1. Developmental Biology
  2. Neuroscience

Differential adhesion regulates neurite placement via a retrograde zippering mechanism

  1. Titas Sengupta
  2. Noelle L Koonce
  3. Nabor Vázquez-Martínez
  4. Mark W Moyle
  5. Leighton H Duncan
  6. Sarah E Emerson
  7. Xiaofei Han
  8. Lin Shao
  9. Yicong Wu
  10. Anthony Santella
  11. Li Fan
  12. Zhirong Bao
  13. William A Mohler
  14. Hari Shroff
  15. Daniel A Colón-Ramos  Is a corresponding author
  1. Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, United States
  2. Laboratory of High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, United States
  3. Developmental Biology Program, Sloan Kettering Institute, United States
  4. Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut Health Center, United States
  5. MBL Fellows, Marine Biological Laboratory, United States
  6. Wu Tsai Institute, Yale University, United States
  7. Instituto de Neurobiología, Recinto de Ciencias Médicas, Universidad de Puerto Rico, Puerto Rico
https://doi.org/10.7554/eLife.71171
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  1. Titas Sengupta
  2. Noelle L Koonce
  3. Nabor Vázquez-Martínez
  4. Mark W Moyle
  5. Leighton H Duncan
  6. Sarah E Emerson
  7. Xiaofei Han
  8. Lin Shao
  9. Yicong Wu
  10. Anthony Santella
  11. Li Fan
  12. Zhirong Bao
  13. William A Mohler
  14. Hari Shroff
  15. Daniel A Colón-Ramos
(2021)
Differential adhesion regulates neurite placement via a retrograde zippering mechanism
eLife 10:e71171.
https://doi.org/10.7554/eLife.71171
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9 figures, 1 table and 2 additional files

Figures

Figure 1 with 7 supplements
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AIB single neurite is placed along two distinct neighborhoods in the nerve ring.

(A) Schematic of an adult/larval C. elegans showing an AIB neuron (cyan) and its posterior (orange) and anterior (magenta) neighborhoods in the head. The AIB neurite has a proximal neurite segment (orange arrow), a posterior-anterior shift at the dorsal midline (dashed line) and a distal neurite segment (magenta arrow; on the other side of the worm, behind the pharynx, which is in gray). The neon-colored outline represents the nerve ring neuropil. The terms ‘proximal’ or ‘distal’ neurite segments refer to the relationship of the neurite segment to the AIB cell body. The neighborhoods in which the ‘proximal’ and ‘distal’ neurite segments are positioned are referred to as the ‘posterior’ or ‘anterior’ neighborhoods, respectively, because of their position along the anterior-posterior axis of the worm. Note that this schematic only shows one neuron of the AIB pair. Cell body is marked with an asterisk. (B) Magnified schematic of AIB and its neighborhoods in (A, C) Representative confocal image showing the lateral view of an AIB neuron labeled with cytoplasmic mCherry (cyan). (D) Representative confocal image showing an AIB neuron labeled with cytoplasmic mCherry (cyan); and RIM motor neuron of the anterior neighborhood labeled with cytoplasmic GFP (magenta) in lateral view. Note the colocalization of the AIB distal neurite (but not the proximal neurite) with the anterior neighborhood marker RIM (compare with E). (E) As (D), but with AIB (cyan) and AWC and ASE sensory neurons of the posterior neighborhood (orange). Note the colocalization of the AIB proximal neurite (but not the distal neurite) with the posterior neighborhood markers AWC and ASE (compare with D). (F–J) Same as A–E but in axial view indicated by the arrow in (F). The worm head is tilted in this view to make the two neurite segments in the two neighborhoods visible. Note shift in H (arrows), corresponding to AIB neurite shifting neighborhoods (compare I and J). (K,L) Volumetric reconstruction from the JSH electron microscopy connectome dataset (White et al., 1986) of AIBL (K), and AIBL overlaid on nerve ring strata (L), in lateral view, with S2 and S3 strata (named as in Moyle et al., 2021), containing anterior and posterior neighborhoods, respectively. (M) Volumetric reconstruction of AIBL and AIBR in axial view (from the JSH dataset White et al., 1986). Note the shift in neighborhoods by AIBL and AIBR, at the dorsal midline (dashed line), forms a chiasm (also see Figure 1—figure supplement 1). (N) Schematic of M highlighting the AIB neighborhoods for context and the dorsal midline with a dashed line (AIB neighborhoods, synaptic polarity and resulting network properties also shown in Figure 1—figure supplement 2). Scale bar = 10 μm for A–J and 3 μm for K–N.

Figure 1—figure supplement 1
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Neurite positions of the bilaterally symmetric AIB neurons.

(A-C) Pseudo-colored confocal maximum intensity projections showing AIBL (A), AIBR (B) and merge (C). Orange and magenta arrows indicate positions of the proximal and distal neurites, positioned in the posterior and anterior neighborhoods, respectively (see Figure 1). Note that the proximal and distal neurites of AIBL and AIBR completely overlap in the lateral view, consistent with what would be expected based on their positions and projections from the EM reconstructions (in D–I; White et al., 1986). Scale bar = 10 μm applies A-C. (D-F) EM and in vivo fluorescent microscopy views of the AIBL-AIBR neuron pair. (D) Schematic of the AIBL-AIBR neuron pair in the context of the nerve ring (light neon); (E) Pseudo-colored confocal maximum intensity projections of AIBL (cyan) and AIBR (yellow) (3D projection of this dataset shown in Figure 1—video 4); (F) Volumetric reconstructions of AIBL and AIBR from segmented EM datasets (JSH, White et al., 1986). (G–I) As D–F, but axial view. In all images (D–I), arrowheads indicate the posterior-anterior shift of the two neurons crossing each other to form a chiasm. The gray circle in G depicts the pharynx. Scale bars = 10 μm in E,H, and 3 μm in F,I.

Figure 1—figure supplement 2
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AIB contacts, synaptic distribution and network properties enable its function as a connector hub neuron.

(A) Volumetric reconstruction of AIBL (cyan), a posterior neighborhood neuron (ASEL, orange) and an anterior neighborhood neuron (RIMR, magenta) from an L1 (5 hr post hatching) and an adult connectome dataset (45 hr post hatching, Witvliet et al., 2021). Scale bars = 3 μm. Note that ASEL contacts AIBL exclusively in the posterior neighborhood, and RIMR contacts AIBL exclusively in the anterior neighborhood. AIB is similarly positioned into the same neighborhoods in all available connectome datasets examined (which span all larval developmental stages; data not shown; White et al., 1986; Witvliet et al., 2021). The observation that AIB is already positioned in the two neighborhoods in the L1 stage indicates that AIB placement occurs during embryogenesis. (B,C) Axial view of the AIB neurite and neuron-neuron contact areas between AIBL and anterior neighborhood neuron, RIMR (B) and AIBL and posterior neighborhood neuron, ASEL (C) from the segmented EM dataset of the L4 stage animal JSH (Brittin et al., 2018; White et al., 1986). Contacts are colored red (see Methods) and overlaid on 3D volumetric reconstructions of the AIB neurite (cyan). Orange and magenta arrows indicate the posterior and anterior neighborhoods respectively. Scale bar = 3 μm, applies to B,C. (D) Volumetric reconstruction of AIBR from the JSH electron microscopy connectome dataset (White et al., 1986) in lateral view. Postsynaptic (red) and presynaptic (yellow) regions of the neurite, based on synaptic connectivity maps of AIBR, are indicated. Note that the postsynaptic and presynaptic regions coincide with the proximal and distal segment of AIB. Arrowhead points to the chiasm. Scale bar = 1 μm. (E–G) Representative confocal image showing a lateral view of an AIB neuron with postsynaptic sites (red, labeled by GLR-1:GFP, E) and presynaptic sites (yellow, labeled by mCh:RAB-3, F). (G) is a merge of E and F. Note the opposite polarity in the posterior and anterior neighborhoods (indicated by the orange and magenta arrows respectively) Scale bar = 10 μm, applies to E-G. (H) From the available segmented serial section EM and neuron-neuron contact data (Brittin et al., 2021; Moyle et al., 2021; White et al., 1986; Witvliet et al., 2021), neuron-neuron adjacency matrices were generated as depicted in the schematic. (I) Cosine similarity plot for AIBR. The cosine similarity values (Han et al., 2012) of AIB contacts between each pair of connectome datasets (White et al., 1986; Witvliet et al., 2021) are plotted as a heat map with the color bar indicating the values corresponding to the shades. The similarity values are >0.5 for all pairs of connectome datasets, indicating positive correlation between distribution of AIB contacts in different datasets. This suggests that distribution of AIB contacts with other neurons is largely established in early larval stages (L1) and maintained through development. The labels assigned to the connectome datasets comprise of the developmental stage and name of the animal sectioned (all times are measured post hatching) (White et al., 1986; Witvliet et al., 2021). (J) Box plot (10–90 percentile) of betweenness centrality values for an L1 (5 hr post hatching) connectome dataset and an adult connectome dataset (45 hr post hatching, Witvliet et al., 2021). The gray dots represent neurons whose centrality values lie above the 90-percentile mark or below the 10-percentile mark. The betweenness centrality values (see Methods) for AIBL and AIBR (indicated by cyan and yellow stars respectively) lie above the 90-percentile mark in both datasets. High betweenness centrality being a standard property of rich-club neurons (Towlson et al., 2013), this indicates that contacts of AIBL and AIBR exhibit rich-club features from early developmental stages (L1) to adulthood.

