrosine kinase Ret and members of its Gfrα coreceptors. Analysis of Ret in brachial LMC neurons revealed striking differences in expression levels along the rostrocaudal axis (Figure 3A–B, S3A–B). In rostral Hox5+ LMC neurons, MNs expressed low levels of Ret mRNA and protein relative to the caudal Hoxc8+ domain which expressed high levels at e12.5 (Figure 3A,B,E,F).

Figure 3. Expression of Ret and Gfrα Genes in LMC Neurons.

Figure 3

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(A–G) Expression patterns of Ret, Gfrα1 and Gfrα3 in MNs along the rostrocaudal axis, aligned with expression of Hoxa5, Hoxc8, Meis1/2, Foxp1, Pea3 and Scip at e12.5. (H–N) Expression of Ret, Gfrα1 and Gfrα4 in MNs aligned with Hoxa5, Hoxc8, Meis1/2, Foxp1, Pea3 and Scip in chick at st29. (O) Summary of the expression profiles of mouse (m) and chick (c) Ret, mGfrα1, cGfrα1, mGfrα3 and cGfrα4 along the rostrocaudal axis. Expression domains of Hoxa5, Hoxc8, Meis1/2, Hoxc6, Pea3 and Scip are depicted for positional comparison. (P) Summary of the intrasegmental expression profile of Ret, mGfrα1, cGfrα1, mGfrα3 and cGfrα4 in brachial MNs. See also Figure S3.

Ret signaling is mediated by Gfrα receptors, which upon ligand binding dimerize and activate specific downstream signaling cascades. Selective expression of Gfrα genes has been reported in subsets of MNs (Gould and Oppenheim, 2004; Gu and Kania, 2010; Homma et al., 2003), and we therefore assessed the expression pattern of each of the four murine Gfrα receptors. Gfrα1 expression was broadly expressed by brachial MNs at e12.5, with elevated levels observed within the caudal LMC (Figure 3C). Gfrα3 was restricted to a caudal subpopulation of LMC neurons that overlapped with the position of Scip+ MNs (Figure 3D, G, S3C). Gfrα2 was weakly expressed by brachial MNs while Gfrα4 was not detected (Figure S3D and data not shown).

Comparison of Ret and Gfrα expression between mouse and chick revealed largely conserved profiles, with a few notable differences (Figure 2O,P, S3F). As in mouse, high levels of Ret mRNA and protein were detected in caudal Hoxc8+ LMC neurons in chick, while lower levels were present in rostral Hoxa5+ MNs (Figure 3H,I,L,M). Gfrα1 displayed a more restricted pattern in chick than in mouse, with high levels of expression overlapping with the position of Pea3+ MNs (Figure 3J,N, S3F). In contrast to mouse, Gfrα2 expression was present in a lateral population of brachial MNs in chick, while Gfrα3 was not detected (Figure S3E and data not shown). In chick Gfrα4 expression was similar to mouse Gfrα3, occupying a position overlapping with Scip+ LMC neurons (Figure 3K,N, S3F).

Ret and Gfrα3 are Downstream Targets of Hox Proteins in LMC Neurons

To determine whether the MN organization and connectivity defects observed in Hox mutants are due to loss of Ret/Gfrα signaling components, we analyzed Hox mutant mice for changes in Ret and Gfrα expression. We assessed Ret mRNA in Hoxc6, Hoxc8MNΔ and HoxC mutant animals and compared expression with control littermates at e12.5. We also quantified the levels of Ret protein in mutant lines and control littermates. This analysis revealed a pronounced reduction in both Ret mRNA and protein levels in the posterior Hoxc8+ region in Hoxc6, Hoxc8, and HoxC cluster mutants (Figure 4A–I). Ret expression was present in caudal LMC neurons, but reduced to levels comparable to those observed in rostral Hox5+ neurons.

Figure 4. Multiple Hox Genes Establish Ret and Gfrα Profiles in LMC neurons.

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(A–H) Analysis of Ret expression in Hox mutant mice at e12.5. Ret levels are attenuated in Hoxc6, Hoxc8 and HoxC mutants within caudal LMC neurons. Ret mRNA and protein levels are shown in the indicated mutants and control littermates. Panels C, F and I show quantification of Ret antibody immunofluorescence from serial sections along the rostrocaudal axis (see Experimental Procedures). Data are shown as mean +/− S.E.M. (J–O) Gfrα3 expression is markedly downregulated in Hoxc8 and HoxC mutants but preserved in Hoxc6 mutants. See also Figure S4.

