Protocadherin-Mediated Cell Repulsion Controls the Central Topography and Efferent Projections of the Abducens Nucleus

Article

Protocadherin-Mediated Cell Repulsion Controls the Central Topography and Efferent Projections of the Abducens Nucleus Graphical Abstract

Authors Kazuhide Asakawa, Koichi Kawakami

Correspondence [email protected] (K.A.), [email protected] (K.K.)

In Brief Cranial neuromuscular systems have been relatively unexplored, because of their restricted tissue accessibility. Using zebrafish, Asakawa and Kawakami develop a model system to analyze cranial neuromuscular connectivity and find that protocadherin-mediated repulsion plays crucial roles for connecting the abducens nucleus to the lateral rectus muscle.

Highlights d

The abducens nucleus and its connection to the lateral rectus are visualized

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Mafba, but not Sall4, is essential for abducens motor neuron development in zebrafish

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Abducens motor neurons clump when Pcdh17 is perturbed

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Protocadherin-mediated repulsive force connects the brain and the eye muscle

Asakawa & Kawakami, 2018, Cell Reports 24, 1562–1572 August 7, 2018 ª 2018 The Author(s). https://doi.org/10.1016/j.celrep.2018.07.024

Cell Reports

Article Protocadherin-Mediated Cell Repulsion Controls the Central Topography and Efferent Projections of the Abducens Nucleus Kazuhide Asakawa1,2,3,* and Koichi Kawakami1,2,* 1Division

of Molecular and Developmental Biology, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan of Genetics, Graduate University for Advanced Studies (SOKENDAI), 1111 Yata, Mishima, Shizuoka 411-8540, Japan 3Lead Contact *Correspondence: [email protected] (K.A.), [email protected] (K.K.) https://doi.org/10.1016/j.celrep.2018.07.024 2Department

SUMMARY

Cranial motor nuclei in the brainstem innervate diverse types of head and neck muscles. Failure in establishing these neuromuscular connections causes congenital cranial dysinnervation disorders (CCDDs) characterized by abnormal craniofacial movements. However, mechanisms that link cranial motor nuclei to target muscles are poorly understood at the molecular level. Here, we report that protocadherin-mediated repulsion mediates neuromuscular connection in the ocular motor system in zebrafish. We identify pools of abducens motor neurons that are topographically arranged according to soma size and convergently innervate a single muscle. Disruptions of Duane retraction syndrome-associated transcription factors reveal that these neurons require Mafba/MAFB, but not Sall4/SALL4, for differentiation. Furthermore, genetic perturbations of Pcdh17/protocadherin-17 result in defective axon growth and soma clumping, thereby abolishing neuromuscular connectivity. Our results suggest that protocadherin-mediated repulsion forms the central topography and efferent projection pattern of the abducens nucleus following Mafba-dependent specification and imply potential involvement of protocadherins in CCDD etiology. INTRODUCTION Diverse movements of the head, face, and eyes are regulated by cranial motor nuclei in the brainstem via complex efferent nerve pathways (Gilland and Baker, 2005; Guthrie, 2007). In adult vertebrates, cranial motor nuclei are typically organized in rostrocaudal columns and include motor neurons originating from transverse hindbrain segments known as rhombomeres established during the embryonic period (Lumsden and Keynes, 1989). For individual cranial motor neurons to connect with appropriate muscle fibers, not only specification but also subsequent axon growth and guidance must be regulated

with remarkable precision. Concurrently, developing motor neurons with similar functions cluster together into a discrete nucleus, and neurons within nuclear subdivisions collectively projected onto peripheral target muscles often in a topographic manner. Such individual- and collective-level regulation of cranial motor neuron development has been intensively explored by analyzing causative genes for human congenital cranial dysinnervation disorders (CCDDs) and by investigating tractable cranial motor systems in experimental animal models (Astick et al., 2014; Chilton and Guthrie, 2016; Whitman and Engle, 2017). Nevertheless, these mechanisms remain incompletely understood. Abducens motor neurons control abduction of the eyes by innervating the lateral rectus muscle. Whereas the abducens nerve targets a single muscle, the abducens nucleus exhibits diverse distribution patterns in the hindbrain among vertebrate taxa. For instance, it is located in both rhombomere 5 (r5) and r6 in teleosts, reptiles, and birds but is restricted to r5 in mammals and to r6 in elasmobranchs. The abducens nucleus originating from a single rhombomere includes multiple types of €ttnermotor neurons that are topographically distributed (Bu Ennever et al., 2001) and projects an efferent nerve consisting of structurally segregated sub-fibers (da Silva Costa et al., 2011; Peng et al., 2010). The lateral rectus muscle also contains structural subdivisions, the global (inner) and orbital (outer) layers with differential content of physiologically distinct myofibers, including those mediating tonic or phasic contraction (Spencer and Porter, 2006). Neither individual-level nor collective-level connectivity between these subdivisions of abducens nucleus and lateral rectus muscle has been fully described, mainly because of the limited anatomical accessibility of the entire neuronal projection. The anatomy and physiological significance of the segmentally separated teleostean and avian abducens nucleus also remain poorly understood, despite intensive investigations (Pastor et al., 1991; Sterling and Gestrin, 1975). The specification of the abducens motor neurons relies on Hox-dependent positional values (Gaufo et al., 2003; Manzanares et al., 1997; Prince et al., 1998). Following specification, axons of abducens motor neurons are guided by extrinsic directional cues, including semaphorin and ephrin, through activation of downstream signaling pathways that modulate cytoskeletal dynamics (Ferrario et al., 2012; Nugent et al., 2017). Consistent

