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. Author manuscript; available in PMC: 2018 Apr 24.
Published in final edited form as: Dev Cell. 2017 Apr 24;41(2):195–203.e3. doi: 10.1016/j.devcel.2017.03.024

PCP and SAX-3/Robo pathways cooperate to regulate convergent-extension-based nerve cord assembly in C. elegans

Pavak K Shah 5,#, Mathew Tanner 1,2,#, Ismar Kovacevic 5, Aysha Rankin 1,2, Teagan E Marshall 5, Nathaniel Noblett 1,2, Nhan Nguyen Tran 5, Tony Roenspies 1, Jeffrey Hung 1,2, Zheqian Chen 2, Cristina Slatculescu 1, Theodore J Perkins 4, Zhirong Bao 5,*, Antonio Colavita 1,2,3,*
PMCID: PMC5469364  NIHMSID: NIHMS865558  PMID: 28441532

SUMMARY

Formation and resolution of multicellular rosettes can drive convergent-extension (CE) type cell rearrangements during tissue morphogenesis. Rosette dynamics are regulated by both Planar Cell Polarity (PCP)-dependent and independent pathways. Here we show that CE is involved in ventral nerve cord (VNC) assembly in C. elegans. We show that a VANG-1/Van Gogh and PRKL-1/Prickle containing PCP pathway and a Slit-independent SAX-3/Robo pathway cooperate to regulate, via rosette intermediaries, the intercalation of post-mitotic neuronal cell bodies during VNC formation. We show that VANG-1 and SAX-3 are localized to contracting edges and rosette foci and act to specify edge contraction during rosette formation and to mediate timely rosette resolution. Simultaneous loss of both pathways severely curtails CE resulting in a shortened, anteriorly-displaced distribution of VNC neurons at hatching. Our results establish rosette-based CE as an evolutionarily conserved mechanism of nerve cord morphogenesis and reveal a role for SAX-3/Robo in this process.

Keywords: Planar Cell Polarity, Convergent-Extension, Vang-1/Van Gogh, Sax-3/Robo

ETOC

Shah, Tanner et al. show that the axon guidance factors SAX-3 and Robo also play a role in organizing the C. elegans ventral nerve cord (VNC) via convergent extension-type cell rearrangements. SAX-3 and Robo cooperate with the planar cell polarity pathway to promote intercalation of neuronal cell bodies during VNC formation.

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INTRODUCTION

Convergent-extension (CE) movements, which consist of regulated cell intercalation behaviors that act to narrow tissues along one axis while elongating them along the perpendicular axis, contribute to many developmental processes including axial elongation, organogenesis and neural tube closure (Devenport, 2016). The formation and resolution of multicellular rosette structures has emerged as a key cellular mechanism driving polarized cell intercalation during CE (Blankenship et al., 2006; Lienkamp et al., 2012; Williams et al., 2014). These movements require the planar polarized organization of actomyosin-based contractile forces and remodeling of adhesive structures at specific cell interfaces to promote rosette assembly and subsequent cell intercalation along the axis of elongation during rosette resolution (Harding et al., 2014; Walck-Shannon and Hardin, 2014). Several molecular pathways participate in this process. The highly conserved planar cell polarity (PCP) pathway that includes orthologs of the Drosophila genes Van Gogh and Prickle is involved in the spatial regulation of cytoskeletal elements and polarized cell movements in a variety of developmental contexts (Devenport, 2016). These include CE, where PCP has been shown to spatially regulate cell neighbor exchanges including those mediated through rosettes (Lienkamp et al., 2012; Nishimura et al., 2012; Williams et al., 2014). For example, the shortened neural tube in mouse Vangl2 mutants is in part attributable to both delays in rosette resolution and loss of spatially-biased cell intercalations during rosette resolution (Williams et al., 2014). The protein tyrosine kinase PTK7 also regulates planar polarized cell neighbor exchanges during CE, including those involving rosettes, but likely acts in parallel with Vang/PCP signaling (Lee et al., 2012; Williams et al., 2014). In the Drosophila embryo, where rosette-based cell movements were first identified and shown to be involved in germband elongation, a PCP-independent pathway containing Toll family receptors acts to establish a polarized actomyosin cytoarchitecture (Blankenship et al., 2006; Pare et al., 2014). These findings highlight the diversity of molecular pathways involved in rosette-mediated cell intercalations.

In C. elegans, components of the classic PCP pathway, such as vang-1/Van Gogh, prkl-1/Prickle or fmi-1/Flamingo, have been shown to function in axon navigation (Najarro et al., 2012; Sanchez-Alvarez et al., 2011; Steimel et al., 2010), long range migration of single cells (Mentink et al., 2014), orienting asymmetric cell division (Green et al., 2008) and tube formation (Asan et al., 2016; Hoffmann et al., 2010) but have not been linked to CE-like behaviors such as dorsal epidermal cell intercalation (Walck-Shannon and Hardin, 2014).

