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. 2024 Mar 6;137(5):jcs261509. doi: 10.1242/jcs.261509

TIAM-1 regulates polarized protrusions during dorsal intercalation in the Caenorhabditis elegans embryo through both its GEF and N-terminal domains

Yuyun Zhu 1,*, Zoe Tesone 2,*, Minyi Tan 3, Jeff Hardin 1,2,3,‡
PMCID: PMC10949065  PMID: 38345070

ABSTRACT

Mediolateral cell intercalation is a morphogenetic strategy used throughout animal development to reshape tissues. Dorsal intercalation in the Caenorhabditis elegans embryo involves the mediolateral intercalation of two rows of dorsal epidermal cells to create a single row that straddles the dorsal midline, and thus is a simple model to study cell intercalation. Polarized protrusive activity during dorsal intercalation requires the C. elegans Rac and RhoG orthologs CED-10 and MIG-2, but how these GTPases are regulated during intercalation has not been thoroughly investigated. In this study, we characterized the role of the Rac-specific guanine nucleotide exchange factor (GEF) TIAM-1 in regulating actin-based protrusive dynamics during dorsal intercalation. We found that TIAM-1 can promote formation of the main medial lamellipodial protrusion extended by intercalating cells through its canonical GEF function, whereas its N-terminal domains function to negatively regulate the generation of ectopic filiform protrusions around the periphery of intercalating cells. We also show that the guidance receptor UNC-5 inhibits these ectopic filiform protrusions in dorsal epidermal cells and that this effect is in part mediated via TIAM-1. These results expand the network of proteins that regulate basolateral protrusive activity during directed rearrangement of epithelial cells in animal embryos.

Keywords: Caenorhabditis elegans, Cell rearrangement, Convergent extension, Morphogenesis, TIAM-1, UNC-5

Summary: The Rac-GEF TIAM-1 promotes contralateral migration via its GEF domain, whereas its N-terminal domains suppress ectopic filiform protrusions around the periphery during mediolateral intercalation of dorsal epidermal cells in the Caenorhabditis elegans embryo.

INTRODUCTION

During embryonic development, cell fate specification and cellular movements work in tandem to achieve the extensive morphological changes that occur as the body plan emerges. One type of cell rearrangement, convergent extension (CE), is a ubiquitous morphogenetic movement that elongates a tissue along one axis while narrowing it along the orthogonal axis (Huebner and Wallingford, 2018; Walck-Shannon and Hardin, 2014). CE or its variants drive morphogenetic movements during gastrulation (Solnica-Krezel and Sepich, 2012), neurulation (Williams et al., 2014) and shape organogenesis in tissues such as the vertebrate lateral line and the mammalian cochlea (Driver et al., 2017; Sutherland et al., 2020). Because of its early involvement during embryonic development, failure of CE is often accompanied by severe congenital abnormalities, including craniorachischisis and spina bifida (Nikolopoulou et al., 2017).

Many studies of epithelial CE have focused on germband extension in Drosophila. During this process, cells shorten their anterior–posterior boundaries and extend their dorsoventral boundaries as they undergo oriented neighbor exchange (Bertet et al., 2004; Kong et al., 2017; Pilot and Lecuit, 2005). In contrast to the apical junctional dynamics that appear to drive Drosophila germband extension, other examples of epithelial CE rely on basolateral protrusive activity to accomplish cell rearrangement (Walck-Shannon and Hardin, 2014). Examples include the ascidian notochord, the sea urchin archenteron and the mammalian neural plate (Munro and Odell, 2002a,b; Sutherland et al., 2020; Williams et al., 2014). How basolateral protrusive activity in epithelia is controlled during tissue rearrangement is, therefore, an important question in morphogenesis.

A useful system for identifying previously unreported, conserved, functionally relevant molecular pathways that regulate basolateral protrusive activity during epithelial cell rearrangement is the dorsal epidermis of the Caenorhabditis elegans embryo. Dorsal epidermal cells undergo a movement known as dorsal intercalation, in which a primary, medially directed basolateral protrusion extended by each cell drives mediolateral intercalation (Walck-Shannon et al., 2015; Williams-Masson et al., 1998). The WAVE complex is constitutively required for protrusion-triggered intercalation (Patel et al., 2008; Walck-Shannon et al., 2015). Our previous work also showed that the C. elegans Rac family proteins CED-10 and MIG-2 promote protrusive activity in dorsal epidermal cells. In their active state, CED-10 and MIG-2 lead to the polymerization and nucleation of actin filaments and subsequent formation of lamellipodia via the WAVE and WASP complexes (Walck-Shannon et al., 2015). We also previously identified an upstream guanine nucleotide exchange factor (GEF), UNC-73 (Trio in mammals), which acts via its Rac-GEF domain to regulate Rac family proteins (Walck-Shannon et al., 2015), but other potential upstream regulators have not been thoroughly investigated.