1562 Cell Reports 24, 1562–1572, August 7, 2018 ª 2018 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

with these molecular mechanisms, the causative genetic loci for Duane retraction syndrome (DRS), a congenital type of strabismus, encode transcription factors for hindbrain patterning (HOXA1 and MAFB) (Park et al., 2016; Tischfield et al., 2005) and a Rac-GAP modulating cytoskeletal dynamics (CHN1/a2chimaerin) (Miyake et al., 2008). In addition, DRS mutations are found in the gene encoding SALL4 transcription factor, but the contribution of SALL4 to DRS pathology is unknown at the molecular level (Al-Baradie et al., 2002; Kohlhase et al., 2002). Currently, molecular mechanisms that spatially organize multiple types of abducens motor neurons within a nucleus and connect each motor neuron subtype to appropriate target myofibers are unknown. Cadherin superfamily cell adhesion molecules are differentially expressed in cranial motor nuclei, and a specific type of cranial motor neuron can be mispositioned upon global misexpression of cadherin-20 in chick (Astick et al., 2014; Liu et al., 2015). However, whether and how cadherin superfamily genes contribute to formation of cranial neuromuscular connectivity is unknown. The genetic amenability and translucency of zebrafish (Danio rerio) are uniquely suited for studying cellular and genetic bases of ocular motor pathway (Asakawa et al., 2012; Clark et al., 2013; Greaney et al., 2017; Higashijima et al., 2000; Lyons et al., 2010; Ma et al., 2014). Here, we identify pools of abducens motor neurons located in r5 and r6 using the cis-elements of the mnr2b/ mnx2b homeobox gene. These abducens motor neurons are topographically arranged according to soma size along the dorsoventral axis and require the DRS-associated hindbrain patterning transcription factor Mafba for differentiation. Furthermore, perturbations of Pcdh17/protocadherin-17 by gene knockout and dominant-negative approaches result in the clumping of abducens motor neuron somata and axons, abrogating the central topography and axon growth. On the basis of these observations, we propose a mechanism for establishing collective-level cranial neuromuscular connectivity in which protocadherin-mediated repulsion promotes the formation of topographic organization, as well as its efferent projection, of the motor nucleus. RESULTS Visualizing Neuromuscular Connections between the Abducens Nucleus and Lateral Rectus Muscle in Zebrafish To visualize neuromuscular connections between the abducens nucleus and lateral rectus muscle, we first generated an improved zebrafish Gal4 driver for abducens motor neurons using a bacterial artificial chromosome (BAC) carrying the hsp70l promoter-linked Gal4FF (Gal4) in the mnr2b/mnx2b locus (Tg[mnr2b-hs:Gal4]) (Figures 1A, 1B, and S1) (Asakawa et al., 2013). In Tg[mnr2b-hs:Gal4]; Tg[UAS:GFP] larvae, the eGFP-expressing cells were bilaterally aligned in both r5 and r6 from 2 days post-fertilization (dpf) (Figures 1C and 1D). In each of these rhombomere hemisegments, the eGFP-expressing cells were segregated dorsoventrally into two clusters (Figures 1E and 1F). The nerve bundles emanating from these eGFP-expressing cell clusters extended ipsilaterally and terminated in the vicinity of the caudal eye globe, showing

that the eGFP-expressing cells included abducens motor neurons. Next, to determine the efferent innervation pattern of these eGFP-expressing abducens motor neurons, we counterstained the lateral rectus muscle by constructing a transgene driving expression of td-Tomato-tagged acetylcholine receptor (AChR) d subunit (tdT-chrnd) from the actc1b promoter (Figures 1A, 1G, S2A). The tdT-chrnd signal revealed both gathered and scattered AChR clusters in the middle and lateral regions of the muscle, respectively, as well as the entire muscle with the weaker signal in the plasma membrane starting at 3 dpf onward (Figure 1H). The eGFP-labeled axon terminals were in close apposition to both the gathered and scattered AChRs (Figure 1G) and distributed more densely in the outer muscle (i.e. distal to the eye globe) than the inner muscle (proximal to the eye globe) (Figure 1J). The biased terminal distribution coincided with the distribution of the presynaptic vesicle protein synaptobrevin/VAMP (Figures 1I and S2B), suggesting that the eGFP-expressing abducens motor neurons primarily innervate the outer muscle. To examine the potential inner-outer muscle differentiation, we screened approximately 1,000 Gal4 gene trap lines (Asakawa et al., 2013) and identified two showing distinct lateral rectus expression. The Tg[gSAIzGFFM773B] line selectively labeled the outer muscle by trapping the troponin T type 1 gene (tnnt1) expressed in slow myofiber, while the Tg[gSAIzGFFM1068A] line displayed broad Gal4 expression by trapping the troponin T type3 gene (tnnt3b) expressed in fast myofibers (Figure S3), suggesting that the outer muscle includes a characteristic of slow fibers, as is the case in primates (Spencer and Porter, 2006). Taken together, we concluded that Tg[mnr2b-hs:Gal4] labeled abducens motor neurons that were anatomically organized into dorsal and ventral clusters and that these neurons preferentially innervated the outer lateral rectus muscle, including both slow and fast fibers. Henceforth, we refer to the abducens motor neurons labeled with Tg[mnr2b-hs:Gal4] as mnr2b-ABNs. mnr2b-ABNs Form Soma Size Topography Independently of Target Myofiber Type and Birth Order The heterogeneous morphology of the axon terminals suggests that mnr2b-ABNs include multiple neuronal subtypes (Figure 2A). To investigate differences among individual mnr2-ABNs, we injected the UAS:eGFP plasmid into Tg[mnr2b-hs:Gal4] embryos at the one-cell stage for sparse labeling of individual neurons. Out of approximately 900 injected larvae, we identified 40 single somata of mnr2b-ABNs in r5 and r6, in addition to one descending interneuron (data not shown). Of these, 6 located in the ventral hindbrain were the only labeled mnr2b-ABNs in a hemibrain and revealed that the morphologies of axon terminals could be categorized into two main classes. The first class included mnr2b-ABNs with a terminal possessing linearly spaced boutons (‘‘en grappe’’ terminals), likely innervating multiply innervated muscle fibers (MIFs) with slow fiber properties (Figures 2B and 2C). The second class included mnr2b-ABNs with a single club-shaped terminal likely innervating singly innervated muscle fibers (SIFs) with fast fiber properties (Figures 2D and 2E). These terminals accumulated in the putative central en plaque endplate zone (CPEZ) in the middle of the muscle as in higher vertebrates