We also examined the expression of Gfrα genes in Hox mutants. Expression of Gfrα3 was markedly decreased in Hoxc8MNΔ and HoxC−/− embryos, but was grossly normal in Hoxc6 mutants (Figure 4J–O, S4B). Expression of Gfrα1 was largely unaffected in Hoxc6, Hoxc8MNΔ, and HoxC mutants at e12.5 (Figure S4A,B). These results show that Hox genes contribute to the differential expression of Ret in LMC neurons along the rostrocaudal axis, and are essential for the intrasegmental pattern of Gfrα3.

Hox Network Interactions Determine the Profiles of Ret/Gfrα Genes in MNs

Our analyses indicate that multiple Hox genes are necessary to define Ret/Gfrα expression in LMC neurons, but also raise the question of how interactions amongst Hox proteins contribute to their specific patterns. We first determined whether Hox genes are sufficient to induce Ret and Gfrα expression outside their normal domains. We misexpressed Hox genes in the neural tubes of stage (st) 13 chick embryos using the chicken β-actin (CAGGs) promoter, and analyzed Ret, Gfrα1 and Gfrα4 expression at st27. We found that both Hoxc6 and Hoxc8 are sufficient to induce high levels of Ret at thoracic levels, where Ret levels are normally low (Figure 5A,D,E,H and S5A,C). In contrast misexpression of the rostrally expressed Hox genes, Hoxc4 and Hoxa5, did not induce Ret at any level (Figure S5I–L).

Figure 5. Hox and Meis Interactions Govern Ret Expression in LMC Neurons.

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(A–D) Electroporation of Hoxc6-pCAGGs induces Ret and Pea3 expression at thoracic levels and Gfrα1 at rostral cervical levels. Hoxc6 and Isl1/2 costaining is shown to indicate electroporated MNs. Arrow in panel A indicates electroporated side of embryo. (E–H) Electroporation of Hoxc8-pCAGGs induces Ret, Gfrα4 and Scip expression at thoracic levels. (I–L) Misexpression of Meis1 represses Ret, Gfrα1 and Gfrα4 in caudal LMC neurons. (M–R) Expression of Meis1/2 and Lhx1 in chick and mouse lumbar LMC neurons. In both chick and mouse Meis1/2 expression is reduced in lateral Lhx1+ MNs, which express high levels of Ret. (S–V) Electroporation of Hoxc10 and Hoxd10 induces expression of Ret at thoracic levels. (W,X) Meis1 misexpression represses Ret in lumbar LMC neurons. (Y) Summary of Ret regulation by Hox/Hox co-factors in brachial LMC neurons. (Z) Summary showing regulation of Ret by Hox/Hox co-factors at lumbar levels. See also Figure S5.

Both Hoxc6 and Hoxc8 can impose an LMC fate on thoracic MNs through induction of the transcription factor Foxp1 (Dasen et al., 2008; Lacombe et al., 2013). The elevated levels of Ret observed upon thoracic Hoxc6 and Hoxc8 misexpression therefore could be indirect, due to the actions of Foxp1. To determine if Foxp1 can elevate Ret expression independent of Hox genes, we misexpressed Foxp1 at rostral cervical and thoracic levels. After misexpression of Foxp1 elevated Ret expression was not observed at either level (Figure S5G,H and data not shown). These results indicate that Foxp1 alone is insufficient to promote the high levels of Ret observed in caudal LMC neurons.

Hoxc6 is expressed in rostral Hox5+ LMC neurons, yet is presumably incapable of promoting high Ret levels in this region. We tested whether factors present in rostral LMC neurons contribute to the reduced levels of Ret. Misexpression of Meis1 in caudal Hoxc8+ LMC neurons, where Ret levels are relatively high, resulted in a marked inhibition of Ret expression (Figure 5I,L and S5E,F). In contrast, misexpression of Hoxc4, Hoxa5, Pbx1 and Pbx3 did not alter Ret expression in caudal LMC neurons (Figure S5M–P and data not shown).