Here, we establish a role for C. elegans TIAM-1, a Rac-specific GEF, in regulating protrusive activity during dorsal intercalation. In tiam-1 mutants lacking GEF activity, dorsal epidermal cells generated less robust primary protrusions during intercalation, leading to significantly increased intercalation time. We also identified a previously unreported function for the N-terminal domains of TIAM-1 in inhibiting the formation of ectopic filiform protrusions at the cell periphery, which at least in part act independently of the GEF domain. Taken together, our data establish a role for TIAM-1 in regulating polarized protrusive activity during dorsal intercalation through both its GEF and N-terminal domains.

RESULTS AND DISCUSSION

tiam-1 is required for normal temporal progression of dorsal intercalation

Dorsal intercalation is driven by the mediolateral protrusive activity of dorsal epidermal cells (see Fig. 1C, wildtype). Our previous work identified the C. elegans Rac-GTPase orthologs CED-10 and MIG-2 as partially redundant regulators of protrusion formation via activation of the downstream Arp2/3 activators, WVE-1 (WAVE) and WSP-1 (WASP), respectively (Walck-Shannon et al., 2015). We showed previously that UNC-73 functions as a Rac-GEF in promoting protrusion formation. We also showed that, in the case of loss of function for the protein CRML-1 (CARMIL), excessive production of filiform (thin, thread-like) protrusions around the periphery of intercalating cells correlated with longer intercalation times. This effect could be offset by loss of unc-73 function, indicating that Rac-GEF activity is necessary for ectopic filiform protrusion production, but that it negatively effects intercalation (Walck-Shannon et al., 2015). Given the relatively strong protrusion defects in ced-10 and mig-2 null mutants compared with unc-73 mutants specifically lacking Rac-GEF activity (Walck-Shannon et al., 2015), however, we hypothesized that additional Rac-GEFs might regulate this pathway. There are two other Rac-GEF orthologs in C. elegans that show specificity for CED-10 and MIG-2: VAV-1 (VAV1) and TIAM-1 (Tiam1) (Demarco et al., 2012; Norman et al., 2005; Vettel et al., 2012). In mammals, the function of both VAV1 and Tiam1 have been extensively studied in the immune system. Disrupted expression of both proteins is tightly associated with uncontrolled cell invasion and metastasis (Bartolomé et al., 2006; Boissier and Huynh-Do, 2014; Gérard et al., 2009; Izumi et al., 2019; Liu et al., 2007; Tybulewicz et al., 2003). Their worm orthologs, VAV-1 and TIAM-1, have been shown to function in axonal outgrowth and guidance (Demarco et al., 2012; Fry et al., 2014; Lin et al., 2022; Malartre et al., 2010; Tang et al., 2019). However, our previous work indicated that knockdown of vav-1 by RNAi had no effect on dorsal intercalation (Walck-Shannon et al., 2015). We therefore focused on TIAM-1, a multidomain protein including an N-terminal myristoylation domain, an EVH1-like domain, a PDZ domain and a GEF domain composed of Dbl homology (DH) and Pleckstrin homology (PH) subdomains (Fig. 1A) (Demarco et al., 2012).

Fig. 1.

Fig. 1.

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TIAM-1 is required for normal dorsal intercalation. (A) Protein domain map of C. elegans TIAM-1. Domain annotations: MYR, myristoylation site; EVH1-like, Ena/Vasp homology; DH, Dbl homology; PH, pleckstrin homology. The asterisk labels the mutation site of the allele tiam-1T548F in the DH domain. The black line labels the deleted region in tiam-1null. (B) Intercalation time in tiam-1T548F and tiam-1null mutants was significantly longer than in wild type. Box plots show the 25–75th percentiles, whiskers show the minimum and maximum values and the median is marked with a line. ***P<0.001, unpaired two-tailed Student's t-test. (C) DIC images of dorsal intercalation in wild-type (top), tiam-1T548F (middle) and tiam-1null (bottom) embryos (dorsal views). Anterior is to the left, posterior to the right; right and left posterior dorsal cells are pseudocolored in cyan and yellow, respectively. White arrows point to the leading edge of right-hand dorsal cells at t=60 min. Cells in wild-type embryos almost made contact with contralateral seam cells, whereas cells in mutants only intercalated halfway. Images are representative of at least 25 embryos from at least eight mounts per genotype. Scale bar: 10 µm.