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€ttner-Ennever et al., 2001). The en grappe and en plaque ter(Bu minals were also visible when dorsal mnr2b-ABNs alone were labeled (Figure 2F), although the terminals were thinner and somata were smaller compared with ventral mnr2b-ABNs (Figure 2G). These observations suggest that the dorsoventral topography of mnr2b-ABNs reflects soma size rather than target myofiber type. To determine whether this soma size topography is defined by the temporal order of neuronal differentiation, as suggested by mnr2b-labeled spinal motor neurons, which consist of earlyborn larger neurons located more dorsally and late-born smaller neurons located more ventrally (Asakawa et al., 2013), we discriminated early- from late-differentiating mnr2b-ABNs using the photoconvertible protein Kaede. In Tg[mnr2bhs:Gal4]; Tg[UAS:Kaede] larvae, mnr2b-ABNs first appeared as a single-cell cluster in a hemisegment prior to dorsoventral segregation (Figures 2H and 2I) (Asakawa et al., 2012). The early differentiating mnr2b-ABNs were marked with red fluorescence by photoconverted Kaede at 44 hr post fertilization (hpf), and the subsequent distribution pattern along the dorsoventral axis was examined at 72 hpf (Figure 2J). Unexpectedly, we found that the early differentiating mnr2b-ABNs were distributed in both the dorsal and ventral clusters (Figures 2K and 2L), suggesting that larger and smaller mnr2b-ABNs concurrently differentiate before topographic segregation. Taken together, we conclude that mnr2b-ABNs in a rhombomere hemisegment are topographically arranged on the basis of soma size rather than target fiber type and neuronal birth order. mnr2b-ABNs in r5 and r6 Form Convergent Efferent Projections In the above experiments, we did not find any major differences in soma distribution pattern or developmental timing between mnr2b-ABNs in r5 and r6, but it remains uncertain whether mnr2b-ABNs in r5 and r6 show differences in efferent innervation patterns. To address this question, we developed a Gal4/Cre intersection approach to visualize mnr2b-ABNs in each of these rhombomeres by eGFP. We created two dual-color intersection UAS reporter lines. One reporter, Tg[UAS:loxRloxG], labeled r5 with eGFP via a ubiquitous Gal4 driver when the floxed mRFP1 was removed from r5 by the egr2b/Krox20-Cre transgene (Tg[egr2b-Cre]) (Figures 3A–3C), while the other reporter,

Figure 1. The Neuromuscular Connection between mnr2b-ABNs and the Lateral Rectus Muscle (A) Structure of Tg[mnr2b-hs:Gal4] and Tg[actc1b:tdT-chrnd]. The hsp70l promoter-Gal4FF-polyA-Kmr cassette was inserted downstream of the 50 UTR of mnr2b. tdT-chrnd is driven by the actc1b-hsp70l tandem promoter. (B) Lateral view of Tg[mnr2b-hs:Gal4]; Tg[UAS:GFP] larvae at 5 dpf. (C) Axonal projection of the eGFP-labeled abducens motor neurons. The dorsal cell cluster was omitted for clarity of the projections.

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(D–F) Dorsal (D), lateral (E), and rear (F) views of the eGFP cell clusters. The brackets indicate the dorsal cell cluster. (G) Dorsal view of lateral rectus muscles (LR) at 5 dpf. (H) Gathered (double arrowhead) and scattered (bracket) AChRs labeled by tdT-chrnd on the right LR in (G). (I) Lateral rectus muscle of Tg[mnr2b-hs:Gal4]; Tg[UAS:V2V]; Tg[actc1b:tdTchrnd] larva at 5 dpf. (J) The fluorescence intensities of the gathered tdT-chrnd and associated eGFP signals measured for each inner and outer halves of the muscle. N = 10 neuromuscular connections from five animals. *p = 0.024. ns, not significant. In (G)–(J), rostral is to the top. Scale bars indicate 500 bp in (A), 250 mm in (B), 50 mm in (C)–(F), (H), and (I), and 20 mm in (J). The vertical dashed lines indicate the midline. Hereafter, double white and black arrowheads indicate the presumptive initial contact sites with the lateral rectus muscle, r5, and r6, respectively, unless otherwise stated. D, dorsal; LL, left-lateral; R, rostral.

Figure 2. Soma Size Topography of mnr2bABNs (A) Left abducens nerve terminal of Tg[mnr2bhs:Gal4]; Tg[UAS:GFP] larva at 5 dpf. (B–E) Axon terminals (orange) and somata (blue) of single abducens motor neurons. (F) Two neighboring abducens motor neurons on the dorsal side of r5, possessing either an en grappe-type (left arrowhead) or en plaque-type (right arrowhead) terminal, respectively. (G) The soma size of the dorsal and ventral cells in r5 and r6. N = 40 cells from 22 animals. *p < 0.05 (t test). (H and I) The lateral (H) and dorsal (I) views of an mnr2b-ABNs in the Tg[mnr2b-hs:Gal4]; Tg[UAS:Kaede] embryo at 44 hpf. (J) Birth-order labeling of mnr2b-ABNs and possible segregation patterns of early-born neurons along the dorsoventral axis (magenta in I, II, and III). Green fluorescence of Kaede that appears after photoconversion is indicated as post-Kaede. (K and L) Dorsal (K) and ventral (L) sections of r5 and r6 in the photoconverted Tg[mnr2b-hs:Gal4]; Tg[UAS:Kaede] larvae at 72 hpf. Scale bars indicate 50 mm in (A)–(F) and 20 mm in (H), (I), (K), and (L).