Here we show that a rosette-based CE mechanism is involved in neuronal intercalation during embryogenesis to assemble the VNC of C. elegans. This process involves a Vang/PCP pathway and sax-3/Robo, a single pass transmembrane protein that has not been previously implicated in rosette dynamics or CE. We find that PCP and sax-3/Robo cooperate to specify edge contraction during rosette assembly and act in a partially redundant manner to mediate rosette resolution and single cell intercalation.

RESULTS and DISCUSSION

Convergent Extension Underlies C. elegans VNC Assembly

During embryogenesis, DA, DB and DD class motor neurons arise from left and right lineages and undergo movements towards the presumptive midline where they intercalate into a single file line running along the anterior-posterior (AP) axis of the animal by the time of hatching (Sulston et al., 1983). This morphogenetic process, in particular how the VNC neurons intercalate to form a single file, has not been well characterized.

To elucidate how the VNC is assembled, we performed 3D fluorescence time-lapse imaging (Hallam et al., 2000; Murray et al., 2012) on a subset of the VNC neurons including DA1–5 and DD1–6. We observed that VNC assembly occurs in three phases (Figure 1A). First, as the neurons migrate towards the ventral midline, constrictions of cell-cell contacts occur on both the left and right sides. These edge contractions are accompanied by intercalations among the VNC neurons, which in turn leads to a stereotypical alternating sequence of DAs and DDs on each side before they meet at the midline (Figure 1B, color key). For all figures showing images of the 2nd rosette, the 0′ time point always corresponds to the first observation of the engaged rosette. Second, upon the meeting at the ventral midline, these neurons form a set of multicellular rosettes through the constriction of cell-cell contacts (Figures 1B and 1C). Figures 1B and 1C were taken of different embryos. The 0′ time point in each shows the same rosette in two different embryos with the same cells engaged in rosettes with identical topologies, we observed the structure of this rosette to be highly stereotyped (N=16). These rosettes form along the midline, involving cells from both sides. Interestingly, rosette formation follows a temporal sequence along the AP axis, with the anterior-most rosette forming first and the posterior-most rosette forming last. Finally, as rosettes resolve, single-cell intercalations occur. These intercalations are not accompanied by constrictions of cell contacts or further rosette formation. Instead, the cells appear to protrude at the leading edge of movement. These single-cell intercalations lead the neurons to cross the midline and eventually form a single file along the ventral midline prior to the onset of embryonic twitching (Figures 1C–E). These three phases occur simultaneously with ventral enclosure and constriction of the epiderm which has been shown to depend on forces generated in the VNC neurons (Wernike et al., 2016). Taken together, our observations show that the C. elegans VNC undergoes CE to assemble into a linear structure.

Figure 1. Convergent extension of the ventral nerve cord.

Figure 1

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(A) Max projections of C. elegans embryos expressing cnd-1p::PH::RFP. Red contours outline the embryo. White dashed box marks the VNC. Scale bar, 10 μm. (B) Single slices showing three distinct rosettes observed during CE in the VNC. White arrow points to the vertex of the most anterior rosette in which only 3 cells are labeled. Red dashed boundary outlines the rosettes. Accompanying schematics and color key identifies the specific neurons in each rosette as well as which arise from the right (R) and left (L) lineages. Asterisk marks RIGL. The color scheme is used in the rosette schematics and 3D reconstructions. Scale bar, 5 μm. (C) Example assembly and resolution dynamics of the central rosette identified in (B) imaged in a different embryo. Dashed circle outlines the rosette. In the corresponding schematics magenta lines mark shrinking cell-cell contacts and cyan marks newly formed cell contacts. Colored circles identify each cell according to the legend provided in (B). Scale bar, 5 μm. (D) 3D reconstruction of DA and DD neurons (same color scheme as in panel (B) during rosette resolution (0′–6′) and subsequent single-cell intercalations beginning with DD3 (9′–19.5′). White arrowheads highlight intercalating cells. (E) 3D reconstruction showing final linear configuration of DA and DD neurons in the VNC prior to the onset of twitching, color scheme same as in (B).

A PCP-Related Pathway Regulates VNC Neuron Positions

We then asked whether genes in the PCP pathway were required for VNC assembly. We found that mutations in the core PCP genes vang-1/Van Gogh and prkl-1/Prickle display strong defects in the position of DD neurons in newly hatched L1 worms (Figures 2A–C and 2F). Specifically, we defined the DD1 position as 0% AP position and the anus as 100% AP position. In vang-1 and prkl-1 mutants, the relative mean positions of DD2–6 along the VNC were found to be more anteriorly displaced compared to wild type (wt) animals (Figures 2C and 2F). DD spacing defects were also apparent relative to DA and DB neurons at later larval stages (Figures 2G and 2H). These defects were unlikely to be a consequence of obvious cell fate changes as expression patterns from DD (flp-13) or DA and DB (unc-129) specific reporters in vang-1 and prkl-1 mutants appeared normal (Figure 2).

Figure 2. PCP genes and sax-3 act in parallel genetic pathways to regulate neuron position in the ventral nerve cord.