Although Hoxc6 and Hoxc8 were both capable of elevating Ret levels, they each promoted distinct patterns of Gfrα expression and MN pool identities. Hoxc6, but not Hoxc8, induced expression of Gfrα1 in cervical MNs, where expression of this receptor is normally low (Figure 5B, S5D). This result is consistent with the observation that in chick Gfrα1 is restricted to the Hoxc6+, Pea3+ MN pool, but excluded from Scip+ MNs. In addition, Hoxc8 was able to induce expression of Gfrα4 at thoracic levels (Figure 5F, S5B), while Meis1 repressed both Gfrα1 and Gfrα4 at caudal brachial levels (Figure 5J,K). Hoxc6 and Hoxc8 also promoted distinct MN pool fates, with Hoxc6 inducing Pea3 expression and Hoxc8 inducing Scip at thoracic levels (Figure 5C,G) (Lacombe et al., 2013).

These results indicate that regulatory interactions amongst Hoxc6, Hoxc8, and Meis1 define the patterns of Ret, Gfrα1 and Gfrα4 in brachial LMC neurons (Figure 5Y). Hoxc6 and Hoxc8 are sufficient to induce elevated Ret levels, while Meis1 dampens Ret expression in rostral LMC neurons. Hoxc6 promotes expression of Gfrα1 within MN pools defined by Pea3 expression, while Hoxc8 promotes Gfrα4 and a Scip+ MN pool identity.

Hox and Meis Regulatory Interactions Control Ret Expression in Lumbar LMC Neurons

Our analyses of Ret gene regulation at brachial levels are seemingly at odds with studies in lumbar LMC neurons, where Ret is essential during dorsoventral projection decisions (Kramer et al., 2006). Dorsally-projecting lateral LMC (LMCl) neurons express high levels of Ret while medial LMCm neurons express low levels. In the absence of Ret, LMCl motor axons fail to pursue a dorsal trajectory along the peroneal nerve (Bonanomi et al., 2012; Kramer et al., 2006). To determine whether Ret expression in lumbar LMC neurons is defined by similar Hox-regulatory interactions, we assessed whether Hox genes expressed at lumbar levels can induce Ret expression. We found that after misexpression of the lumbar LMC determinants Hoxc10 and Hoxd10 elevated levels of Ret were detected in thoracic MNs (Figure 5S–V).

Because Meis1 is involved in dampening Ret expression in rostral brachial MNs, we asked whether it is also involved in restricting Ret to lumbar LMCl neurons. In both mouse and chick Meis1 expression was restricted to the LMCm at lumbar levels and only weakly detected in LMCl neurons (Figure 5M–R). Moreover, misexpression of Meis1 at lumbar levels repressed Ret expression in LMCl neurons (Figure 5W,X). These observations indicate that the pattern of Ret expression in brachial and lumbar LMC neurons is defined by a common Hox/Meis regulatory strategy (Figure 5Y,Z).

Ret and Gfrα1 Activities Bypass the Requirement for Hox Genes in Pea3 Induction

What is the functional significance of the profile of Ret and Gfrα genes during MN differentiation? The ligand for Ret/Gfrα1 receptors, Gdnf, is broadly expressed in the periphery (Haase et al., 2002; Jung et al., 2010), suggesting that MN responsiveness to peripheral signals is constrained by LMC-intrinsic factors. To test this we asked whether elevating Ret and Gfrα1 outside their normal domains affects MN pool differentiation. We expressed Ret and Gfrα1 by in ovo electroporation at rostral brachial and thoracic levels, where expression of these genes is normally low, and assessed expression of Pea3, a known target of Gdnf-Ret/Gfrα1 signaling (Haase et al., 2002). Elevating Ret expression at thoracic levels, but not in rostral LMC neurons, generated ectopic Pea3+ MNs (Figure 6A–D, S6E). The selective induction of Pea3 at thoracic levels appears to stem from the fact that thoracic MNs in chick also express Gfrα1 (Figure S6A), creating a condition where MNs now express both Ret and Gfrα1. Consistent with this idea; upon coexpression of Ret and Gfrα1 in either rostral brachial or thoracic MNs we observed induction of Pea3 expression (Figure 6I–N, Q). In contrast, expression of Gfrα1 in either rostral LMC or thoracic segments was insufficient to induce Pea3 (Figure 6E–H).

Figure 6. Ret and Gfrα1 Patterns Facilitate Pea3 Induction in MN Pools.