Tg[UAS:loxGloxR], labeled r6 with eGFP because of Cre-mediated removal of the floxed eGFP in r5 (Figures 3D and 3E). We first labeled mnr2b-ABNs in r5 with eGFP by combining Tg[mnr2b-hs:Gal4], Tg[UAS:loxRloxG], Tg[egr2b-Cre], and Tg[actc1b:tdT-chrnd] transgenes (Figures 3J and 3K). In the quadruple-transgenic fish, the axon terminals were detected both in the CPEZ and around the caudal end of the eye globes (Figures 3L and 3M), indicating that the r5 subnucleus includes both en grappe- and en plaque-type mnr2b-ABNs. This axon terminal distribution was quite similar to that of all mnr2b-ABNs in r5 and r6 (Figures 3F–3I). Next, we labeled mnr2b-ABNs in r6 with eGFP using the Tg[UAS:loxGloxR] reporter (Figures 3N and 3O) and found that the r6 subnucleus also included mnr2bABNs with both en grappe and en plaque terminal morphologies (Figures 3P and 3Q). Collectively, these results suggest that mnr2b-ABNs in r5 and r6 contain similar types of mnr2b-ABNs and form convergent efferent projections onto the lateral rectus muscle. The intersectional labeling also clearly revealed an elaborate dendritic network of mnr2b-ABNs in r5 emanating laterally and then trespassing into r6 (Figures 3J and 3N), implying that mnr2b-ABNs in r5 and r6 have overlapping lateral dendritic fields. All mnr2b-ABNs Require mafba, but Not sall4, for Specification To elucidate the molecular mechanisms underlying the topographic organization and efferent projection patterns of

mnr2b-ABNs, we investigated the effects of DRS gene knockout on mnr2b-ABN development. Haploinsufficient and dominant-negative mutations of the transcription factor gene MAFB cause DRS in humans (Park et al., 2016), and mutations of mouse Mafb (also known as Kreisler or Krml1) result in the absence or hypoplasia of abducens nerve, recapitulating DRS pathology (McKay et al., 1994; Park et al., 2016). To address how the zebrafish ortholog of MAFB (mafba/valentino) contributes to mnr2b-ABN development, we used a novel mafba mutant (valM35A-Gal4) in which its only exon was disrupted by the Gal4 gene trap insert Tg[gSAIzGFFM35A] (Figure 4A) (Asakawa et al., 2013). As reported in the val mutant (Ma et al., 2014; Moens et al., 1996), the homozygous valM35A-Gal4/ M35A-Gal4 embryos formed a vestigial r5 and r6 with diminished rostrocaudal length at 24 hpf (Figures 4B–4E), suggesting that valM35A-Gal4 is a null allele. The enhancer trap line for mnr2b, Tg[mnr2b-GFP] (Asakawa et al., 2012), was crossed with valM35A-Gal4 to visualize mnr2b-ABNs expressing eGFP in a Gal4-independent manner (Figure S1). We found that not only the axons but also somata of mnr2b-ABNs were entirely absent in both r5 and r6 of valM35A-Gal4/ M35A-Gal4 homozygotes at 4–6 dpf (n = 5 animals; Figures 4F and 4G). Furthermore, all mnr2b-ABNs expressed olig2, which is essential for abducens motor neurons specification, using Tg[olig2:egfp] marker (Zannino and Appel, 2009) (Figures 4H–4K). Collectively, these observations indicated that mnr2b-ABNs require mafba for specification and are descendants of olig2-expressing neural progenitors in both r5 and r6. Haploinsufficiency of SALL4 transcription factor causes a specific type of DRS characterized by abducens nerve hypoplasia

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Figure 3. mnr2b-ABNs in r5 and r6 Form Convergent Efferent Projections (A) Structure of Tg[egr2b-Cre]. The Cre-polyA-Kmr cassette was inserted downstream of the 50 UTR of egr2b. (B and C) eGFP expression in a Tg[SAGFF73A]; Tg[egr2b-Cre]; Tg[UAS:loxRloxG] embryo at 20 hpf (C). Tg[SAGFF73A] transgene ubiquitously drives Gal4 from zfand5b promoter (B) (Asakawa and Kawakami, 2009). (D and E) EGFP expression (D) driven by the combination of Tg[SAGFF73A], Tg[egr2b-Cre], and Tg[UAS:loxGloxR] transgenes (E). (F, J, and N) Dorsal views of r5 and r6 in larvae carrying the transgenes indicated above at 5 dpf. (G, K, and O) Schematic illustrations of Gal4- and Cre-mediated expression of eGFP and mRFP1 in r5 and r6. (H, I, L, M, P, and Q) Axon terminals of mnr2bABNs innervating the left lateral rectus muscle. Tg [UAS:loxG] is generated by spontaneous excision of the floxed mRFP1 from Tg[UAS:loxRloxG] in the germline. The orange arrowheads indicate the terminal and boutons of en grappe terminals. The double arrowheads show the CPEZ. In (Q), mRFP1 signal in mnr2b-ABNs in r6 is below detection limit. Scale bars indicate 200 bp in (A) and 50 mm in (C), (E), (F), and (H).