Figure 2

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(A) Schematic of DD, DA, and DB neuron positions in the ventral nerve cord (VNC), retrovesicular ganglion (RVG) and preanal ganglion (PAG). (B–E) Representative images of L1 stage DD1–6 (arrowheads) in wt and mutant backgrounds labeled with a ynIs37[flp-13p::gfp] reporter. (F) Quantification of DD2–4 positions relative to DD1 in L1 stage wt and mutant worms. Neurons (color coded as indicated along top) are plotted along AP axis where DD1 marks the 0% AP position and the anus is the 100% AP position. Means and 95% confidence intervals are shown for each DD neuron with DD2–6 neurons in mutants compared to the corresponding wt neuron (N=40–53 worms). *p≤0.01; **p≤0.001, using the two-tailed t-test. (G–J) Representative images of L4 stage DD (green arrowheads) and DA/DB (red arrowheads) neurons in wt and mutant backgrounds visualized with a zyIs27[flp-13p::gfp unc-129p::mCherry] reporter. (K and L) Representative images of L1 stage DD and DA/DB neurons in wt and sax-3(zy5); prkl-1(ok3182) double mutant visualized with zyls27. (M and N) Representative fluorescent and Nomarski images of L1 stage nervous system in wt and vang-1(ok1142) sax-3(zy5) double mutant visualized with the pan-neuronal otIs173[rgef-1p::mCherry] reporter. Bracket indicates region in double mutant devoid of neuronal cell bodies. All scale bars, 50 μm.

sax-3/Robo Acts in Parallel to PCP to Distribute Neurons Along the VNC

sax-3(zy5), a mutation in the sole worm Robo ortholog (Zallen et al., 1998), identified in a separate genetic screen for axon guidance mutants was found to exhibit the same DD position defects as in the PCP mutants (Figures 2D, 2F, 2I, and S1). zy5, a nonsense allele (Q987stop) that is predicted to generate a protein with a truncated cytoplasmic domain (Figure S2A), displays significantly less of the lethality, body morphology and head neuronal structural defects (Ghenea et al., 2005; Kennerdell et al., 2009) that are hallmarks of the sax-3(ky123) null phenotype (Figures S2B–F). Both zy5 and ky123 worms displayed similar VNC defects (Figure S1A).

To investigate the genetic interactions between PCP and sax-3 pathways, we examined neuron positioning in double mutants. Strikingly, vang-1(ok1142) sax-3(zy5) and sax-3(zy5); prkl-1(ok3182) double mutants displayed much more exaggerated phenotypes in which DD, DA and DB neurons were located at more extreme anterior positions compared to single mutants (Figures 2E, 2F and 2J). Embryonically-derived VNC neurons were preferentially more affected than post-embryonically derived neurons (Figure S3). However, despite the severe position defects, most DD, DA and DB neurons still aligned in a relatively single file (Figures 2K and 2L). Axons also ran along the full length of the VNC between grossly normal head and tail ganglions (N>100 worms) (Figures 2M and 2N) suggesting that the main function of the PCP and sax-3 pathways in regulating VNC assembly is in regulating cell positions instead of the nerve tracts. Not surprisingly given the drastically altered VNC landscape, double mutants were strongly uncoordinated. Interestingly, loss of slt-1, the sole member of the Slit family of Robo ligands in worms (Hao et al., 2001) did not display DD position defects on its own or more severe defects in vang-1 and prkl-1 double mutants (Figure S1B). Taken together, these findings suggest that a PCP pathway and a slit-independent sax-3/Robo pathway act in parallel to regulate the proper placement of VNC neurons.

VANG-1, PRKL-1 and SAX-3 Act in Embryonic VNC Neurons

Given that VNC assembly in the embryo involves CE and that PCP and Robo mutants show neuronal positioning defects in the VNC, we asked whether these pathways act in the VNC to regulate CE. vang-1, prkl-1 and sax-3 have been found to be expressed in both neuronal and the adjacent epidermal tissue (Bernadskaya et al., 2012; Ghenea et al., 2005; Sanchez-Alvarez et al., 2011). Therefore, to narrow their site of action we expressed these proteins from neuronal or epidermal specific promoters and assessed their ability to rescue DD position defects in mutant animals. We found that only neuronally expressed vang-1, prkl-1 and sax-3 were capable of rescuing anteriorly displaced DD neurons indicating that these genes act in neurons to mediate VNC assembly (Figures 3A–C).

Figure 3. Localization of PCP components, SAX-3/ROBO and NMY-2 during rosette assembly.