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(A–D) Misexpression of Ret under pCAGGs induces Pea3 expression in thoracic MNs but not rostral brachial LMC neuron. (E–H) Gfrα1 missexpression fails to induce Pea3 expression in rostral brachial and thoracic MNs. (I–N) Co-expression of Ret and Gfrα1 is sufficient to induce Pea3 at both rostral brachial and thoracic levels. (O–P) After induction of Pea3 by Ret and Gfrα1 there is a reduction in the number of Scip+ LMC neurons. (Q) Quantification showing increased numbers of Pea3+ and reduced Scip+ MNs along the rostrocaudal axis after Ret and Gfrα1 misexpression. Numbers are representative of electroporation experiments with efficiencies of greater than 50%. (R) Diagram summarizing the relationship between Ret, Gfrα1, Scip and Pea3 expression, as well as the effects of Ret and Gfrα1 misexpression on the differentiation of Pea3+ and Scip+ MN pools. See also Figure S6.

Induction of Pea3 after misexpression of Ret and Gfrα1 did not lead to ectopic expression of LMC-associated transcription factors such as Hoxc6, Hoxc8, or Foxp1 (Figure S6B–D). These observations indicate that Pea3 induction can occur independently of an earlier LMC specification program, implying that a key function of Hox genes is to establish the domain in which the Pea3+ MN pool can be specified. High levels of Ret in combination with Gfrα1 expression appears therefore to define the competence of MNs to express Pea3 (Figure 6R). Given that in Pea3 mutants there is a selective loss in terminal branches at the CM muscle (Livet et al., 2002), these results suggest the CM innervation defects observed in Hoxc8 and Hoxc6 mutants are due to the loss of Pea3 induction by Ret/Gfrα1 signaling.

Ret and Gfrα3 Mutants Display Innervation Defects Similar to Hox Mutants

To assess whether attenuation of Ret/Gfrα signaling contributes to the MN defects observed in Hox mutants, we analyzed mice lacking Ret and Gfra3. Previous analysis of Ret mutants revealed essential roles in the innervation of dorsal hindlimb muscles (Bonanomi et al., 2012; Kramer et al., 2006), and Gdnf and Gfrα1 mutants have been shown to be required for the induction of Pea3 at brachial levels (Haase et al., 2002). However the role of Ret in the specification and connectivity of LMC neurons has not been fully established. In Ret mutants expression of Hoxc6, Hoxc8, and Foxp1 was preserved at e12.5 and e13.5, consistent with a function downstream of Hox genes (Figure S7A–C and data not shown). However, there was a marked reduction in the number of MNs expressing Pea3 (Figure 7A, B). Interestingly, the total number of Scip+ MNs was increased by 38% in Ret−/− embryos, leading to a ventral expansion of this pool (Figure 7C, D). Conversely, generating ectopic Pea3 neurons, via misexpression of Ret and Gfrα1, led to a marked reduction in the number of Scip+ neurons (Figure 6O–Q). These results indicate that the establishment of the normal distribution of Pea3+ and Scip+ MNs relies on Ret function.

Figure 7. Ret and Gfrα3 are Required for MN Pool Differentiation and Connectivity.

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(A) Pea3 expression is reduced in brachial LMC neurons in Ret mutants at e12.5. (B) Quantification of the number and distribution of Pea3+ MNs in Ret−/− and control mice. (C) The number of Scip+ MNs increases in Ret−/− mice. (D) Quantification of the number and distribution of Scip+ MNs. (E,F) Wholemount GFP staining showing defects in CM muscle innervation in Ret−/− embryos (arrow) at e12.5. (G,H) Forelimb innervation at e13.5 showing failure of ulnar MNs to project distally in Ret−/− embryos (arrow). Arrows in control diagram indicate region where nerve diameters were measured. (I–K) Disorganization of MNs in Ret−/− mice shown by retrograde labeling of MNs projecting along the medial anterior thoracic, median and ulnar nerves. (L,M) Forelimb innervation pattern in Gfrα3+/+ and Gfrα3−/− mice at e13.5 showing thinning of the median and ulnar nerves. Scale-bars represent 500 μm. (N) Quantification of muscle nerve diameters expressed as a ratio between the radial and ulnar nerves in Ret and Gfrα3 mutants and controls at e13.5. See also Figure S7.