and radial ray defects (also known as Okihiro syndrome/Duaneradial ray syndrome) (Al-Baradie et al., 2002; Demer et al., 2007; Kohlhase et al., 2002). To investigate functions of the zebrafish ortholog of SALL4 (sall4) in mnr2b-ABN development, we generated a frameshift mutation in the middle region of sall4 (sall4d11), the region where causative nucleotide insertions and deletions have been found in Okihiro syndrome patients, by using CRISPR-Cas9 method (Figures S4A and S4B). The homozygous sall4-d11 larvae were lethal and exhibited morphological abnormalities, including cardiac and eye edema, smaller eyes, and shortened bodies (Figures S4C and S4D). To our surprise, however, sall4-d11 homozygotes developed normal mnr2bABN clusters in r5 and r6 (Figures S4F–S4J), as well as neuromuscular connection between mnr2b-ABNs and the lateral rectus muscle at 4–6 dpf (Figures S4E and S4H), indicating that sall4 is not involved in the development of mnr2b-ABNs in zebrafish. pcdh17 Knockout Causes Abnormal Axonal Extension and Disrupts the Nuclear Topography of mnr2b-ABNs The cell-adhesion molecule Pcdh17/protochadherin-17 is expressed in the abducens nucleus of adult zebrafish (Liu et al., 2015), and its mouse ortholog Pcdh17 regulates the axonal extension of amygdala neurons (Hayashi et al., 2014) and synaptogenesis in corticobasal ganglia circuits (Hoshina et al., 2013).

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In zebrafish, pcdh17 is also expressed as early as 1.5–6 hpf, and expression becomes enhanced in specific tissues, including the hindbrain, during larval stages (Biswas and Jontes, 2009; Liu et al., 2009). Therefore, we hypothesized that Pcdh17 may play a key role in axon projection of mnr2bABNs. By double fluorescent in situ hybridization, we detected enhanced pcdh17 expression in a subset of mnr2b-ABNs in r5 and r6 as well as weaker widespread expression across the hindbrain at 40–44 hpf (Figures 5A, S5A, and S5B). Using the CRISPR-Cas9 method, we then created a pcdh17 mutant encoding a truncated Pcdh17 protein due to two neighboring deletions (75 and 2 bp) in the middle of the extracellular domain (pcdh17-d77; Figure 5B). We found that the pcdh17-d77 mutation exhibited maternal effects because, among the offspring of pcdh17-d77 incrosses, 31% of embryos including pcdh17d77 homozygotes, heterozygotes, and wild-type siblings displayed early developmental abnormalities, such as gastrulation defect (Figure S5C). Moreover, 65% of the embryos that did not show such early defects failed to inflate the swim bladder on time at 5 dpf, with an occasional formation of cardiac edema. Similar developmental abnormalities were observed among the offspring from pcdh17-d77 female outcrosses and, to a lesser extent, from pcdh17-d77 male outcrosses, suggesting that both maternal and zygotic Pcdh17 are required for normal development. In pcdh17-d77 homozygous as well as heterozygous larvae, we observed reduced axon growth and misguided axon projection of mnr2b-ABNs in approximately 20% of nerves at 3 dpf (Figures 5C and 5D). Furthermore, the distribution pattern of mnr2b-ABN somata was shifted significantly to the dorsal

side in pcdh17-d77 homozygous larvae at 3 dpf (Figures 5E and 5F). These observations indicate that pcdh17 is involved in the axon growth and nuclear formation of mnr2b-ABNs, although the earlier and global effect of the pcdh17-d77 mutation precluded a rigorous evaluation of pcdh17 expressed within mnr2b-ABNs.

Figure 4. All mnr2b-ABNs Require mafba for Differentiation (A) The mafba/valentino locus in Tg[gSAIzGFFM35A] line (valM35A-Gal4 allele). Tg[gSAIzGFFM35A] (shown as M35A) is inserted in the coding exon of mafba (filled red box). (B–E) Lateral (B and C) and dorsal (D and E) views of the hindbrain areas of Tg[UAS:GFP]; valM35A-Gal4/+ and Tg[UAS:GFP]; valM35A-Gal4/M35A-Gal4 embryos at 24 hpf. The yellow brackets indicate the vestigial r5 and r6. Dashed lines and ovals show the midline and otic vesicle (ov), respectively. (F and G) Dorsal views of the hindbrain and first spinal segment of Tg[mnr2bGFP]; valM35A-Gal4/+ (F) and Tg[mnr2b-GFP]; valM35A-Gal4/M35A-Gal4 (G) larvae at 80 hpf. The brackets and arrowheads indicate the dorsal and ventral mnr2bABNs, respectively. Asterisks in (G) show eGFP-expressing cells unrelated to mnr2b-ABNs. smn; spinal motor neuron; vr1; ventral root from the first spinal segment. (H) Dorsal view of r5 and r6 in Tg[olig2:egfp]; Tg[mnr2b-hs:Gal4]; Tg [UAS:mRFP1] at 3 dpf. (I and J) A single confocal section of the dorsal hindbrain in (H). (I) Arrows show mnr2b-ABNs (magenta) expressing eGFP derived from Tg[olig2:egfp] (J).