Figure 3

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(A–C) Neuronal but not epidermal-specific expression of (A) vang-1, (B) prkl-1 and (C) sax-3 rescue the anterior displacement of DD neurons in the corresponding mutant backgrounds. DD neuron quantification and color scheme as described in Figure 2F. Each DD2–6 neuron in transgenic worms (TG+) are compared to their corresponding neuron in non-transgenic (TG-) siblings. Means and 95% confidence intervals are shown for each DD neuron. *p≤0.01; **p≤0.001, using the two-tailed t-test. (N=44–62 worms). (D–G) Partial max projections of volumes acquired of embryos expressing the cnd-1 membrane label and functional GFP fusions with (D) PRKL-1 (zyls33), (E) VANG-1 (zy60), (F) NMY-2 (cp13) and (G) SAX-3 (zyls43) during rosette formation. Scale bar is 5 μm in (D), 2 μm in (E–G). Dashed ovals in early time point mark a contracting cell contact, dashed circles at later time point marks the resulting rosette vertex. (H) Enrichment of VANG-1::GFP and NMY-2::GFP at the contracting cell-cell contact during rosette assembly relative to mean GFP levels at non contracting cell contacts. (N=3) (I) Enrichment of VANG-1::GFP and NMY-2::GFP at the rosette center after rosette formation relative to mean GFP levels at the pair-wise cell contacts between all cells in the rosette. (N=3) Error bars show standard error.

Protein localization during VNC assembly was examined using transgenic lines expressing functional PRKL-1 and SAX-3-GFP fusions (Ghenea et al., 2005; Sanchez-Alvarez et al., 2011) and a CRISPR/Cas9-mediated GFP insertion at the N-terminus of the native vang-1 locus (Figure S1B). We found that vang-1, prkl-1 and sax-3 are expressed in VNC neurons. However, in contrast to Prickle localization in other CE models (Jiang et al., 2005; Narimatsu et al., 2009; Yin et al., 2008), PRKL-1 (N=6) was not observed to exhibit recognizable membrane or polarized sub-cellular localization. Relatively uniform PRKL-1 cytoplasmic localization was observed in VNC neurons (Figure 3D). On the other hand, VANG-1 (N=9) was observed to localize to cell membranes and is enriched at contracting cell contacts (Figure 3E). Additionally, VANG-1 is localized at rosette foci. These localization dynamics resemble that of endogenous myosin II (NMY-2) (Dickinson et al., 2013) which is also found on contracting cell contacts and rosette foci (Figure 3F). Our SAX-3::GFP transgene showed low levels of expression and GFP signal, making it difficult to quantify. However, in a small fraction of embryos with relatively high signal (N=2), the localization patterns resemble that of VANG-1 qualitatively (Figure 3G). VANG-1 and NMY-2 both exhibited approximately 4-fold enrichment at contracting edges versus non-contracting edges (Figure 3H) and approximately 7-fold enrichment at the rosette center relative to the planar contacts between cells engaged in the rosette (Figure 3I).

PCP and sax-3/Robo Pathways are Required for Proper Cell Intercalation and Rosette Resolution During CE

In vang-1(ok1142), prkl-1(ok3182) and sax-3(zy5) mutants, VNC neurons fail to fully intercalate into a single file prior to the onset of twitching (Figure 4A). These neurons also exhibit persistent ectopic cell-cell contacts. Strikingly, sax-3(zy5); prkl-1(ok3182) double mutants exhibit a significantly more severe phenotype manifesting as a nearly complete failure to intercalate across the ventral midline by the onset of twitching (Figure 4A).

Figure 4. Phenotypes of vang-1, prkl-1 and sax-3/Robo mutants in rosette resolution and orienting cell intercalation.

Figure 4

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(A) Max projections and 3D reconstructions showing the embryonic VNC in representative wt and mutant embryos expressing cnd-1p::PH::RFP at the 1.5-fold stage. White arrows highlight ectopic cell contacts not observed in the wt at this stage. Color key used is the same as in Figure 2. Scale bar, 5 μm. (B) Schematic highlighting the location of expanded views (dashed box) showing a stereotyped neighbor exchange between posterior DA and DD neurons in wt and mutant embryos expressing cnd-1p::PH::RFP. Red dot marks DA5, blue dot marks DD4 and purple dot marks DD6. Red arrows point to the contraction of the posterior edge of DD4, white arrows point to the contraction of the anterior edge of DD4. Schematics illustrate the corresponding wt and mutant behaviors. C) Frequency with which the anterior edge of DD3/4 was observed to contract in wt and mutants. D) Relative enrichment of VANG-1::GFP at the posterior edge of DD3/4 vs the anterior edge of DD3/4 in embryos immediately prior to contraction as in (B) in wt (N=5) and prkl-1(ok3182) (N=4) embryos. Error bars show standard error. E) Examples of varied rosette resolution dynamics observed in wt, vang-1(ok1142), prkl-1(ok3182), sax-3(zy5) and sax-3(zy5);prkl-1(ok3182) embryos expressing cnd-1p::PH::RFP. Red dashed line outlines the cells participating in the rosette. F) Box-and-whisker plots showing the distribution of observed rosette lifetimes in wt (N=7), vang-1(ok1142) (N=9), prkl-1(ok3182) (N=10) sax-3(zy5) (N=5) and sax-3(zy5);prkl-1(ok3182) (N=4) embryos. Red line marks the median, whiskers encompass 0–100% of range.