Figure 1—figure supplement 2—source data 1

Pairwise cosine similarity values between the EM connectome datasets (White et al., 1986; Witvliet et al., 2021) analyzed in Figure 1—figure supplement 2I.

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Figure 1—figure supplement 2—source data 2

Betweenness centrality values for all neurons in the EM connectome datasets (Witvliet et al., 2021) analyzed in Figure 1—figure supplement 2J.

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Figure 1—video 1
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Neighborhoods of AIB, related to Figure 1, Figure 1—figure supplement 1 and Figure 1—figure supplement 2.

3D volumetric reconstruction of AIBR (cyan), posterior neighborhood neuron AWCR (orange) and anterior neighborhood neuron, RIML (magenta) from segmented EM micrographs from the JSH EM dataset (White et al., 1986). Scale bar = 2 μm.

Figure 1—video 2
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AIB and anterior neighborhood neuron, RIM, related to Figure 1 and Figure 1—figure supplement 2.

3D projection confocal image of AIB (cyan) and the RIM neurons (magenta) of the anterior neighborhood. The RIM neurites show extensive overlap with the AIB distal neurite. This video was made from the same image as in Figure 1D and I. Scale bar = 10 μm.

Figure 1—video 3
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AIB and posterior neighborhood neurons, related to Figure 1 and Figure 1—figure supplement 2.

3D projection confocal image of AIB (cyan) and posterior neighborhood amphid sensory neurons (orange). The posterior neighborhood neurons show extensive overlap with the AIB proximal neurite. This video was made from the same image as in Figure 1E and J. Scale bar = 10 μm.

Figure 1—video 4
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The bilaterally symmetric AIBL and AIBR neurons, related to Figure 1 and Figure 1—figure supplement 1.

3D projection confocal image of the AIB neuron pair, AIBL (cyan) and AIBR (yellow), pseudocolored to distinguish the AIBL and AIBR neurites and their chiasm at the dorsal midline (in the video, the crossover at the very top). The maximum intensity projections in Figure 1—figure supplement 1E and H are from this same dataset. Scale bar = 10 μm.

Figure 1—video 5
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Polarized localization of AIB presynaptic sites, related to Figure 1—figure supplement 2.

3D projection confocal image of AIBR (cyan) with presynaptic sites labeled with a CLA-1 reporter (yellow) (Xuan et al., 2017). Posterior and anterior neighborhoods are marked in the lateral and axial views with orange and magenta arrows, respectively. Note localization of CLA-1 (yellow) specifically along the distal neurite. Scale bar = 10 μm.

Figure 2 with 13 supplements
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A retrograde zippering mechanism positions the AIB neurites in the anterior neighborhood during embryonic development.

A, Schematic of axial view of the AIB neuron pair: AIBL (cyan) and AIBR (yellow) in the context of the nerve ring (light neon) and the pharynx (grey), with posterior neighborhood labeled (orange) and the dashed line representing the dorsal midline where the AIB chiasm is present in adults (see Figure 1). Dotted box represents region in B’-F’, and dotted box in G. B,F, Time-lapse showing initial placement of AIBL and AIBR in the posterior neighborhood and their subsequent separation from this neighborhood. Images are deconvolved diSPIM maximum intensity projections obtained from developing embryos. Neurons were individually pseudocolored to distinguish them (see Methods). The dorsal half of the nerve ring (dotted box in A) are magnified in B’-F’. B’’-F’’ are schematic diagrams representing the images in B-F. Dashed vertical lines midline. Note in (B, B’, B’’), the AIBL and AIBR neurites approaching the dorsal midlinerepresent the dorsal in the posterior neighborhood. In (C, C’, C’’), AIBL and AIBR have met at the dorsal midline and continue growing along each other, past the midline. The latter part of the neurite, past the midline, becomes the future distal neurite. (D, D’, D’’) shows the tip of the AIBL future distal neurite moving away from the posterior neighborhood and its counterpart, AIBR. The arrowhead indicates the point of separation of the AIBL distal neurite and the AIBR proximal neurite. (E, E’, E’’) shows further separation of the two neurites and by (F, F’, F”), they have completely separated. The arrowheads in (E, E’, E’’) and (F, F’, F’’) also indicate the junction between the separating AIBL distal neurite and the AIBR proximal neurite. A similar sequence of events is visualized at higher spatial resolution in Figure 2—figure supplement 1 using triple-view line scanning confocal microscopy (Figure 2—figure supplement 1). G, G’, Confocal micrograph of a postembryonic L4 animal (axial view) showing the relationship between AIBL and AIBR. The region in the box represents the dorsal part of the nerve ring, magnified in G’. H, Axial view schematic of one AIB neuron (cyan) in the context of the anterior neighborhood marker, the RIM neuron (magenta), the nerve ring (light neon) and the pharynx (grey). I-K, Time-lapse showing placement of the AIB neurite (cyan) relative to the anterior neighborhood (magenta). As in B-F, images are deconvolved diSPIM maximum intensity projections and the neurons were pseudocolored. The dorsal half of the nerve ring (dotted box in H) are magnified in I’-K’. Dashed line indicates dorsal midline (where the shift, or chiasm, in the adult is positioned, see Figure 1). I’’-K’’ are schematic diagrams representing the images in I-K. Note in (I, I’, I”), the tip of the AIB neurite encounters the RIM neurite in the anterior neighborhood (green arrowhead). In (J, J’, J’’), the AIB distal neurite has partially aligned along the RIM neurites. The green arrowhead now indicates point of initial encounter of the two neurites (same as in I’), and the red arrowhead indicates the retrograde zippering event bringing the AIB and RIM neurons together in the anterior neighborhood. In (K, K’, K”) the two neurites have zippered up to the dorsal midline. Arrow in J’ indicates direction of zippering. L, Confocal micrograph of a postembryonic L4 animal in axial view showing the final position of AIB with respect to the anterior neighborhood. The same image as Figure 1I was used here for reference. The region in the dotted box represents dorsal part of the nerve ring, magnified in (L’). M, Schematic highlights the steps by which the AIB distal neurite is repositioned to a new neighborhood – (i) exit from the posterior neighborhood and (ii) retrograde zippering onto the anterior neighborhood with intermediate partially zippered states and completely zippered states. Scale bar = 10 µm for B-G and I-L. Scale bar = 2 µm for B’-G’ and I’-L’ Times are in m.p.f. (minutes post fertilization).

Figure 2—figure supplement 1
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Images of AIBL and AIBR unzippering with and without pseudocoloring.

(A-E) diSPIM maximum intensity projections of AIBL and AIBR showing unzippering. The corresponding pseudocolored images (A’-E’) have been used in Figure 2B–F.

Figure 2—figure supplement 2
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Strategies for labeling and visualizing AIB outgrowth dynamics in early embryos.