Protocadherin-Mediated Repulsion Regulates the Topographic Organization and Axon Projection of mnr2b-ABNs To investigate cell-autonomous functions of Pcdh17 in mnr2bABNs, we then used the Gal4/UAS system to express a dominant-negative Pcdh17 mutant lacking the cytoplasmic domain, which normally serves to mediate signaling with cytoskeletal regulators (Pcdh17-DCP) (Hayashi et al., 2014) (Figure 6A). A plasmid in which mRFP1-tagged pcdh17-DCP (pcdh17-DCPmRFP1) and eGFP were placed on either side of UAS (Figure 6A) was injected into Tg[mnr2b-hs:Gal4] embryos to label mnr2bABNs expressing pcdh17-DCP-mRFP1 with eGFP. We found that the eGFP-labeled mnr2b-ABN axons formed clumps either at the shaft or terminal region in approximately 40% of nerves (Figures 6C and 6D; 7 of 18 nerves, nine animals), while this clumping phenotype was not observed in axons expressing the full-length Pcdh17 (pcdh17-FL-mRFP1) (Figure 6B; 0 of 9 nerves, six animals), suggesting that Pcdh17 is cell-autonomously required for axon growth of mnr2b-ABNs. Axonal clumping is indicative of loss of self-avoidance mediated by protocadherin homophilic interactions (Hoshina et al., 2013; Mountoufaris et al., 2017). We reasoned that if protocadherin-mediated self-avoidance were required for growth of mnr2b-ABN axons, then the whole axon bundle would clump because of persistent homophilic interactions when pcdh17-DCP-mRFP1 was expressed in all mnr2b-ABNs. Strikingly, when pcdh17-DCP-mRFP1 and eGFP were bidirectionally expressed from a transgene (Figure 6A), the eGFP-labeled axons emanating from r5 and r6 were heavily clumped together around the ventral side of the r5/r6 boundary, and rostral extension was completely inhibited (Figures 6G and 6H). Moreover, the somata of mnr2b-ABNs expressing pcdh17-DCP-mRFP1 were tightly clustered on the dorsal side of the hindbrain and rarely observed on the ventral side. The clumping of axons and somata was not observed when pcdh17-FL-mRFP1 was overexpressed from a transgene (Figures 6E–6G), although misguided axon projections and defasciculation were observed at a low frequency (Figures 6I and S6B). The pcdh17-DCP-dependent clumping required membrane targeting because removal of the transmembrane domain from pcdh17-DCP (pcdh17-DCPT) completely abolished the clumping effect (Figures S6C and S6D). Taken together, these observations suggest that protocadherin-mediated repulsion is required not only for axon growth but also for migration of abducens motor neurons. Intriguingly, pcdh17-DCP-mRFP1 expression in the spinal motor neurons driven by Tg[mnr2b-hs:Gal4] resulted in similar clumping of (K) Percentage of the mRFP1-expressing cells that expressed eGFP. In total, 78 and 87 cells in r5 and r6, respectively, were examined in five larvae. Scale bars indicate 200 bp in (A), 100 mm in (B), 50 mm in (D)–(H), and 5 mm in (I) and (J).

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Figure 5. pcdh17-d77 Mutation Causes Defective Axon Growth and Guidance, as Well as Soma Topography Formation (A) Fluorescence in situ hybridization for EGFP and pcdh17 in the left hemisegments of r5 and r6 of the Tg[mnr2b-hs:Gal4]; Tg[UAS:GFP] larva at 40 hpf. Arrows indicate stronger pcdh17 signal overlapping EGFP signal. (B) The pcdh17-d77 allele contains two deletions (red) in the extracellular domain 3 (EC3) generating a premature stop codon (green). The underlined cgg indicates the protospacer adjacent motif (PAM). EC, TM, and CP stand for extracellular, transmembrane, and cytoplasmic domains, respectively. (C) The axon growth (yellow) and pathfinding (red) defects of mnr2b-ABNs at 3 dpf. The black and orange dashed lines demarcate the eye and otic vesicle (ov), respectively. (D) Frequency of abnormal axonal projection in wild-type (WT), heterozygous (d77/+), and homozygous (d77/d77) mutant larvae. (E) Dorsal shift of mnr2b-ABN somata by pcdh17d77 mutation. The dorsal index is defined by the ratio of the soma areas in the dorsal half of the nucleus (s[D]) to those of all mnr2b-ABNs (s[D] + s [V]) in a rostrocaudally projected image. ns, p = 0.15; *p = 0.0144 (unpaired t test). Error bars represent SD. (F) Nuclear organization mnr2b-ABNs (left) and dorsal and frontal views of mnr2b-ABN somata at 3 dpf. Scale bars indicate 20 mm in (A) and (F) and 50 mm in (C).

axons and somata and disrupted the dorsal localization of larger neurons in the spinal cord (Figure S6E), suggesting that protocadherin-mediated repulsion commonly operates in somatic motor neurons to promote axon growth and formation of soma topography. On the other hand, pcdh17-DCP-mRFP1 expression in r5, r6, and spinal interneurons did not cause gross anatomical alteration (Figure S7), suggesting that Pcdh17-mediated repulsion operates in a cell-type-specific manner. DISCUSSION Our analyses revealed that the abducens nerve and lateral rectus muscle of zebrafish include evolutionarily conserved cell types in higher vertebrates, such as motor neurons with en grappe and en plaque axon terminals and myofibers arranged in longitudinal subcompartments (Figure 7A). Despite these similarities at the cellular level, it was unclear how these motor neurons are spatially arranged and connected to subcompartments of the target muscle given that the abducens nucleus is segmentally iterated in teleosts. Single-cell, birth order, and Gal4/Cre intersectional labeling approaches indicated that mnr2b-ABNs in r5 and r6 are similar in neuronal type, differentiation timing, and efferent innervation pattern. In addition, the lateral dendrites of mnr2b-ABNs are shared beyond the rhombomeric border, suggesting that these abducens nuclei may receive common premotor inputs. Therefore, we conclude that the segmentally iterated teleostean abducens nuclei contain anatomically and

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functionally homologous motor neurons (Cabrera et al., 1992; Pastor et al., 1991). A potential advantage of having such a homologous set of neurons is that the dynamic range of the lateral rectus muscle, and hence that of lateral eye movement, could be expanded by an increase in recruitable motor units (Fuchs and Luschei, 1970), which may be beneficial for survival of lateraleyed animals such as fish. Our results, however, do not exclude the possibility that the projections from abducens nuclei in r5 and r6 are topographic within the inner or outer muscle compartment, because the positional difference between these nuclei may cause a temporal difference in the onset of innervation, potentially generating a local innervation topography. The findings that mnr2b-ABNs required Mafba for differentiation and that virtually all mnr2b-ABNs were the descendants of olig2+ progenitors suggest that Mafba-Olig2-Mnx pathway serves as a common genetic cascade for abducens motor neuron differentiation across vertebrates (Figure 7B) (Lu et al., 2002; McKay et al., 1997; Thaler et al., 1999; Zhou and Anderson, 2002). Robust activation of hoxb3/Hoxb3 appears necessary for development of the abducens nucleus as hoxb3/ Hoxb3 upregulation occurs in both r5 and r6 of zebrafish, but only in r5 of mice (Gaufo et al., 2003; Manzanares et al., 1997; Prince et al., 1998). Considering that Mafba/MafB, which can directly activate hoxb3/Hoxb3 (Manzanares et al., 1997), is expressed in both r5 and r6 of zebrafish and mice, the lack of an abducens nucleus in r6 of higher vertebrates may have been caused by extinction of Mafba-mediated Hoxb3 activation