We further examined how these mutants affect rosette dynamics and cell intercalation. We found that these mutants affect the spatial distribution of contracting edges. For example, normally the posterior edge of DD4 contracts, leading DA5 to intercalate between DD4 and DD6 and form an alternating pattern of DAs and DDs (Figure 4B). The same applies to the left side regarding DA4, DD3 and DD5. In the mutants, the anterior edge of DD3/4 contracts (Figure 4B). This phenotype is observed for vang-1(ok1142) (6 edges out of 22), prkl-1(ok3182) (6 out of 26) and sax-3(zy5) (1 out of 12) (Figure 4C). In some embryos where the anterior edge of DD3/4 contracts, the posterior edge of DD3/4 fails to contract, but in others it also contracts as in the wt (Figure 4B). These results suggest that the PCP and sax-3 pathways are required to specify contracting edges, and consequently the spatial pattern of cell intercalation. More specifically, the results support a model where localization of VANG-1 and SAX-3 specify contracting cell-cell contacts by promoting actomyosin contraction. In wt embryos, VANG-1 is roughly 2-fold enriched on the posterior edge of DD4 relative to the anterior edge just prior to the contraction of the cell contact between DD4 and DA5 (N=5, Figure 4D). In prkl-1(ok3182) embryos where the anterior edge of DD3/4 contracts and the posterior edge fails to contract this polarized localization pattern is reversed with nearly 2-fold higher levels of VANG-1 on the anterior edge relative to the posterior edge (N=4, Figure 4D). NMY-2 accumulates on the ectopically contracting contacts, but not on the contacts that fail to contract (Figure S4). Taken together, these results suggest that prkl-1 regulates the polarity of VANG-1 and NMY-2 localization and thus the proper specification of contracting cell edges during CE.

All vang-1, prkl-1 and sax-3 mutants exhibited significant delays and defects in rosette resolution and subsequent single-cell intercalation (Figures 4E and 4F). In the wt, the central rosette resolves in less than 5 minutes after assembly (N=7 Figure 4F). vang-1(ok1142) (N=9, p=0.001), prkl-1(ok3182) (N=10, p=0.007) and sax-3(zy5) (N=5, p=0.013) single mutants all exhibit significantly longer rosette lifetimes. In the sax-3(zy5); prkl-1(ok3182) double mutants, the rosette persists even longer: for greater than 20 minutes in all observed cases (N=4, p=0.0003, Figure 4F). These results are similar to the delays in rosette resolution observed during neurulation in vangl2 deficient mice (Williams et al., 2014). Additionally, in some cases, the persistent rosettes show features of unstable rosette resolution (Paré et al., 2014) wherein the rosette partially resolves and reforms (for example vang-1 and sax-3 in Figure 4E). Instances of unstable resolution were observed in all mutant alleles (data not shown). In cases of unstable resolution, we observed no single cell intercalations until the rosette was fully resolved. We thus scored rosette lifetimes in Figure 4F as the time between the first observation of the rosette and either the final observation of the rosette or the beginning of twitching which precluded further imaging, whichever occurred first. Therefore, in addition to specifying the spatial pattern of contracting cell-cell contacts during rosette formation, the PCP and sax-3 pathways act in parallel to promote rosette resolution.

The failure of rosette resolution in PCP and sax-3 mutants appears to prevent timely single-cell intercalations. Given that VNC neurons in these mutants form a single file at hatching (Figures 2K and 2L), these cells must intercalate at a later stage. We were not able to characterize the late intercalation due to technical difficulties of imaging after embryonic movement starts. We also postulate that upon intercalation into a single file, VNC neurons may undergo an additional CE-independent process that contributes to proper neuronal spacing in the VNC, given the significant space between neuronal cell bodies along the VNC at hatching. It is interesting to speculate that our finding that DD position defects in vang-1; prkl-1 double mutants are significantly less severe than prkl-1 single mutants (Figure S1C) may involve genetic interactions during the later stage to achieve proper neuronal spacing prior to hatching.

Our principal finding that sax-3/Robo, better known for its role in guiding growth cone and cell migration (Blockus and Chédotal, 2016) is required for rosette dynamics and CE was unexpected. The observation that it resembles the PCP pathway in every aspect of VNC assembly including localization dynamics, edge contraction phenotypes, unstable/persistent rosettes and final neuron position suggests that these pathways utilize similar mechanisms and may regulate common downstream targets. These pathways could promote proper cell neighbor exchanges by regulating actomyosin contraction (Murray et al., 2010; Wang et al., 2013) or cell adhesivity (Nagaoka et al., 2014; Oteiza et al., 2010; Rhee et al., 2002; Tatin et al., 2013). Indeed, regulation of cell adhesion may be particularly important in the latter stages of VNC assembly as neurons show tight opposition of cell contacts during rosette formation but eventually detach to intercalate as individual cells after rosette resolution.