(A), Schematic of the ZIF-1/1-ZF1 degradation system used for subtractive fluorescent labeling of specific neurons in embryos (Armenti et al., 2014), also see Materials and methods. Briefly we used lim-4p, expressed in sublateral neurons (Santella et al., 2015) to express ZIF-1 and unc-42p, expressed in the sublateral neurons + AIB + ASH (http://promoters.wormguides.org) to express ZF1-tagged PH:GFP. This results in degradation of PH:GFP from the sublateral neurons, resulting in cell-specific labeling of AIB and/or the ASH neurons. (B) diSPIM image showing unc-42p-driven membrane-tethered PH:GFP expression, without ZIF-1/ZF1 mediated degradation, in neurons of the embryonic nerve ring. Distinction of AIB neurite outgrowth dynamics is not possible in this background due to abundant labeling. Scale bar = 10 μm applies to B,C. (C) diSPIM image showing the nerve ring of an embryo expressing the ZIF-1/ZF1 degradation construct strategy outlined in A. The identity of AIB was confirmed by colocalization and lineaging as described (Moyle et al., 2021). (D,E) Imaging methods that we established and implemented for the investigation of neurodevelopmental events in C. elegans embryos. (D) A triple-view line-scanning confocal microscope that provides enhanced (twofold) axial resolution compared to conventional confocal microscopy (Wu et al., 2021). To image AIB in living nematode embryos, we additionally created a two-step deep learning framework that denoises the raw data, enabling us to turn down the illumination intensity ~30 fold, offering more gentle imaging than conventional confocal microscopy. (E) Dual-view inverted selective plane illumination microscopy (diSPIM), a light-sheet microscopy technique for long-term imaging of AIB neurite development (Kumar et al., 2014; Wu et al., 2013). Deconvolution and fusion of images from orthogonal views result in isotropic spatial resolution. ( F–H), Time-lapse showing relative positions of AIBL (pseudo-colored in cyan) and AIBR (pseudocolored in yellow) in the embryonic nerve ring. Images are reconstructions derived from triple-view line-scanning confocal microscopy, which used a deep learning algorithm for denoising and deconvolving all three views. The dashed white lines represent the dorsal midline of the nerve ring. The dotted boxes represent the dorsal half of the nerve ring and are magnified in F’-H’. F’’-H’’ are schematic diagrams representing the images in (F–H). In (F,F’F’’), the neurites are initially positioned in the same neighborhood. In (G,G’,G’’) they have separated partially from the tip up to a point along their lengths (arrow). In (H,H’,H’’) they have separated completely up to the dorsal midline (arrowhead). The high spatial resolution allows us to clearly distinguish the two neurites and determine their relative positions reliably, confirming results in Figure 2 and enabling detailed quantifications. Scale bar = 2 μm in F, applies to G,H, and 1 μm in F’, applies to G’,H’. (I) Schematic showing AIBL and AIBR in the context of the nerve ring (light neon), pharynx (gray) and the anterior and posterior neighborhood (magenta and orange regions). AIBL exits the posterior neighborhood (direction of outgrowth indicated by black arrow) and cuts through the nerve ring to meet the anterior neighborhood. α is the angle of exit. AIBR also exits similarly. We also measured β: the angle between tangents drawn at the point of downward bend of the nerve ring in the posterior neighborhood, as indicated in schematic (from embryos in which posterior neighborhood neurons are labeled by nphp-4p:PH:GFP). (J) Scatter plot of α and β values (n = 6, 3 AIBL and 3 AIBR neurons measured from three embryos for each of α and β). Unpaired two-tailed t test indicates no significant difference (n.s.) between α and β values (P = 0.1368). The AIB distal neurite therefore exits tangentially from the posterior neighborhood, consistent with AIB losing adhesion in this neighborhood, growing straight instead of following the arc of the nerve ring and crossing the nerve ring toward its eventual encounter with the RIM neuron in the anterior neighborhood.

Figure 2—video 1
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Selective AIB labeling by Zif-1/ZF1-mediated degradation, related to Figure 2 and Figure 2—figure supplement 2.

diSPIM maximum intensity projection images of embryos, labeled with membrane-tagged GFP (expression driven by an unc-42 promoter, see Materials and methods, Armenti et al., 2014) without (left) and with (right) Zif-1-ZF1-mediated degradation. The dataset with the Zif-1-ZF1 degradation was used in Figure 2—figure supplement 1C. Scale bar = 10 μm.

Figure 2—video 2
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diSPIM single-view z-stacks for Figure 2B.

This video represents the raw z-stack (acquired from a single diSPIM objective) for the representative timepoint 455 m.p.f. Scale bar = 10 microns.

Figure 2—video 3
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diSPIM single-view z-stack for Figure 2C.

This video represents the raw z-stack (acquired from a single diSPIM objective) for the representative timepoint 480 m.p.f. Scale bar = 10 microns.

Figure 2—video 4
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diSPIM single-view z-stack for Figure 2D.

This video represents the raw z-stack (acquired from a single diSPIM objective) for the representative timepoint 505 m.p.f. Scale bar = 10 microns.

Figure 2—video 5
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diSPIM single-view z-stack for Figure 2E.

This video represents the raw z-stack (acquired from a single diSPIM objective) for the representative timepoint 520 m.p.f. Scale bar = 10 microns.

Figure 2—video 6
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diSPIM single-view z-stack for Figure 2F.

This video represents the raw z-stack (acquired from a single diSPIM objective) for the representative timepoint 530 m.p.f. Scale bar = 10 microns.

Figure 2—video 7
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3D projection of deconvolved, fused diSPIM image for timepoint 455 m.p.f. (Figure 2B).

Scale bar = 10 microns.

Figure 2—video 8
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3D projection of deconvolved, fused diSPIM image for timepoint 480 m.p.f. (Figure 2C).

Scale bar = 10 microns.

Figure 2—video 9
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3D projection of deconvolved, fused diSPIM image for timepoint 505 m.p.f. (Figure 2D).

Scale bar = 10 microns.

Figure 2—video 10
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3D projection of deconvolved, fused diSPIM image for timepoint 520 m.p.f. (Figure 2E).

Scale bar = 10 microns.

Figure 2—video 11
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3D projection of deconvolved, fused diSPIM image for timepoint 530 m.p.f. (Figure 2).

Scale bar = 10 microns.

Figure 3 with 1 supplement
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Biophysical modeling of AIB developmental dynamics is consistent with differential adhesion leading to retrograde zippering.

(A) Axial view schematic of a single AIB neuron during transition of its neurite between the posterior (orange) and anterior (magenta) neighborhoods. (B,B’) Magnified schematic of dotted inset in (A) showing the AIB neurite (cyan) during its transition from the posterior to the anterior neighborhood. The lengths of the neurite positioned in the posterior and anterior neighborhoods are denoted by Lp and La, respectively. The velocity with which the AIB neurite zippers onto the anterior neighborhood is denoted by vzip , and the velocity with which it unzippers from the posterior neighborhood is denoted by vunzip . At the junction between the neurite and the two neighborhoods, that is at the zippering and unzippering forks, tension and adhesion forces act on the neurite (see B’, Appendix 1 and Appendix 1—figure 1). B', Schematic of AIB neurite zippering to the anterior neighborhood. Adhesion Santerior acts in the direction of zippering (and therefore in the direction of the zippering velocity vzip) and favors zippering. Tension Tanterior acts in the opposite direction, disfavoring zippering. (C) Plot of position vs. time of the AIB neurite in both neighborhoods in synchronized embryos at the indicated timepoints on the x-axis ( ± 5 mins). Plot shows mean of Lp (n = 4) and La (n = 3) values at different timepoints. Note zippering from the anterior neighborhood and unzippering from the posterior neighborhood take place in the same time window and are inversely related (between 500–545 m.p.f.). Quantifications were done from three embryos for each of La and Lp. See Figure 3—figure supplement 1 for the individual Lp and La values at each timepoint. (D) Plot of zippering velocities vs time (n = 3) for the indicated timepoints on the x-axis ( ± 5 mins). Note a tenfold increase in velocity mid-way through zippering (530 m.p.f.) m.p.f. = minutes post fertilization. Error bars represent standard error of the mean (S.E.M.), The three embryo datasets used for measuring La values in (C) were used to calculate zippering velocities. For C and D, n represents the number of AIB neurites quantified.

Figure 3—figure supplement 1
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Individual data points from Figure 3C.

(A,B) Plots showing individual measurements of Lp (A) and La (B) (see Methods for details). The Lp (A) and La (B) values at each timepoint were averaged to produce the orange and magenta curves (respectively) in the plot in Figure 3C. Each point represents a single AIB neurite. Lp was quantified from four different embryos and La from three different embryos.

Figure 4 with 1 supplement
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SYG-1 and SYG-2 are required for precise placement of the AIB neurite in the anterior neighborhood.