We next analyzed the pattern of limb innervation in Ret mutants. Similar to Hoxc6−/−, Hoxc8MNΔ, and Pea3−/− mice (Livet et al., 2002), Ret−/− embryos displayed a marked loss in axonal arbors at the CM and ALD muscles (Figure 7E–H). Projections along the ulnar and median nerves were present in Ret−/−:Hb9::GFP mice, but displayed aberrant branches along the ulnar nerve, resulting in thinning of this nerve distally (Figure 7G,H,N). Retrograde tracing of MNs projecting along the median and ulnar nerves labeled MNs positioned more ventrally, as observed in Hoxc8 mutants (Figure 7J,K). In contrast, tracing from the medial anterior thoracic nerve in Ret mutants labeled MNs positioned more dorsally, similar to Hoxc6 and Hoxc8 mutants (Figure 7I).

We next analyzed mice lacking Gfrα3, which is restricted to Scip+ MNs. In Gfrα3;Hb9::GFP mice both the median and ulnar nerves were thinner at e13.5 (Figure 7L–N), similar to Hoxc8MNΔ mice. However, in contrast to Hoxc8MNΔ and Ret mutant mice, retrograde labeling from the median and ulnar nerves in Gfrα3 mutants indicated a grossly normal organization (Figure S7D, E). Given that in Hoxc8 mutants median and ulnar MNs are disorganized, these results indicate that Gfrα3 mutation does not phenocopy loss of Hoxc8, and that Hox proteins have additional targets required for motor pool clustering. The number of Scip+; Foxp1+ MNs was unchanged in Gfrα3 mutants (Figure S7F), suggesting that the innervation defects are due to an inability of motor axons to fully extend within the limb. Collectively these results indicate that Ret and Gfrα3 mutants display limb innervation defects similar to Hox mutants.

Discussion

Networks of transcription factors are critical for neuronal subtype diversification, but how cell fate determinants orchestrate expression of surface receptors is poorly understood. In this study we found that Hox genes are essential for the organization and peripheral connectivity of MNs targeting the forelimb, and identified Ret and Gfrα genes as key targets of their actions. We discuss these findings in the context of transcriptional and signaling networks conferring MN-muscle target specificity, and consider the possible mechanisms contributing to the diversification of limb innervation programs in tetrapods.

Hox Genes Govern MN Diversity and Peripheral Innervation Pattern

Hox genes are essential for neuronal specification in the hindbrain and spinal cord (Philippidou and Dasen, 2013; Tumpel et al., 2009), but the extent to which they define the connectivity of limb-innervating MNs is unclear. LMC neurons segregate into ~50 MN pools, each pool defined by its connectivity to a single limb muscle. While certain MN pools express subtype-restricted factors, whether a selective transcriptional code defines each subtype is unknown. We found that MNs extending along the nine major forelimb nerve pathways are defined by restricted patterns of Hox proteins and Hox cofactors. Hox5 genes define MNs that target proximal/anterior limb regions, Hoxc8 distal/posterior regions, with additional Hox genes and cofactors contributing to MN diversity and connectivity within these broad domains. In principle, additional layers of target specificity could be imparted by differences in Hox protein levels between MN pools, which may grade surface receptor expression. In support of this idea, level-dependent activities of Hox proteins in MNs have been demonstrated during the innervation of leg muscles in Drosophila (Baek et al., 2013).

Consistent with roles in establishing the identity and connectivity of LMC neurons, removal of Hox genes leads to defects in the pattern of limb innervation, and dissolves the normal topographic relationship between MN position and target specificity. Mutation of individual Hox genes causes a deterioration of MN organization and peripheral target specificity along multiple nerves. However, with the exception of the CM and ALD muscles, most targets appear to receive at least some innervation in the absence of Hox genes. These observations are in agreement with analysis of Foxp1 mutants, where Hox-dependent LMC programs are lost, and MNs target limb muscles in a stochastic manner (Dasen et al., 2008; Rousso et al., 2008). Therefore in the absence of Hox genes motor axons can continue to pursue trajectories along the available paths within the limb, but appear to lose the ability to select appropriate targets.

The most severe deficits after removal of Hox genes are apparent upon assessment of the relationship between MN position and target specificity. Hox mutants display specific defects in the normal topographic organization and position of MN pools. In Hoxc8 mutants MNs targeting the distal forelimb are disorganized within the LMC, and a subset of MNs lacking Hoxc8 target proximal forelimb muscles normally supplied by Hox5+ populations. These observations suggest that in Hoxc8 mutants there is a partial transformation of LMC neurons to a Hox5+ fate and connectivity pattern. Although it is unclear whether similar fate transformations occur in each of the Hox mutants we analyzed, their phenotypes likely reflect loss of guidance systems that dictate the selection of one peripheral innervation pathway over another.