Our observations on VANG-1 localization are consistent with previous findings that PCP components act to specify actomyosin contraction during CE (Nishimura et al., 2012; Yin et al., 2008). Interestingly, our findings contrast with the localization pattern of Vangl2 during CE in zebrafish and Xenopus. In zebrafish, Vangl2 shows asymmetric membrane localization but not during gastrulation stages when CE movements are most pronounced, even though these movements are misoriented in trilobite/Vangl2 mutants (Roszko et al., 2015). In the Xenopus neural plate, Vangl2 is localized to anterior membranes but, instead of a reversal in localization in prickle mutants, this asymmetrical distribution is lost when Prickle activity is disrupted (Ossipova et al., 2015). These differences may simply be a function of species-specific variations in molecular or cellular mechanisms that regulate polarized CE behaviors. Indeed, in contrast to its prominent role in vertebrate CE, loss of fmi-1, the sole worm ortholog of Flamingo/Celsr (Steimel et al., 2010), results in relatively mild VNC neuron positioning defects and does not enhance position defects in vang-1, prkl-1 or sax-3 mutants (Figure S1B).

However, it is not clear what confines VANG-1 and SAX-3 to the AP axis in DD4 or its preference to the posterior edge vs anterior. One possibility is the Wnt/Frizzled pathway, which is known to regulate AP polarity during many aspects of C. elegans development (Sawa and Korswagen, 2013), CE-based cell movements more generally (Sokol, 2015), as well as Vang localization during CE in other systems (Ossipova et al., 2015). However, the Wnt/Frizzled pathway has not been shown to localize SAX-3/Robo.

In summary, we have shown that the assembly of the C. elegans VNC involves a rosette-based CE mechanism that involves cooperation between a PCP pathway and a Slit-independent sax-3/Robo pathway to regulate cell neighbor exchanges, rosette dynamics and consequent cell intercalation during VNC formation. This demonstrates that CE is used in the development of the central nerve cord outside vertebrates, and suggests a deep evolutionary root in the morphogenesis of the central nervous system. Furthermore, our results suggest that multiple polarity systems may cooperate to orchestrate the collective cell behaviors in CE.

STAR METHODS

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Antonio Colavita (acolavita@ohri.ca).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

C. elegans Strains and Genetics

C. elegans strains were cultured at either 20°C or 25°C as indicated using standard methods. The Bristol N2 strain was used as wild type along with the following alleles and transgenes: LGX: vang-1(ok1142), vang-1(tm1422), vang-1(zy60[gfp::vang-1]), sax-3(zy5), sax-3(ky123), slt-1(eh15), slt-1(ok255), zyIs27[flp-13p::gfp; unc-129p::mCherry; myo-2p::mCherry], zyIs36[cnd-1p::PH::mCherry; myo-2p::mCherry]. LGI: nmy-2(cp13[nmy-2::gfp]), zdIs5[mec-4p::gfp]. LGII: wdIs6[del-1p::gfp]. LGIII: ynIs37[flp-13p::gfp], otIs173[rgef-1p::mCherry]. LGIV: prkl-1(ok3182), prkl-1(zy11), zdIs13[tph-1p::gfp]. LGV: fmi-1(rh308). Unassigned linkage or extrachromosomal: zyIs33[gfp::prkl-1; odr-1::mCherry], zyIs43[sax-3::gfp; odr-1::rfp], wyIs75[unc-47p::mCherry; exp-1p::gfp], zyEx49[unc-33p::vang-1; myo-2p::mCherry], zyEx50[unc-33p::gfp::prkl-1; myo-2p::mCherry], zyEx59[dpy-7p::gfp::vang-1; myo-2p::mCherry], zyEx60[dpy-7p::gfp::prkl-1; myo-2p::mCherry], zyEx63[unc-33p::sax-3; myo-2p::mCherry], quEx100[ajm-1 p::sax-3; odr-1::mCherry].

METHOD DETAILS

Molecular Biology and Transgenes

The cnd-1p::PH::mCherry construct was made using Gibson Assembly to fuse a PCR amplified 5.6 kb cnd-1p::PLCdPH fragment from cnd-1p::PLCdPH::gfp (The PH domain is from rat PLC-delta) and a PCR amplified 4.1 kb mCherry + vector backbone from pCFJ90 (myo-2p::mCherry). cnd-1p::PH::mCherry was injected using standard germline transformation protocols at 10 ng/μl with 5 ng/μl of pCFJ90 co-injection marker and 100 ng/ul pBluescriptII. The extrachromosomal transgene was integrated using UV irradiation (UVP CL-1000 cross-linker, 254 nm, 3.25×104 μJ/cm2) to generate zyIs36.