(A-D) Representative confocal images of AIB (A) and RIM neurons (B) which mark the anterior neighborhood, in a wild-type animal. (C) is a merge of A and B. The dashed box represents the region of contact of AIB with the anterior neighborhood, magnified in (D). The AIB distal neurite colocalizes extensively with the anterior neighborhood in wild-type animals (Arrow in D and Figure 1—figure supplement 2A,B). Cell bodies are marked with an asterisk. (E–L) As A–D but in the syg-1(ky652) (E–H) and syg-2(ky671) (I–L) mutant background. Note the gaps between the AIB distal neurite and the RIM neurites (H,L, arrows), indicating loss of contact between the AIB and the anterior neighborhood in these mutants. (M) Schematic and scatter plot of quantifications of the loss of contacts between AIB and the anterior neighborhood for wild type (n = 42), syg-1(ky652) mutant (n = 40) and syg-2(ky671) animals (n = 49). ‘n’ represents the number of AIB neurites quantified from 21, 20 and 25 animals, respectively. The extent of detachment of the AIB distal neurite, and hence its deviation from the RIM neighborhood, was quantified using the indicated formula (see also Materials and methods). Error bars indicate standard error of the mean (S.E.M.). ****p < 0.0001, **p = 0.0095 (one-way ANOVA with Dunnett’s multiple comparisons test). n represents the number of AIB neurites quantified. Estimated effect size, d = 1.087 for WT vs. syg-1(ky652) and 0.775 for WT vs. syg-2(ky671). For neurites that do not show visible detachment, the precent detachment values = 0 and therefore these datapoints lie on the x-axis. The mean percent deviations include neurites with 0 percent detachment. (N) Quantification of the penetrance of the AIB neurite placement defect as the percentage of animals with normal AIB distal neurite placement in WT, syg-1(ky652), syg-2(ky671), syg-1(ky652);syg-2(ky671) double mutant, inx-1p:syg-2 rescue, inx-1p:syg-1 rescue and SYG-1 cosmid rescue (also see Figure 4—figure supplement 1G-I'). inx-1p is a cell-specific promoter driving expression in AIB (Altun and Chen, 2008). The green and purple bars represent syg-1(ky652) and syg-2(ky671) mutant backgrounds respectively. Numbers on bars represent number of animals examined. ****p < 0.0001 by two-sided Fisher’s exact test between WT and syg-1(ky652), between WT and syg-2(ky671), and between syg-1(ky652) and SYG-1 cosmid rescue, and **p = 0.0055 between syg-2(ky671) and inx-1p:syg-2 rescue. There is no significant difference (abbreviated by n.s.) in penetrance between the syg-1(ky652) and syg-1(ky652);syg-2(ky671) (p = 0.6000) populations and between syg-1(ky652) and the inx-1p:syg-1 animals (p = 0.3558). Scale bar = 10 μm, applies to (A–L).

Figure 4—source data 1

Number of animals of each genotype (in the bar graph in Figure 4N) displaying normal vs aberrant distal neurite placement.

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Figure 4—figure supplement 1
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SYG-1 and SYG-2 regulate placement of the AIB neurite specifically in the anterior neighborhood.

(A) Scatter plot of lengths of the dorsal midline shift (that form a chiasm for the neuron pair, see Figure 1 and Figure 1—figure supplement 1) for wild type (n = 18) and syg-1 (ky652) (n = 18). Error bars indicate standard error of the mean (S.E.M.). ***p = 0.0008 (unpaired two-tailed t-test). In wildtype animals, the chiasm is stereotyped and similar in length across L4 stage animals, as measured from confocal micrographs and displayed in this scatter plot (mean length = 2.97 ± 0.05 μm, n = 18), and electron micrographs (dorsal midline shift length in AIBL and AIBR in electron micrographs of an L4 stage animal, JSH, are 3.01 μm and 3.16 μm, respectively). In syg-1(ky652), the mean length of the chiasm is significantly smaller and is = 1.96 ± 0.27 μm (n = 18). n represents the number of AIB neurons measured from nine wild type and nine syg-1(ky652) animals. Effect size estimate, d = 1.233. (B) Scatter plot of distal neurite lengths for wild type (n = 31) and syg-1(ky652) (n = 30). n represents the number of AIB neurons measured from 16 wild type and 15 syg-1(ky652) animals. (C) Scatter plot of nerve ring width as measured from strains expressing a nerve ring marker cnd-1p:PH:GFP (see Materials and methods), in WT (n = 14) and syg-1(ky652) (n = 14) backgrounds. Two values of nerve ring width were obtained from each animal (one from each side). n = number of animals of each genotype from which measurements were done. For B and C, unpaired two-tailed t test indicates no significant (abbreviated by n.s.) difference (p = 0.0793 and 0.3140, respectively). Error bars indicate standard error of the mean (S.E.M.). (D,E) Representative confocal images of a syg-1(ky652) (D) and a syg-2(ky671) (E) animal with AIB labeled with cytoplasmic mCherry (cyan) and the posterior neighborhood markers, the AWC and ASE neurons labeled with cytoplasmic GFP (orange). Note that the placement of the AIB neurite in the posterior neighborhood is unaffected in syg-1(ky652) and syg-2(ky671). The orange and magenta arrows indicate the positions of the posterior and anterior neighborhoods respectively. Scale bar = 10 μm. (F) Quantification of the minimum perpendicular distances between the AIB proximal and distal neurites in WT (n = 19), syg-1(ky652) (n = 29) and syg-2(ky671) (n = 18). ****p < 0.0001; ***p = 0.0007 (one-way ANOVA with Dunnett’s multiple comparisons test). ‘n’ represents the number of AIB neurons measured from 9, 15, and 9 animals from the WT, syg-1(ky652) and syg-2(ky671) populations, respectively. Effect size estimate, d = 2.239 for WT and syg-1(ky652) and 1.148 for WT and syg-2(ky671). G-I’ Representative confocal images of AIB (cyan) and the anterior neighborhood (magenta) in strains with cell-specific SYG-2 expression (G), cell-specific SYG-1 expression (H) and a cosmid containing syg-1 (recapitulating endogenous SYG-1 expression) (I). The dashed boxes in G, H and I represent the region of contact between AIB and the anterior neighborhood, magnified in G’, H’ and I’, respectively. Note complete alignment of the AIB distal neurite with the anterior neighborhood in G’ and I’ and detachment in H’. Scale bar = 10 μm for G,H,I, and 1 μm for G’,H’,I’.

Figure 5 with 8 supplements
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Increased local expression of SYG-1 in the anterior neighborhood coincides with zippering of the AIB neurite onto this neighborhood.

(A-E) Schematic (A) and representative confocal image of a wild-type animal co-expressing (B) a membrane-targeted syg-1 transcriptional reporter (see Materials and methods, Schwarz et al., 2009) and (C) cytoplasmic AIB reporter. Merged image in (D). Since the syg-1 reporter is membrane-targeted, it labels cell body outlines and neurites (B, D). The dashed box or inset in (D) represents the region of overlap between AIB and syg-1-expressing neurites, magnified in (E). Note that the syg-1 reporter shows two bands of expression in the nerve ring (arrows in B and D) which coincide with the posterior and anterior AIB neighborhoods (orange and magenta arrows). Note also that there is no membrane outline corresponding to the AIB cell body (B) we drew a dashed silhouette of the AIB cell body position as determined in (C). Asterisk indicates cell body. (F–I) As B–E, but with a translational SYG-1 reporter. Note the SYG-1 protein shows a similar expression pattern. (J–N) Schematic (J) and time-lapse images (K–N) of SYG-1 translational reporter expression during embryogenesis (460–640 m.p.f.). Images are deconvolved diSPIM maximum intensity projections. The dashed boxes represent the dorsal half of the nerve ring and are magnified in O-R. O’-R’ are schematic diagrams representing the images in (O–R). In (K, O, O’), SYG-1 expression is primarily visible in a single band containing amphid neurites and corresponding to the AIB posterior neighborhood. The magenta dashed line and magenta arrows point to the anterior neighborhood and the orange arrow, to the posterior neighborhood. By 535 m.p.f. (L, P, P’), SYG-1 expression is visible in both the anterior and posterior neighborhoods. In subsequent timepoints (M, Q, Q’, N, R, R’), SYG-1 expression increases in the anterior neighborhood and decreases in the posterior neighborhood, coincident with AIB developmental events that enable its transition from the posterior to the anterior neighborhood (Figure 2B–K). The syg-1 transcriptional reporter shows a similar expression pattern throughout development (Figure 5—figure supplement 1). (S) Plot showing relative enrichment of the syg-1 transcriptional reporter in the anterior neighborhood over time (magenta) overlaid with plot showing percentage of the relocating AIB distal neurite that has zippered onto the anterior neighborhood (blue). Relative enrichment in the anterior neighborhood is defined as the ratio of mean intensity of the syg-1 reporter in the band corresponding to the AIB anterior neighborhood, as compared to that in the posterior neighborhood (see Materials and methods). This value is calculated starting at a timepoint when syg-1 reporter expression becomes visible in the anterior neighborhood and averaged for four embryos. The relative enrichment values plotted represent values calculated at the indicated developmental times on the x-axis ( ± 10 mins). The reported values of ‘% AIB zippered’ are averaged across the three independent embryo datasets used for the plots in Figure 3. Note similar SYG-1 expression dynamics to zippering dynamics in AIB. Error bars represent standard error of the mean (S.E.M.). See Figure 5—figure supplement 5 for the individual values of syg-1 anterior enrichment and ‘% AIB zippered’. Scale bar = 10 μm, applies to B–D, (F–H) and K–N. Scale bar = 2 μm in E, I and O–R. Times are in m.p.f. (minutes post fertilization).

Figure 5—figure supplement 1
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Spatiotemporal regulation of syg-1 transcriptional reporter expression during embryogenesis.