Hox Target Effectors in Neuronal Fate Specification and Axon Guidance

The ability of motor axons to navigate towards and innervate peripheral targets with precision relies upon the selective expression of cell surface receptors. Combinatorial sets of transcription factors are known to be essential for neuronal subtype diversification, but how intrinsic factors shape MN identities and innervation pattern is poorly defined. Studies in Drosophila have shown that the transcription factor Hb9 acts in ventrally projecting MNs to regulate expression of the Robo2 guidance receptor, while in dorsally projecting MNs, Eve2 controls expression of the Unc5 gene (Santiago et al., 2014; Zarin et al., 2014a). In vertebrates, motor axon guidance in the limb bud is known to require the activities of a variety of genes, including Ret, Epha4, Ephb1, Frizzled3, Npn1, and Celsr3 (Bonanomi and Pfaff, 2010; Hua et al., 2013; Santiago and Bashaw, 2014). However, beyond the contribution of Lim HD proteins to the selection of dorsal and ventral limb trajectories, how fate determinants orchestrate MN connectivity is poorly understood.

We found that Hox/Hox cofactor interactions define the spatial profiles of Ret and Gfrα genes in MNs, and signaling through Ret/Gfrα receptors is essential in Hox-dependent programs of MN differentiation and connectivity. During MN pool specification, activation of Ret/Gfrα1 signaling by Gdnf is required for Pea3 expression in a subset of LMC neurons (Haase et al., 2002). Application of Gdnf throughout the spinal cord however induces Pea3 only within the caudal LMC populations that would have expressed it normally. This suggests the existence of intrinsic systems that limit MN responses to Gdnf. Our results indicate that Hox/Meis interactions define the pattern of Ret and Gfra1 in caudal LMC neurons, thereby restricting the ability of peripheral cues to induce Pea3 expression. In support of this idea, elevating Ret and Gfrα1 throughout the spinal cord is sufficient to activate Pea3, bypassing the requirement for Hox genes. Hox genes therefore appear to constrain MN responsiveness to peripheral cues by selectively regulating expression of surface receptors.

In addition to contributing to MN pool diversification, signaling through Ret is essential during motor axon guidance decisions. In the hindlimb, Ret is required for the selection of a dorsal trajectory by LMC axons, acting in part by modulating signaling through Eph receptors (Bonanomi et al., 2012). We found that Hox/Meis regulatory interactions determine the spatial profiles of Ret expression in LMC neurons, and silencing Ret and Gfrα3 causes innervation defects similar to those observed after selective removal of Hox genes. In the absence of Ret, projections towards posterior and distal muscle groups are deteriorated, similar to the innervation defects observed in Hoxc6 and Hoxc8 mutants. These phenotypes appear to be due in part to the loss of Pea3, as Pea3 mutants display a severe reduction in intramuscular branches at the CM muscle (Livet et al., 2002). Our studies show that loss of Ret and Gfrα3 also leads to innervation defects in the distal forelimb, raising the possibility that Ret/Gfrα signaling contributes to guidance decisions independent of Pea3 regulation.

Hox Genes and the Evolution of Limb Innervation Programs

The musculoskeletal system of the forelimb varies significantly amongst vertebrates, raising the question of how MN specification programs evolved to accommodate different motor behaviors. The basic topographical organization of MNs is conserved amongst all tetrapods that have been examined (Fetcho, 1992), and evolutionary modification of Hox activities appears to have contributed to changes in LMC position relative to the limbs (Jung et al., 2014). During the evolution of paired-appendages, the appearance of new muscle groups enabled species to achieve greater complexity in the range of limb movements. This process presumably required an expansion of MN subtype identites from a more simplified ancestral population. The Lim HD code governing dorsoventral projections appears to be present in pectoral fins of zebrafish (Uemura et al., 2005), suggesting this program appeared prior to land invasion. As the number of muscle groups expanded in early vertebrates, Hox5 and Hox8 paralogs could have been coopted to govern the innervation of proximal/anterior and distal/posterior muscles respectively. Subsequently the actions of Hox4, Hox6, and Hox7 genes further diversified MN pools to allow for more selective targeting of limb muscles.