Figure 6. Dominant-Negative Pcdh17 Disrupts Nuclear Topography and Axon Growth of mnr2b-ABNs (A) Bidirectional UAS constructs for expression of the mRFP1-tagged full length (pcdh17-FL-mRFP1) and dominant-negative Pcdh17 (pcdh17-DCPmRFP1). (B and C) Dorsal views of the right brain hemispheres of Tg[mnr2b-hs:Gal4] larvae injected with the UAS constructs for the full-length pcdh17 (B) or pcdh17-DCP (C). In (C), the nerve terminal and axon clump are indicated by an arrow and a red bracket, respectively. (D) Frequency of the axon clumping. For pcdh17FL and pcdh17-DCP, 9 nerves from 6 animals and 18 nerves from 11 animals were examined, respectively. (E–G) Dorsal (left, right) and rear (middle) views of r5 and r6 at 3 dpf. The orange arrows and double arrowheads indicate the normal and clumped axons, respectively. (H and I) Frequency of abnormal axon growth (H) and fasciculation (I). Numbers in the histograms indicate the numbers of nerves examined. NA, not applicable because of severe axon growth defect. (J and K) Schematic illustrations of normal (wild-type and pcdh17-FL) (J) and abnormal (pcdh17-DCP) (K) nuclear formation and axon growth of mnr2b-ABNs. Scale bars indicate 50 mm in (B) and (C) and 20 mm in (E)–(G).

specifically in r6 by an as yet unknown mechanism (Manzanares et al., 1997). Our conclusion that Mafba is essential for the differentiation of mnr2b-ABNs is in accordance with the observation that the abducens nucleus recognized by the zn-5 antibody is rare or completely absent in the val mutant (Moens et al., 1996) but is inconsistent with the finding that, in the val mutant, the olig2 enhancer trap line labels the abducens/VI nerve with a similar diameter to wild-type siblings, which lead to the proposal that the abducens nucleus exists in the val mutant and is likely to have similar size as the wild-type (Ma et al., 2014). Although such Mafba-independent abducens motor neuron is indeed identifiable by retrograde labeling (Ma et al., 2014), the entire size of such neuronal population needs to be determined based not only on nerve diameter but also on soma positioning and target muscle, as loss of Mafba could lead to ectopic neuronal differentiation through alteration of Hox3 expression (Gaufo et al., 2003; Manzanares et al., 1997). In

addition, whether such Mafba-independent abducens motor neuron is solely responsible for the horizontal eye movement performed by the val mutant (Ma et al., 2014) needs to be determined, given that the lateral rectus muscle is ectopically innervated by other ocular motor neurons when abducens motor neuron development is disrupted by knocking out MafB in mice (Park et al., 2016). The topographic organization of the zebrafish abducens nucleus along the dorsoventral axis is based on soma size, but not on birth order, highlighting similarities and differences between abducens and spinal motor neurons, both of which are categorized as somatic motor neurons. From a functional point of view, soma size topography of the spinal motor column, where large and early-born neurons are located more dorsally, matches recruitment order during locomotion (McLean et al., 2007; Menelaou et al., 2014). How the soma size topography of abducens motor neurons relates to recruitment pattern during eye movement control is an important question to be addressed in the future (Robinson, 1970). From a developmental perspective, the soma clumping of the abducens and spinal motor neurons by Pcdh17-DCP expression argues that Pcdh17-dependent migration of larger motor neurons is a major driving force for the topographic soma distribution. These clumping phenotypes are likely due to loss of self-avoidance mediated by the

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Figure 7. Architecture and Developmental Mechanism of the Neuromuscular Connection between mnr2b-ABNs and the Lateral Rectus Muscle (A) mnr2b-ABNs with en plaque-type terminal (SIFMN) innervate the CPEZ in the lateral rectus muscle, while ones with en grappe-type terminals (MIF-MN) innervate the outer lateral rectus muscle. The outer layer contains SIF expressing the fast muscle-specific troponin T type3 (tnnt3b) and MIFs expressing the slow muscle-specific troponin T type 1 (tnnt1). (B) mnr2b-ABNs in the right hemisegment of r5 and r6 form a convergent projection to the lateral rectus muscle. In each of r5 and r6, mnr2b-ABNs with smaller and larger somata are clustered dorsally (D) and ventrally (V), respectively. The lateral dendrites of mnr2b-ABNs in r5 and r6 overlap each other. In both r5 and r6, Mafba activates the Olig2Mnr2b/Mnx cascade, possibly via Hoxb3, for differentiation of mnr2b-ABNs. Protocadherinmediated repulsive force (right) supports ventral migration and axon growth of mnr2b-ABNs.