(A-D) Time-lapse images of syg-1 reporter expression during embryogenesis (450–630 m.p.f.). Images are deconvolved diSPIM maximum intensity projections. The dashed boxes represent the dorsal half of the nerve ring and are magnified in E-H. E’-H’ are schematic diagrams representing the images in E–H. In (A,E,E’), syg-1 expression is primarily visible in a single band containing amphid neurites, and therefore coincident with the AIB posterior neighborhood (indicated with orange arrow). The magenta dashed line and magenta arrows point to the anterior neighborhood. (B,F,F’) show onset of weak syg-1 expression in the anterior neighborhood (white arrow in F) and ingrowth of syg-1-expressing neurites into this neighborhood (white arrowhead, identified as RIM neurons by colocalization, see Figure 5—figure supplement 3). syg-1 expression increases in the anterior neighborhood and decreases in the posterior neighborhood as embryonic development progresses (C,G,G’,D,H,H’), quantified in Figure 5S and similar to the SYG-1 protein reporter (Figure 5J–R’). Scale bar = 10 μm in A–D. Scale bar = 1 μm in E–H. All times are in m.p.f. (minutes post fertilization).

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Neurons expressing SYG-1 in the embryonic nerve ring.

(A,B) Single z-slice of a diSPIM image of an embryo expressing a ubiquitous mCherry histone label (used for lineaging at 430 mpf and as described in Bao et al., 2006) and syg-1p (3.5 kb) driving GFP (A). (B) shows only the syg-1 channel. Scale bar = 10 μm, applies to A and B. (C) Identity of the SYG-1-labeled neurons in the anterior and posterior neighborhoods. Our lineaging analysis are consistent with embryonic transcriptomic dataset previously reported (Packer et al., 2019). Of note, neurons in the posterior neighborhood that exhibit syg-1 expression at the time window (430–550 m.p.f.) (ADLR, ADLL, ASHR, ASHL), coinciding with when lineaging was performed (430 m.p.f.), in the transcriptomic dataset (Packer et al., 2019) show a decrease in syg-1 expression levels at a later developmental window (550–690 m.p.f.), consistent with our observations in Figure 5 and Figure 5—figure supplement 3. The transcriptome analyses also reveal that certain neurons in the anterior neighborhood (e.g. RIB) exhibit a greater than two-fold increase (435.7–1007.8 estimated transcripts per million - Packer et al., 2019) in expression between the earlier (430–550 m.p.f.) and later (550–690 m.p.f.) developmental windows. Other anterior neighborhood neurons, such as RIM, show a decrease in expression levels in the transcriptomic reports (1536.3–1078.2 estimated transcripts per million), but contribute to an increase in SYG-1 in the anterior neighborhood by growing its neurite into the anterior neighborhood at these time windows. (D) EM reconstruction showing areas of contact between AIBL and all its neighboring neurons (blue), and AIBL and SYG-1 expressing neurons (yellow). These data were derived from segmentations of the JSH EM dataset (Brittin et al., 2021; White et al., 1986) (see Materials and methods for how these contact areas or ‘patches’ were created). Inset shows a rotated view to highlight all patches. Note that while SYG-1 is important for placement of the AIB interneuron, not all fasciculating partners of AIB express SYG-1 (we observe that 13 % of AIB fasciculating partners express SYG-1). (E) Similar to D but for AIBR. Scale bar = 2 μm, applies to D and E.

Figure 5—figure supplement 3
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SYG-1 is expressed in the anterior neighborhood RIM neurons.

(A) Schematic of the axial view of an embryo with the red box showing the region in the head containing the nerve ring. (B–D) Deconvolved diSPIM maximum intensity image of (B) membrane-targeted PH:GFP driven by the syg-1 promoter (as in Figure 5—figure supplement 1) and (C) RIM in an embryo. d is a merge of B and C. Note the colocalization of the RIM neurites (arrow) and the RIM cell body (arrowhead) in D with the syg-1 reporter in the anterior neighborhood. (E–I) Expression of the same syg-1 reporter as in B–D but in larval stage three in a lateral view (E). The syg-1 reporter (F) is co-expressed with a cytoplasmic RIM neuron marker (G). (H) is a merge of F and G. The dashed box represents the region of the nerve ring containing the RIM neuron and the syg-1-expressing neurons. Note the RIM neurite colocalizes with the anterior band of syg-1 expression, coincident with the AIB anterior neighborhood (magenta arrow). The white arrowhead in F–H and semi-transparent magenta outline in F indicates colocalization of the RIM cell body with the syg-1 reporter. Scale bar = 10 μm applies to B–I.

Figure 5—figure supplement 4
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The RIM neurons regulate AIB distal neurite placement.

(A) Schematic of the axial view of an embryo where the red box highlights the region in the head where nerve ring neurons are present, and cropped from images of whole embryos, to produce the images in B,C. B,C, diSPIM maximum intensity projections of fluorescently labeled RIM neurons (arrows) in embryos prior to AIB distal neurite placement (~500–550 m.p.f., minutes post fertilization), labeled with inx-19p:GFP (B) or tdc-1p:GFP (C). Scale bar = 10 μm applies to B,C. (D) Schematic showing strategies used for ablation of the RIM neurons in embryos. In strategy 1, a small (p12) and a large (p17) subunit of human Caspase-3 are both expressed by inx-19p, similar to previously described (Chelur and Chalfie, 2007). The inx-19p is expressed in the RIM neurons from 370 m.p.f – the time of their birth. In strategy 2, p12 is expressed by inx-19p and p17 by tdc-1p (tdc-1p is expressed in the RIM neurons ~ 445 m.p.f.). These caspase subunits are therefore expected to reconstitute expression (and induce ablation) in embryonic RIM neurons. (E) Representative confocal image of a wild type L3 animal expressing membrane-targeted PH:GFP in the RIM neurons with RIM-specific promoter gcy-13p. (F,G) As E, but in animals additionally expressing the caspase subunits for (f) ablation strategy one and (G) ablation strategy 2. Note the absence of RIM labeling, indicating successful ablation of the RIM neurons. Scale bar = 10 μm, applies to E-G. H-M, Confocal images showing AIB (labeled with cytoplasmic mCherry; H,K) and RIM neurons (labeled with PH:GFP; I,L) and merged images (J,M) for wild-type animals (H–J) and animals in which RIM was genetically ablated (K–M). RIM ablation was achieved using Strategy 2, see Materials and methods. Magenta dashed line (M) represents the AIB anterior neighborhood. (N) Quantification of the penetrance of the AIB neurite placement defect as the percentage of animals with normal AIB neurite placement in the anterior neighborhood. Strategy one and Strategy two refer to split caspase ablations (Chelur and Chalfie, 2007) using two different combinations of promoters expressed in RIM neurons (see Materials and methods). ****p < 0.0001 (two-sided Fisher’s exact test). Numbers on bars represent number of animals examined. (O) Quantification of the minimum perpendicular distances between the AIB proximal and distal neurites in WT (n = 28) and RIM-ablated populations (n = 10 for strategy one and n = 14 for strategy 2). ****p < 0.0001; **p = 0.0011 (one-way ANOVA with Dunnett’s multiple comparisons test). n represents the number of AIB neurons measured from 14, 5, and 7 animals from the WT, ablation strategy one and ablation strategy two populations respectively. Effect size estimate, d = 2.313 for WT and ablation strategy 1, and 1.19 for WT and ablation strategy 2. (P,Q) Confocal micrographs of animals where AIB (cyan) and the RIM and RIC neurons (magenta) are co-labeled and SYG-1 is expressed specifically in RIM and RIC (see Materials and methods) in a syg-1(ky652) mutant background. The dashed box represents the nerve ring region containing the neurites of AIB, RIM and RIC, and is magnified in P’ and Q’. The AIB distal neurite is positioned along RIM (P’) or along both RIM and RIC (Q’). The yellow arrowheads and yellow arrows point at the RIC neurite and the RIM neurite, respectively. Scale bar = 10 μm for H–M, P,Q and 2 μm for P’ and Q’.

Figure 5—figure supplement 4—source data 1

Number of animals of each genotype (in the bar graph in Figure 5—figure supplement 4N) displaying normal vs aberrant distal neurite placement.

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Figure 5—figure supplement 5
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Individual data points from Figure 5S.

(A,B) Plots showing individual values of syg-1 anterior enrichment (A), as measured from the syg-1 transcriptional reporter and percent AIB neurite zippered (B) (see Materials and methods for details). Individual values at each timepoint in A and B were averaged to produce the magenta and blue curves respectively in the plot in Figure 5S. Each point in A represents a measurement of syg-1 anterior enrichment from one side of the embryo, two values are therefore obtained from each embryo (see Materials and methods). Each point in B represents a single AIB neurite. The syg-1 anterior enrichment values (A) were quantified from four different embryos and percentages of the AIB neurite zippered (B) were quantified from three different embryos.