Comparison of limb innervation programs in mice and chick suggests the existence of diversification programs acting downstream of Hox genes. Both birds and rodents appear to share a common Hox/Ret/Gfrα-based regulatory strategy for specifying Pea3+ MN pools. However, there are significant functional differences between the muscle groups targeted by these neurons. In birds Pea3+ MNs target the pectoralis (Pec), a large muscle providing the major driving force for flexing the wing during flight (Biewener, 2011; Hollyday and Jacobson, 1990). By contrast, in mice most Pea3+ MNs target the cutaneous maximus, a subcutaneous muscle present in fur-bearing mammals, with no known role in locomotion. We found that in chick Gfrα1 is restricted to the Pea3+ pool, while in mice Gfrα1 is broadly expressed by LMC neurons. Differences in the regulation of Gfrα genes by Hox proteins may have contributed to the evolutionary divergence of muscle-specific connectivity programs in birds in mammals.

In summary these studies indicate that Ret and Gfrα genes are key targets of Hox proteins during MN specification and the establishment of limb innervation pattern. Given that one feature of Hox activity is to define Ret levels in MNs, it is plausible that additional target effectors are under similar graded control mechanisms. Regulation of the levels of receptor gene expression could represent a common regulatory strategy through which networks of transcription factors establish neuronal subtype identities and determine target specificity.

Experimental Procedures

Animals

All mouse procedures were approved by the Institutional Animal Care and Use Committee of the New York University School of Medicine.

In situ Hybridization and Immunohistochemistry

Embryos were harvested between e11.5–14.5 and fixed in 4% paraformaldehyde for 1.5–2 hours and processed for in situ hybridization or immunohistochemistry. For wholemount immunohistochemistry, embryos were processed for GFP staining as previously described (De Marco Garcia and Jessell, 2008). Confocal images were obtained with a Zeiss (LSM 700) microscope and analyzed with ImageJ software.

Retrograde Labeling of MNs

Embryos were harvested at e12.5, eviscerated and forelimb nerves were visualized using a MVX10 wide-field fluorescent microscope (Olympus). The nerve of interest was cut and injected with lysine fixable Rhodamine-dextran (Molecular Probes). Embryos were incubated for 3.5–4.5 hours in oxygenated DMEM/F12 (50:50) solution at 30–34°C, fixed, and processed for immunohistochemistry.

In ovo Chick Embryo Electroporations

Electroporations were performed at Hamburger and Hamilton (HH) st12–14 chick embryos as previously described (Dasen et al., 2003). Plasmid concentrations ranged from 100–500 ng/μl and pBKS was used as carrier DNA to achieve a final concentration of 1 μg/μl. Results for each experiment are representative of five or more embryos in which the electroporation efficiency in MNs was >50%.

Quantifications and Statistical Analyses

Cell counting was performed using ImageJ software in at least three embryos per genotype. Cells stained for Foxp1, Pea3, or Scip were counted and aligned by comparison of LMC position between littermates. Nerve thickness was analyzed by measuring the length of a line running perpendicular to the long axis of the nerve. For consistent comparison, measurements were taken in similar regions of the nerves between littermates. Ret fluorescence intensities were measured using ImageJ software as described previously (Gavet and Pines, 2010), with some modifications. For fluorescence measurements, we used the following formula for each section of at least three animals per genotype: Foxp1+ cells signal = sum of the intensity of pixels for Foxp1+ cells. Background signal = average signal per pixel for a region selected just beside Foxp1+ cells. Foxp1+ Ret fluorescence signal = Foxp1+ cell signal – surface selected (number of pixels for the selected area) x background. Statistical significance was determined with the unpaired two-tailed Student’s t-test using Microsoft Excel. *P < 0.05; **P < 0.01; ***P < 0.001.

Supplementary Material

1
2

Highlights.

  • A Hox code defines MNs projecting along the major axes of the limb

  • Ret and Gfrα genes are key targets of Hox proteins in limb-innervating MNs

  • Hox/Hox cofactor interactions govern Ret levels and Gfrα patterns in MNs

  • Loss of Ret and Gfrα3 causes MN defects similar to Hox gene mutations

Acknowledgments

We thank Myungin Baek, Gord Fishell, Heekyung Jung, and Polyxeni Philippidou for comments on the manuscript, and Rocio Rivera for preliminary analyses of Hoxc8 mutants. This work was supported by funding from the NIH (R01 NS062822), and an NIH predoctoral training grant (T32NS086750) to M.M.S.

Footnotes

Author Contributions

C.C., M.M.S. and J.S.D conceived and designed the study. D.H.L provided technical assistance, and J.P.L. generated reagents essential for the studies. C.C. and J.S.D wrote the manuscript.

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