Tissue-specific rescue experiments: The unc-33 promoter was used for pan-neuronal specific expression (Altun-Gultekin et al., 2001). The 1.96 kb unc-33 promoter (5′-tagcaagaag …. tcactggaaa-3′) was PCR amplified from N2 genomic DNA and inserted into a HindIII-BamHI digested unc-4p::gfp::vang-1 construct (Sanchez-Alvarez et al., 2011) (removes the unc-4p::GFP fragment) to generate unc-33p::vang-1 construct. The unc-33p::gfp::prkl-1 construct was generated in a similar manner with the unc-33 promoter replacing the unc-4 promoter in an SphI-SalI digested unc-4p::gfp::prkl-1 construct (Sanchez-Alvarez et al., 2011). The unc-33p::sax-3 construct was made by Gibson assembly of three fragments: A PCR amplified 3.822 kb sax-3 cDNA from IC459 worms containing a quEx102 transgene (Ghenea et al., 2005), the 1.96 kb unc-33 promoter and the unc-54 3′UTR and vector backbone (minus gfp) from pPD95.77. dpy-7 and ajm-1 promoters were used to for pan-epidermal specific expression (Gilleard et al., 1997; Köppen et al., 2001). The dpy-7 promoter (5′-gtgtgatcga …. ttccagataa-3′) was PCR amplified from N2 genomic DNA and inserted into a HindIII-XbaI digested unc-4p::gfp::vang-1 and HindIII-SalI digested unc-4p::gfp::prkl-1 to generate dpy-7p::gfp::vang-1 and dpy-7p::GFP::PRKL-1 constructs respectively. Constructs were sequenced to verify fidelity of PCR. Extrachromosomal transgenes were made by injecting each construct at 10 ng/μl with 5 ng/μl of pCFJ90 co-injection marker and 100 ng/ul pBluescriptII. quEx100[ajm-1p::sax-3; odr-1::rfp] (Ghenea et al., 2005) was used for sax-3 epidermal specific expression.

Localization studies: vang-1(zy60[gfp::vang-1]) was generated using a CRISPR/Cas9 approach to insert GFP in-frame with the N-terminus of endogenous vang-1 using the methods described in (Friedland et al., 2013; Tzur et al., 2013). Overlap fusion PCR was used to generate a U6p::vang-1sgRNA according to instructions in (Friedland et al., 2013) where the N-terminus proximal 5′-ggaaacttccgaaagacacg-3′ sequence was used as the Cas9 target site. The gfp::vang-1 homology-directed repair (HDR) plasmid was made by PCR amplifying a ~3.7 kb fragment containing 1.376 kb upstream of the vang-1 ATG start, an in frame GFP cassette (867 bp) and 1.464 kb downstream of the vang-1 ATG start from a gfp::vang-1 genomic construct described in (Sanchez-Alvarez et al., 2011) and cloned into pBluescript. The PAM (NGG) motif corresponding to the Cas9 target site in the HDR plasmid was altered (using synonymous codon substitutions) by PCR-mediated site-directed mutagenesis. The U6p::vang-1sgRNA, HDR fragment, Cas9 expressing plasmid and pCFJ90 co-injection marker were injected into N2 worms and knock-in animals among progeny isolated by visualization of GFP expression and PCR using GFP and vang-1-specific primers (data not shown). vang-1(zy60[gfp::vang-1]) and zyIs21, a gfp::vang-1 genomic transgene (Sanchez-Alvarez et al., 2011), display similar embryonic and larval expression and localization patterns consistent with CRISPR/Cas9 modification of the endogenous vang-1 locus.

zyIs33 and zyIs43 were made by integrating the gfp::prkl-1 extrachromosomal transgene described in (Sanchez-Alvarez et al., 2011) and quEx168[sax-3::gfp; odr-1::mCherry] described in (Ghenea et al., 2005) using UV irradiation as described above. NMY-2 localization was assessed using nmy-2(cp13[nmy-2::gfp]) (Dickinson et al., 2013).

Measurements of Relative DD Neuron Position along AP-Axis

DD neurons were characterized using ynIs37 at the L1 larval stage in worms grown at 25°C. Worms were individually imaged on a Zeiss AxioPlanII/AxioCam HRm. The positions of the DD neurons were measured from DD1 to the anus using Zeiss AxioVision software and converted to percentage locations (DD1 = 0%, anus = 100%). This data was then used to generate DD neuron positioning scatter plots in Microsoft Excel.

Embryonic Imaging

Embryos were dissected from gravid hermaphrodites in M9 buffer. Embryos were mounted in a 1–2 μL droplet of M9 containing a total of 50–100 20 μm diameter polystyrene beads (Polysciences Inc.) on a coverslip. A smaller coverslip was laid on top and sealed with melted Vaseline to prevent evaporation. For dual-color imaging, both channels were simultaneously acquired on a pair of aligned EM-CCD cameras (C9100-13) on a Zeiss AxioObserver Z1 inverted microscope frame with Yokogawa CSU-X1 spinning disk. Image acquisition was performed using MetaMorph software (Molecular Devices). An Olympus UPLSAPO 60xs silicone oil immersion objective was used for all embryonic imaging with an adapter to enable mounting on a Zeiss body (Thorlabs). Errors in alignment between the two cameras were corrected for by periodically imaging a field of multi-color beads, computing an affine alignment (Preibisch et al., 2014) and applying the transformation to the acquired images using Fiji (Schindelin et al., 2012). To enable higher contrast imaging for protein localization without photobleaching, thinner sections were imaged; typically 11–15 slices in total with 0.75 μm between slices.