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Localization of SYG-2 puncta to the AIB distal neurite.

(A-C) Confocal micrograph of a larval stage four animal showing AIB (cyan, A), a translational fusion marker GFP:SYG-2 (GFP fused to a 16 kb genomic region including 8 kb upstream sequence and the coding region of syg-2 Shen et al., 2004) (yellow, B) and merge (C). Arrowheads in C point to the subset of SYG-2 puncta that colocalize with the AIB distal neurite (co-injection marker odr-1p:RFP labels an amphid neuron in the same channel as AIB (cyan)).

Figure 5—video 1
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Layered expression of SYG-1 in the nerve ring in embryos, related to Figure 5 and Figure 5—figure supplement 1.

3D projection of deconvolved diSPIM images of an embryo expressing the syg-1 transcriptional reporter (syg-1p) (this video is the same as the 529 m.p.f. timepoint in Figure 5—figure supplement 1, also see Materials and methods). Expression of syg-1p is restricted to two nerve ring neighborhoods, corresponding to the AIB posterior and anterior neighborhoods (as indicated in Figure 5). Scale bar = 10 μm.

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Identification of SYG-1-expressing neurons by lineage tracking, related to Figure 5—figure supplement 2.

Video showing z-slices through an embryo with dual labeling of mCherry-labeled histones and syg-1p:GFP. Lineaged cells are annotated across slices. Scale bar = 10 μm. Same embryo as in Figure 5—figure supplement 2.

Figure 6 with 1 supplement
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Ectopic syg-1 expression is sufficient to alter placement of the AIB distal neurite.

(A) Lateral view schematic of a wild-type AIB neuron (cyan) in the context of the posterior (orange) and anterior (magenta) neighborhoods, and the nerve ring (light neon). Higher SYG-1 endogenous expression in the anterior neighborhood represented by yellow arrowhead. (B–C) Confocal image of a wild type animal with AIB (labeled with cytoplasmic mCherry, in cyan) and the posterior neighborhood neurons AWC and ASE (labeled with cytoplasmic GFP, in orange). The dashed box represents the region of contact between AIB and the posterior neighborhood neurons, magnified in (C). Magenta dashed line represents the AIB anterior neighborhood. (D–F) As (A–C), but in the syg-1(ky652) lof (loss of function) mutant background. Note that the distal neurite is positioned away from the posterior neighborhood, as in wild type, although these animals display defects in fasciculation with the anterior neighborhood (as shown in Figure 4). (G–I) As (D–F), but with ectopic overexpression of SYG-1 in the posterior neighborhood neurons. In the schematic (G), expression of SYG-1 in the posterior neighborhood (achieved here using nphp-4p, also see Figure 6—figure supplement 1) is represented by a yellow arrowhead (as in (A), but here in posterior neighborhood). Note that the AIB distal neurite is now abnormally positioned in the posterior neighborhood in which SYG-1 was ectopically expressed (H, I). (J) Schematic (left) and scatter plot quantification (right) of minimum perpendicular distances (dmin, indicated by black double-headed arrow) between the AIB distal neurite and posterior neighborhood neurons in WT (in black, n = 17), syg-1(ky652) (in green, n = 18), and two syg-1(ky652) populations with SYG-1 overexpressed in two different sets of posterior neighborhood neurons via the use of nphp-4p and (in blue) mgl-1bp (in red) (n = 18 and n = 16 respectively). **p = 0.0056 and 0.0070, respectively (one-way ANOVA with Dunnett’s multiple comparisons test). Effect size estimate, d = 1.075 and 1.140, respectively. Error bars indicate standard error of the mean (S.E.M.). n represents the number of AIB neurites quantified. Quantifications were done from nine animals each for WT, syg-1(ky652) and nphp-4p:syg-1; syg-1(ky652) and eight animals for mgl-1bp:syg-1; syg-1(ky652). (K) Quantification of penetrance of the ectopic AIB neurite placement represented as the percentage of animals with the AIB distal neurite partially positioned in the posterior neighborhood in the WT, syg-1(ky652), posterior SYG-1 overexpression strains (colors represent the same strains as in J) and a posterior SYG-1 overexpression strain in syg-2(ky671) background. Numbers on bars represent number of animals examined. ***p = 0.0002 for syg-1(ky652) and nphp-4p expressed SYG-1 and ****p < 0.0001 for syg-1(ky652) and mgl-1bp expressed SYG-1 by two-sided Fisher’s exact test (also see Figure 6—figure supplement 1). Scale bar = 10 μm in B, E and H and 1 μm in C, F, and I. Cell body is marked with an asterisk.

Figure 6—source data 1

Numbers of animals of each genotype (in Figure 6K) displaying ectopic distal neurite placement in the posterior neighborhood.

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Figure 6—figure supplement 1
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A SYG-1-SYG-2 interaction underlies ectopic AIB neurite placement.

(A) Plot showing the percentage of the AIB distal neurite ectopically positioned in the posterior neighborhood in two strains expressing SYG-1 ectopically in the posterior neighborhood. These measurements were done from the same WT, syg-1(ky652), mgl-1bp:syg-1; syg-1(ky652) and nphp-4p:syg-1; syg-1(ky652) as in Figure 6. mgl-1bp and nphp-4p are promoters driving expression specifically in the posterior neighborhood (see Figure 6 and Materials and methods) One-way ANOVA with Dunnett’s multiple comparisons test was performed. **p = 0.0017 for WT and mgl-1bp:syg-1; syg-1(ky652) and for syg-1(ky652) and mgl-1bp:syg-1; syg-1(ky652), and **p = 0.0015 for WT and nphp-4p:syg-1; syg-1(ky652). syg-1(ky652) and nphp-4p:syg-1; syg-1(ky652). Effect size = 1.186 for WT and nphp-4p:syg-1 and nphp-4p:syg-1; syg-1(ky652). Effect size = 1.304 for WT and mgl-1bp:syg-1 and mgl-1bp:syg-1; syg-1(ky652). (B) Schematic of the receptor-ligand pair SYG-1 (green) and SYG-2 (purple). The red dashed box includes the SYG-1 extracellular Ig domains and transmembrane domain (collectively referred to as SYG-1 ecto). The yellow dashed box includes the SYG-1 transmembrane domain and cytoplasmic domains (collectively referred to as the SYG-1 endodomain or SYG-1 endo). (C) Quantification of penetrance of the ectopic AIB neurite placement as the percentage of animals with the AIB distal neurite partially positioned in the posterior neighborhood in the indicated genotypes. ****p < 0.0001 (by two-sided Fisher’s exact test) for syg-1(ky652) and mgl-1bp:syg-1; syg-1(ky652), in which SYG-1 is expressed specifically in the posterior neighborhood (denoted as posterior SYG-1); for mgl-1bp:syg-1; syg-1(ky652) and mgl-1bp:syg-1; syg-1(ky652); syg-2(ky671), and for mgl-1bp:syg-1; syg-1(ky652) and mgl-1bp:syg-1endo; syg-1(ky652) where mgl-1bp:syg-1endo drives expression of the SYG-1 endodomain in the posterior neighborhood. Number on bars represent the number of animals examined. The first four bars are the same as the ones corresponding to these genotypes in Figure 6K. The black, green and purple bars represent WT, syg-1(ky652) and syg-2(ky671) backgrounds, respectively.

Figure 6—figure supplement 1—source data 1

Numbers of animals of each genotype (in Figure 6—figure supplement 1C) displaying ectopic distal neurite placement in the posterior neighborhood.

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Figure 7 with 3 supplements
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AIB neurite placement by retrograde zippering, and presynaptic assembly are coordinated during development.