homophilic interactions between Pcdh17 molecules (Hoshina et al., 2013), as observed when protocadherin function is compromised (Hayashi et al., 2014; Lefebvre et al., 2012; Mountoufaris et al., 2017). Thus, considering the everted configuration of the teleost brain, we propose that protocadherin-mediated repulsion promotes migration of larger motor neurons away from the ventricle and spinal canal (i.e., from the apical side of the hindbrain and spinal cord, respectively) (Holmgren, 1922). The Pcdh17-DCP-dependent axon clumping of abducens and spinal motor neurons further suggests that axon growth is also regulated by protocadherin-mediated repulsion (Figure 7B). Such repulsive forces among motor neurons may help maintain appropriate positional relationships within both the nucleus and nerve bundle, while still allowing individual neurons to respond to extrinsic directional axon guidance cues. Although we focused on pcdh17 in this study, multiple cadherin superfamily molecules present on the cell surface could also be involved in nuclear topography formation and axon growth of abducens motor neurons. In fact, protocadherin pcdh19 is also expressed in the zebrafish abducens nucleus (Liu et al., 2015). Functional redundancy of protocadherins may explain why the pcdh17 knockout produced milder phenotypes than Pcdh17-DCP overexpression. In principle, any gene mutation compromising abducens motor neuron development may contribute to eye movement disorders in humans. Indeed, mutations in MAFB (Park et al., 2016) and HOXA1 (Tischfield et al., 2005), genes required for specification of abducens motor neurons, have been identified in DRS patients. Alternatively, the DRS-associated transcription factor SALL4/sall4 is dispensable for abducens motor neuron specification in mice (Sakaki-Yumoto et al., 2006) and zebrafish, implying a human-specific function of SALL4 in abducens motor neuron development. Our finding that the perturbation of pcdh17

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disrupted soma topography and axon growth of abducens motor neurons predicts that protocadherins are potential genetic candidates for human eye movement disorders. It is worth noting that the mouse Pcdh17 has been identified as a major protocadherin in the MafB-expressing spiral ganglion cells, but not in closely related vestibular ganglion cells lacking MafB expression (Lu et al., 2011), raising the possibility that Pcdh17-mediated cell repulsion is an important cellular outcome of MafB-mediated transcriptional program in health and DRS pathology. Although a pathological link between protocadherins and CCDDs remains to be proven, mutations in protocadherin genes could possibly contribute to CCDDs, given the distinct expression profiles of cadherin superfamily cell adhesion molecules in various cranial motor nuclei (Astick et al., 2014). Last, the ocular neuromuscular pathways in humans display selective vulnerability or resistance to certain types of muscular dystrophies and motor neuron diseases (Kaminski et al., 2002; Yu Wai Man et al., 2005), but mechanisms are relatively unexplored because of restricted tissue accessibility. We envision that the optically and genetically accessible abducens neuromuscular connection uncovered here could be of great help for interrogating pathologies of such diseases, as well. EXPERIMENTAL PROCEDURES Fish This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee (IACUC; approval identification number 24-2) of the National Institute of Genetics (NIG) of Japan, which has an Animal Welfare Assurance on file (assurance number A5561-01) at the Office of Laboratory Animal Welfare of the NIH. Procedures for creating transgenic and mutant lines are shown in Supplemental Experimental Procedures.

Whole-Mount Double Fluorescence In Situ Hybridization Fluorescence in situ hybridization was performed as previously described (Asakawa et al., 2013). An antisense probe for pcdh17 was synthesized with the digoxigenin (DIG) RNA labeling kit (Roche) using nucleotides 1–2148 of the pcdh17 coding sequence (GenBank accession number NM_001160822). An antisense probe for eGFP was synthesized with the fluorescein RNA labeling kit (Roche) using the full length of eGFP. The DIG-labeled pcdh17 and fluorescein-labeled eGFP were detected by TSA Kit#16 Alexa Fluor 647 tyramide and TSA Plus Fluorescein system (NEL74100KT; PerkinElmer), respectively. Kaede Photoconversion In Tg[mnr2b-hs:Gal4]; Tg[UAS:Kaede] larvae (Scott et al., 2007), mnr2b-ABNs expressing Kaede were irradiated by a laser with 405 nm wavelength by using Olympus FV-1000D laser confocal microscope at 44 hpf. Single-Cell Labeling For single-cell labeling of Gal4-expressing cells, 18 pg of pT2KUASGFP plasmid (Asakawa et al., 2008) was injected into a blastomere at the four- or eight-cell stage. Only cells uniquely labeled with eGFP in a hemisphere were analyzed by confocal microscopy. Soma size was defined by measuring the largest cross sectional area by ImageJ software (NIH). Microscopic Analysis For confocal microscopy, a fish was embedded in 1% low-melting agarose (NuSieve GTG Agarose; Lonza) on a Glass Base dish (3010-035; IWAKI) and analyzed by Olympus FV-1000D microscope. Z sections were analyzed with Olympus Fluoview version 2.1b Viewer and Adobe Photoshop CS6. Fluorescence intensity measurements were performed using ImageJ software. DATA AND SOFTWARE AVAILABILITY The data that support the findings in this study are available within the article and in the Supplemental Information and from the corresponding authors upon request. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and one table and can be found with this article online at https://doi.org/10.1016/j.celrep.2018.07.024. ACKNOWLEDGMENTS We would like to thank Kawakami lab members for valuable discussions, M. Okamura for helping with UAS:loxRloxG construction, Drs. B. Appel and H. Baier for sharing fish lines, and A. Ito, M. Suzuki, M. Mizushina, N. Mouri, Y. Kanebako, and F. Mizuno for fish maintenance. This work was supported partly by KAKENHI grants JP15H02370 (K.K.), JP18H04988 (K.K.), JP22700349 (K.A.), and JP23115720 (K.A.), the Uehara Memorial Foundation (K.A.), the Kao Foundation for Arts and Sciences (K.A.), the Mitsubishi Foundation (K.A.), the Daiichi-Sankyo Foundation of Life Science (K.A.), and the National BioResouce Project of the Japan Agency for Medical Research and Development (AMED) (K.K.). AUTHOR CONTRIBUTIONS K.A. conceived the research, designed and performed the experiments, and analyzed the data. K.A. and K.K. performed gene trap screening. K.A. and K.K. wrote the manuscript.

Received: March 1, 2018 Revised: May 31, 2018 Accepted: July 6, 2018 Published: August 7, 2018

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DECLARATION OF INTERESTS

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The authors declare no competing interests.

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