Identification of VNC Neurons

Cell identities were established through automated lineaging and manual curation of BV293 embryos as previously described (Du et al., 2014). The identity and relative positioning of all DA, DB and DD neurons was established in 3 lineaged embryos. The identity of all cell labeled by the cnd-1 membrane label was established by lineaging expressing cells in >10 embryos and were found to exhibit a highly reproducible pattern, clearly evident by the time the mothers of the terminal neurons are born. The identities of DD1–6 and DA2–5 in all subsequent experiments were then established by the pattern in which their mothers were born and manually tracking each cell.

Phenotypic Characterization of sax-3(zy5)

Worms were grown at 20°C for all phenotypic analyses. L4 larvae were picked to new plates overnight. The next day, roughly synchronized embryos were generated by allowing mothers to lay eggs for 1-hour time intervals. Lethality was determined by counting the number of unhatched embryos after 24 hours. L4 larvae were examined for a notch head phenotype as described in (Ghenea et al., 2005) and the nerve ring phenotype as described in (Kennerdell et al., 2009) with the modification that otIs173[rgef-1p::mCherry] was used to visualize nerve ring morphology. AVM and HSN axon guidance defects, visualized using zdIs5[mec-4p::gfp] and zdIs13[tph-1p::gfp] respectively, were scored as any deviation from ventrally-directed axon guidance.

QUANTIFICATION AND STATISTICAL ANALYSIS

Image Analysis

Intensity measurements were made by manually tracing cell-cell contacts of interest using a 3 pixel-wide line segment tool in Fiji with only the membrane-labeled channel visible to the user. The mean per-pixel intensity was used in all comparisons after background subtraction using the median of a 75×75 pixel neighborhood centered on the VNC neurons as the background estimate. Cell boundaries for rosette schematics were manually traced in Adobe Illustrator over corresponding fluorescence images. 3D reconstructions were made by manually tracing cell outlines in Imaris (Bitplane) and were performed on volumes acquired in 35 slices with 0.75 μm spacing between slices. Since the central rosette was observed to resolve transiently and reform in some mutant embryos, rosette lifetime was defined as the time between the initial formation of the rosette and the last observation of the same rosette before the onset of twitching. In cases where the rosette was observed to be engaged at the onset of twitching the lifetime was scored as the time between the initial formation and the onset of twitching.

DD neuron positioning scatter plots

For DD neuron positioning scatter plots, DD neuron means and errors were plotted for each neuron within each worm strain. Error bars represent a 95% confidence interval, or +/− 1.96 * Standard Error. Two-tailed, two-sample t-tests were performed for each DD neuron between condition and control strain worms, with significance indicated by * (p ≤ 0.01) or ** (p ≤ 0.001). The control strain for each comparison is indicated in the figure legends. This work was done within Microsoft Excel. Statistical methods are also indicated in the figure legends.

sax-3(zy5) phenotype quantification

Error bars in Figure S2B–F represent +/− the Standard Error of Proportion. Significance was assessed using Fisher exact tests, and indicated by *** (p ≤ 0.001) or ns (not significant). This work was done within Microsoft Excel. Statistical methods are also indicated in the figure legends.

Supplementary Material

supplement

HIGHLIGHTS.

Rosette-based convergent-extension is conserved in central nerve cord assembly prkl-1/vang-1 and sax-3 regulate convergent-extension and neuron positions in the VNC VANG-1 and SAX-3 are localized to contracting edges and rosette foci PCP and sax-3 specify contracting edges and are required for rosette resolution

Acknowledgments

This work was supported by CIHR Grant 123513 and NSERC Discovery Grant 312460-2012 to A.C., NIH grants (R01 GM097576 and R24 OD016474) to Z.B. and the MSK Cancer Center Support/Core Grant (P30 CA008748). We thank Dr. Jennifer Zallen and Justin Evans for comments on the manuscript and Dr. Monica Colaiacovo for CRISPR/Cas9 reagents. Some strains were provided by the CGC, Dr. Kang Shen, Dr. Shohei Mitani (National Bioresource Project, Japan) and the Gene Knockout Consortium (University of British Columbia and the University of Oklahoma). We thank Wormbase.

Footnotes

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AUTHOR CONTRIBUTIONS

We describe contributions to the paper with the authors listed in alphabetical order. Conceptualization, A.C and Z.B. Methodology, A.C., M.T., P.K.S., T.J.P. and Z.B. Investigation, A.R., C.S., I.K., J.H., M.T., N.N., P.K.S., T.R., Z.C. Data Curation, N.N.T., P.K.S., T.E.M. Formal Analysis, P.K.S and T.J.P, Writing - Original Draft, A.C., M.T., P.K.S. and Z.B. Writing - Review & Editing, A.C., M.T., P.K.S. and Z.B. Funding Acquisition, A.C. and Z.B. Supervision, A.C. and Z.B.

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Supplementary Materials

supplement
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