(A) Axial view schematic of the AIB neurons (cyan) with presynaptic protein RAB-3 (yellow) puncta along the distal neurite. Arrowhead indicates the tip of the distal neurite and arrow/dashed line indicate the dorsal midline. (B–E) Time-lapse imaging of RAB-3 localization in AIB during embryogenesis. (B–E) are merged diSPIM maximum intensity projections of AIB labeled with membrane-tagged mCherry (cyan) and AIB presynaptic sites labeled with GFP:RAB-3 (yellow), at different timepoints during embryogenesis. (B’-E’) represent the GFP:RAB-3 channel for images in B–E). Note in (B, B’) and (C, C’) that the RAB-3 signal in the neurite is localized exclusively near the neurite tip. As development progresses, there is more RAB-3 signal throughout the neurite from the tip up to the midline (in (D, D’) and (E, E’). Therefore, RAB-3 becomes progressively enriched from the tip up to the midline during development, and the timing for this process correlates, with a slight delay, with the developmental timing of AIB zippering (Figure 2I–K). Arrowhead and arrow, as in (A), indicate the tip of the distal neurite and the region of the neurite near the dorsal midline (dashed vertical line) respectively. Scale bar = 10 μm applies (B–E) and (B’-E’). (F–I) Straightened distal neurites from AIB (corresponding to the region in (B–E) which is marked by the arrowhead (AIB tip) and arrows (dorsal midline)). Note presynaptic assembly, as imaged by RAB-3 accumulation, from the tip of the neurite towards the midline of AIB, reminiscent of the zippering event (Figure 2). Scale bar = 1 μm. (J) Plot showing average RAB-3, CLA-1 and SYD-2 intensities along the AIB distal neurite over time (yellow, orange and green, respectively) and percentage of the relocating AIB distal neurite that has zippered onto the anterior neighborhood (blue). See Figure 7—figure supplement 1 for images of CLA-1 developmental dynamics in AIB. The intensities in the plot represent values calculated at the indicated developmental times on the x-axis ( ± 10 min). The reported values of ‘% AIB zippered’ are averaged are the same as in Figure 5S. Note that RAB-3, CLA-1 and SYD-2 intensity start increasing from after completion of zippering (540 m.p.f.). Error bars represent standard error of the mean (S.E.M.). See Figure 7—figure supplement 2 for individual RAB-3, CLA-1 and SYD-2 intensity values. Times are in m.p.f. (minutes post fertilization). (K) Schematic model showing progressive retrograde zippering leading to placement of the AIB neurite along two different layers. This is accompanied by a switch in SYG-1 expression between layers, and synaptic protein localization in a retrograde order along the neurite, resembling the order of zippering.

Figure 7—figure supplement 1
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Presynaptic proteins populate the distal neurite following zippering-mediated neurite placement.

(A) Schematic of the lateral view of an AIB neuron (cyan) with presynaptic sites labeled with active zone protein CLA-1 (yellow). (B–E) Time-lapse imaging of CLA-1 localization in AIB during embryogenesis. (B–E) are merged diSPIM maximum intensity projections of AIB labeled with membrane-tagged mCherry (cyan) and AIB presynaptic sites labeled with GFP:CLA-1 (yellow), at different timepoints during embryogenesis. As development progresses, there is more CLA-1 signal throughout the neurite from the tip up to the midline, similar to the time-course of RAB-3 localization (Figure 7). Arrowhead and arrow, indicate the tip of the distal neurite and the region of the neurite near the dorsal midline respectively. Scale bar = 10 μm applies (B–E). (F) Straightened distal neurites from AIB (corresponding to the region in (B–E) which is marked by the arrowhead (AIB tip) and arrows (dorsal midline)). Scale bar = 2 μm. Times are in m.p.f. (minutes post fertilization). (G–K) Representative confocal image of a syd-2(ola341) animal co-expressing (H) cytoplasmic mCherry and (I) GFP:RAB-3 in AIB for simultaneous visualization of AIB morphology and presynaptic sites. (J) is a merge of (H) and (I). The region of the AIB neurite bound by the dashed box in (J) is magnified in (K). Note altered distribution of presynaptic protein RAB-3. RAB-3 is enriched near the tip instead of being localized all along the distal neurite. Scale bar = 10 μm in (H–J) and 3 μm in (K).(K) Representative confocal image of a syd-2(ola341) animal with AIB labeled with cytoplasmic mCherry (cyan) and the distal neighborhood neuron, RIM, labeled with cytoplasmic GFP (magenta). Note that although distribution of RAB-3 is altered (H–K), the placement of the AIB neurite in the RIM-containing distal neighborhood is unaltered in syd-2(ola341). Scale bar = 10 μm.

Figure 7—figure supplement 2
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Individual data points from Figure 7J.

A-C Plots showing SYD-2 (A), RAB-3 (B), and CLA-1 (C) mean intensities over time from individual neurites. These values were averaged to obtain the intensity plot in Figure 7J. SYD-2, RAB-3 and CLA-1 intensities were calculated from 2, 3, and 4 different embryos, respectively.

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Presynaptic protein distribution phenotype in the AIB neurite of syg-1(ky652).

(A-F) Schematic (A) and representative confocal image of the AIB neurite (B), AIB presynaptic sites (C) and neurite of the postsynaptic partner, the RIM neuron in the anterior neighborhood (D). (E) is a merged image. The dashed box represents the region of contact between the AIB and RIM neurites, magnified in F. (G–L) As (A–F) but in the syg-1(ky652) mutant background. Note the gaps between the AIB distal neurite and the RIM neurites (L) and reduced localization of RAB-3 along the AIB neurite in the region where it is detached from RIM (white arrows in L). Scale bar = 10 μm in A-E, G-K and 1 μm in F,L. (M) Line intensity plot showing RAB-3 fluorescence intensity (yellow) and AIB-RIM contact (magenta) along the length of the AIB distal neurite in G-L. Note the peaks in the two line profiles align, indicating higher RAB-3 intensity at points of AIB-RIM contact. (N) Box plot (10–90 percentile) showing mean intensities of RAB-3 in adhered and detached regions in syg-1(ky652) mutant neurites that exhibit detachment between AIB and RIM (n = 12, where n = number of animals). **p = 0.0003 (unpaired two-tailed t-test). Effect size, d = 1.643.

Figure 7—figure supplement 3—source data 1

Mean fluorescence intensities of RAB-3 in adhered and detached regions of the AIB distal neurite in syg-1(ky652) (Figure 7—figure supplement 3N).

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SYG-1 regulates neighborhood-specific placement of AVE.

(A) Volumetric reconstruction of command interneuron AVER (green) and AIBR (cyan) from the segmented JSH EM dataset (Brittin et al., 2018; White et al., 1986). Note the similarity in morphology of the two neurons. The distal neurite of AIB and the proximal neurite of AVE lie at the same position (indicated by magenta arrow). The arrowheads indicate the dorsal shift that forms the chiasm in AIB and AVE. Scale bar = 5 μm, also applies to B. (B,C) Volumetric reconstruction of the AVE neurons (green) (B) and the AIB neurons (cyan) (C) in the context of the nerve ring strata S2 (purple) and S3 (orange). Note the placement of the AVE proximal neurite along the border of S2 and S3, and the AVE distal neurite at the anterior boundary of S2 (the anterior boundary abuts S1, not shown here). Note the placement of the AIB distal neurite, also at the S2/S3 border, similar to the AVE proximal neurite. The dashed lines indicate the layer borders. The yellow, magenta and orange arrows correspond to the S1/S2, S2/S3, and S3/S4 borders respectively (S4 not shown here). Scale bar in C = 5 μm. (D) Schematic of the lateral view of AVE (green) in the context of its neighborhoods: proximal (magenta) and distal (yellow), with the nerve ring (light brown) and pharynx (gray). Black arrowhead indicates a posterior-anterior chiasm. The magenta and yellow arrows indicate the positions of the AVE proximal and AVE distal neighborhoods, respectively and coincide with the S2/S3 and S1/S2 borders, respectively (see B). Note that while the design principles of AVE are similar to those of rich-club interneuron AIB, their positions in the nerve ring, and the strata they connect, are different – the AVE neurite is placed more anteriorly by one stratum compared to AIB. E,F,F’, Confocal image of wild-type animal with AVE and RIM co-labeled. The magenta and yellow arrows indicate the positions of the AVE proximal and AVE distal neighborhoods, respectively. White arrowhead indicates AVE chiasm, corresponding to its anterior shift. Dashed box shows region of contact of the AVE and RIM neurites, magnified in F. (F’) is a schematic of the image in (F). Scale bar corresponds to 10 μm in E and 1 μm in F. Scale bars in E and F apply to G and H, respectively. Cell bodies are marked with an asterisk. G,H,H’, As E,F,F’ but in syg-1(ky652) mutant background. Note the gap between the AVE proximal neurite and the RIM neurites (G,H,H’) and defect in the dorsal midline shift. (I) Scatter plot showing quantification of the loss of contacts between the AVE and RIM neurites. The extent of detachment of the AVE proximal neurites from RIM, and hence its deviation from the RIM neighborhood, was quantified using the indicated formula in Figure 4M (also see STAR Methods). Scatter plot depicts % detachment values for wild type (n = 22) and syg-1(ky652) (n = 16) calculated from 11 and 8 animals respectively. Error bars indicate standard error of the mean (S.E.M.). **P = 0.002 (unpaired two-tailed t-test). Effect size d = 1.002. (J) Quantification of length of the posterior-anterior shift, quantified for each AVE neurite, for WT (n = 32) and syg-1(ky652) mutants (n = 40) and displayed as a scatter plot. These were calculated from 16 and 20 animals respectively from WT and syg-1(ky652). Error bars indicate standard error of the mean (S.E.M.). ***p = 0.0001 (unpaired two-tailed t-test). Effect size d = 1.003. n represents the number of AIB neurites quantified.

Appendix 1—figure 1
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Biophysics of retrograde zippering and unzippering.

Force balance at the zippering and unzippering points.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain (C. elegans)ujIs113[pie-1p::mCherry::H2B::pie-1 3'UTR+ nhr-2p::his-24::mCherry::let-858 3'UTR+ unc-119(+)];IIDuncan et al., 2019BV276Strain available from D. Colón-Ramos lab
Strain (C. elegans)ujIs113;oyIs48[Pceh-36::GFP, lin-15(+)];Vgift from John MurrayJIM158Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaIs67[DACR2245 at 40 ng/uL + DACR1412 at 30 ng/uL + DACR218 at 30 ng/uL];XThis paperDCR5516Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex3394[DACR2796 at 60 ng/uL + DACR2651 at 60 ng/uL + DACR218 at 30 ng/uL]This paperDCR5761Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex3666[DACR199 at 2 ng/uL + DACR218 at 30 ng/uL];olaIs67This paperDCR6222Strain available from D. Colón-Ramos lab
Strain (C. elegans)oyIs48;olaIs67This paperDCR6301Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4624[DACR3149 at 10 ng/uL + DACR218 at 30 ng/uL];olaIs67This paperDCR7648Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaIs68[DACR2245 at 40 ng/uL + DACR1412 at 30 ng/uL + DACR218 at 30 ng/uL]This paperDCR5517Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaIs68;syg-1(ky652)This paperDCR8220Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaIs68;syg-1(ok3640)This paperDCR8486Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4624;olaIs68;syg-1(ky652)This paperDCR8183Strain available from D. Colón-Ramos lab
Strain (C. elegans)kyIs235;kyEx679;syg-1(ky652)This paperCX5862Strain available from D. Colón-Ramos lab
Strain (C. elegans)kyEx679;olaIs68;syg-1(ky652)This paperDCR8180Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4624;kyEx679;olaIs68;syg-1(ky652)This paperDCR8489Strain available from D. Colón-Ramos lab
Strain, strain background (C. elegans)olaIs68;syg-2(ky671)This paperDCR6767Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4624;olaIs68;syg-2(ky671)This paperDCR8468Strain available from D. Colón-Ramos lab
Strain (C. elegans)oyIs48; olaIs68;syg-1(ky652)This paperDCR8488Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex5120[DACR3529 at 30 ng/uL + DACR1412 at 30 ng/uL + DACR218 at 30 ng/uL]This paperDCR8440Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex5063[DACR3492 at 25 ng/uL + DACR3505 at 40 ng/uL + DACR2312 at 25 ng/uL + DACR20 at 25 ng/uL];olaIs67This paperDCR8365Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4071[DACR2637 at 15 ng/uL + DACR218 at 30 ng/uL];olaIs67This paperDCR6814Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4130[DACR2704 at 100 ng/uL + DACR218 at 50 ng/uL];ujIs113This paperDCR6920Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4052[DACR2607 at 100 ng/uL + DACR2609 at 25 ng/uL + DACR218 at 30 ng/uL];olaIs67This paperDCR6782Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4054[DACR2607 at 100 ng/uL + DACR2609 at 25 ng/uL + DACR218 at 30 ng/uL];olaIs67This paperDCR6784Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex3388[DACR2371 at 75 ng/uL + DACR2404 at 30 ng/uL + DACR218 at 30 ng/uL]This paperDCR5730Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4618[DACR2607 at 100 ng/uL + DACR2609 at 25 ng/uL + DACR2863 at 25 ng/uL + DACR218 at 30 ng/uL];olaIs67This paperDCR7642Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4619[DACR2607 at 100 ng/uL + DACR2609 at 25 ng/uL + DACR2863 at 25 ng/uL + DACR218 at 30 ng/uL];olaIs67This paperDCR7643Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex3949[DACR2607 at 100 ng/uL + DACR2609 at 25 ng/uL + DACR2351 at 25 ng/uL + DACR218 at 30 ng/uL]Moyle et al., 2021DCR6633Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex2887[DACR2245 at 100 ng/uL + DACR2404 at 30 ng/uL + DACR218 at 30 ng/uL]This paperDCR4894Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex3570[DACR2481 at 10 ng/uL + DACR218 at 50 ng/uL];ujIs113This paperDCR6082Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex5105[DACR3605 at 50 ng/uL + DACR218 at 30 ng/uL]This paperDCR8421Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaIs117[DACR3502 at 30 ng/uL + DACR20 at 25 ng/uL];olaIs68This paperDCR8347Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaIs117;olaIs68;syg-1(ky652)This paperDCR8350Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex5059[DACR3503 at 10 ng/uL + DACR20 at 25 ng/uL];olaIs68;syg-1(ky652)This paperDCR8361Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex5050[DACR3698 at 30 ng/uL + DACR20 at 25 ng/uL];olaIs68;syg-1(ky652)This paperDCR8352Strain available from D. Colón-Ramos lab
Strain (C. elegans)oyIs48; olaex5059; olaIs68; syg-1(ky652)This paperDCR8470Strain available from D. Colón-Ramos lab
Strain (C. elegans)oyIs48; olaIs117; olaIs68; syg-1(ky652)This paperDCR8472Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex4087[DACR1412 at 30 ng/uL + DACR2618 at 50 ng/uL + DACR218 at 30 ng/uL]This paperDCR6841Strain available from D. Colón-Ramos lab
Strain (C. elegans)oyIs48;olaIs68;syg-2(ky671);This paperDCR8758Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaEx5279[DACR3527 at 30 ng/uL + DACR20 at 25 ng/uL]; olaIs68; syg-1(ky652)This paperDCR8762Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaEx5276 [DACR3780 at 5 ng/ul + DACR1412 at 20 ng/uL + DACR218 at 30 ng/uL]This paperDCR8759Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaEx5281[DACR3888 at 30 ng/uL + DACR20 at 30 ng/uL]; olaIs68; syg-2(ky671)This paperDCR8764Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaEx5283[DACR3781 at 30 ng/uL + DACR20 at 25 ng/uL]; olaIs68;syg-1(ky652)This paperDCR8766Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaIs117; olaIs68; syg-1(ky652); syg-2(ky671)This paperDCR8767Strain available from D. Colón-Ramos lab
Strain (C. elegans)zbIs3[cnd-1p::PH::GFP]Fan et al., 2019BV293Strain available from D. Colón-Ramos lab
Strain (C. elegans)zbIs3;olaIs68;syg-1(ky652)This paperDCR8772Strain available from D. Colón-Ramos lab
Strain (C. elegans)kyex684[syg-2:GFP]Shen et al., 2004TV6006Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex5347[DACR1412 at 30 ng/uL + DACR218 at 30 ng/uL]This paperDCR8922Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex5332[DACR3901 at 125 ng/uL + DACR2404 at 75 ng/uL + DACR218 at 30 ng/uL]This paperDCR8894Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex5340[DACR3890 at 100 ng/uL + DACR2404 at 75 ng/uL + DACR218 at 30 ng/uL]This paperDCR8908Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex5144[DACR3492 at 50 ng/uL + DACR3493 at 50 ng/uL + DACR218 at 30 ng/uL];olaIs67This paperDCR8469Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex5195[DACR3529 at 30 ng/uL + DACR218 at 30 ng/uL];ujIs113This paperDCR8626Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaIs67;syd-2(ola341)This paperDCR6756Strain available from D. Colón-Ramos lab
Strain (C. elegans)olaex3666; olaIs67;syd-2(ola341)This paperDCR6842Strain available from D. Colón-Ramos lab
Strain (C. elegans)hdIs32 [glr-1::DsRed2]. gvEx173 [opt-3::GFP+ rol-6(su1006)]CGCNC1750Strain available from D. Colón-Ramos lab
Strain (C. elegans)gvex173;syg-1(ky652)This paperDCR8907Strain available from D. Colón-Ramos lab

Additional files

Supplementary file 1

Plasmids generated and used in this study.

https://cdn.elifesciences.org/articles/71171/elife-71171-supp1-v2.docx
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https://cdn.elifesciences.org/articles/71171/elife-71171-transrepform1-v2.pdf

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  1. Titas Sengupta
  2. Noelle L Koonce
  3. Nabor Vázquez-Martínez
  4. Mark W Moyle
  5. Leighton H Duncan
  6. Sarah E Emerson
  7. Xiaofei Han
  8. Lin Shao
  9. Yicong Wu
  10. Anthony Santella
  11. Li Fan
  12. Zhirong Bao
  13. William A Mohler
  14. Hari Shroff
  15. Daniel A Colón-Ramos
(2021)
Differential adhesion regulates neurite placement via a retrograde zippering mechanism
eLife 10:e71171.
https://doi.org/10.7554/eLife.71171