We first confirmed that tiam-1 is expressed in the dorsal epidermis. Although we could not detect TIAM-1 using an endogenous mNeonGreen (mNG) knock-in strain or via antibody staining of the tagged protein in this line (Y.Z. and J.H., data not shown), we were able to detect tiam-1 transcription in dorsal epidermal cells using a tiam-1 transcriptional reporter strain (Ziel et al., 2009) (Fig. S1). Embryos homozygous for an existing tiam-1 loss-of-function allele, tm1556, as well as embryos from tiam-1(RNAi) hermaphrodites displayed delayed completion of dorsal intercalation (Fig. S3A). The gross morphology of the epidermis appeared normal in tiam-1 loss-of-function embryos, based on differential interference contrast (DIC) movies (Fig. 1C). The intercalation delay was validated by quantifying the intercalation time in both a GEF-dead allele, tiam-1T548F (Tang et al., 2019), and a new tiam-1null allele we generated, tiam-1(syb4244), in which the entire tiam-1 coding region was eliminated using CRISPR/Cas9 genome editing (Fig. 1B,C).

We also examined potential functional redundancy between UNC-73 and TIAM-1 during dorsal intercalation. tiam-1(tm1556);unc-73(rh40) double mutants had a slightly longer intercalation time compared to that of rh40 homozygotes alone (Fig. S2A). tiam-1(syb4244);unc-73(RNAi) null mutants showed a statistically significant difference in number of filiform protrusions as well, especially during early stages of intercalation (Fig. S2B). These results indicate that tiam-1 is required for normal dorsal intercalation. Moreover, given the defects displayed by tiam-1T548F mutants, TIAM-1 appears to function as a Rac-GEF in regulating dorsal intercalation alongside unc-73.

TIAM-1 promotes migration in dorsal epidermal cells

We next assessed in detail the effects of abrogating the Rac-GEF activity of TIAM-1 on protrusive behavior of dorsal epidermal cells. In control embryos, dorsal epidermal cells initially generate unpolarized filiform protrusions; shortly thereafter, as cells become wedge shaped and begin to migrate contralaterally, lateral filiform protrusions are retracted and protrusions are concentrated medially (Fig. 2A, left) (Walck-Shannon et al., 2015). We compared this wild-type sequence of behaviors to that displayed by homozygotes for the GEF-dead allele, tiam-1T548F. tiam-1T548F mutants invariably produced a main medial protrusion; we could not detect a statistically significant difference in the base-to-tip length of the main medial protrusion in tiam-1T548F or other tiam-1 loss-of-function backgrounds (see Fig. S3B). Compared to controls, however, tiam-1T548F mutants showed significantly fewer filiform protrusions during the entire intercalation process (Fig. 2A, quantified in Fig. 2B), suggesting that the main effect of TIAM-1 is on smaller, filiform protrusions.

Fig. 2.

Fig. 2.

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tiam-1 loss-of-function mutants generate fewer protrusions during dorsal intercalation. (A) Mosaic expression of an epidermal-specific F-actin reporter (Plin-26::vab-10::gfp) shows dynamic protrusions during dorsal intercalation in control (left), tiam-1T548F (middle) and tiam-1null (right) embryos. White arrowheads indicate protrusions; yellow lines depict the areas lacking protrusions. Scale bar: 5 µm. (B) Quantification of the number of protrusions generated during early, middle and late stages of dorsal intercalation in control and tiam-1 mutant embryos. Error bars indicate s.e.m. ns, no significant difference; *P<0.05; **P<0.01; ***P<0.001; one-way ANOVA with Tukey's post hoc test.

We next compared tiam-1T548F mutants to homozygotes for a true null allele, tiam-1null. Unexpectedly, although tiam-1null mutants showed fewer filiform protrusions during the early and middle stages of intercalation, they displayed similar levels of filiform protrusive activity compared to that in controls at later stages, i.e. greater levels of filiform protrusive activity than in tiam-1T548F GEF-dead mutants, especially later in dorsal intercalation (Fig. 2A, quantified in Fig. 2B). This result suggested that there might be previously unrecognized inhibitory roles for the N-terminus of TIAM-1.

The N-terminus of TIAM-1 prevents ectopic filiform protrusive activity during dorsal intercalation

Recent work has suggested that the N-terminal domains of TIAM-1 are involved in regulating dendrite development; more specifically, its PDZ domain promotes 4° dendrite branch formation during growth cone morphogenesis (Lin et al., 2022; Tang et al., 2019). We therefore next set out to examine the role of the TIAM-1 N-terminus during dorsal intercalation. Using CRISPR/Cas9, we introduced specific mutations into the tiam-1 locus, including (1) tiam-1ΔN, which removes all N-terminal domains, leaving only the GEF domain intact; (2) tiam-1ΔPDZ; and (3) tiam-1ΔPDZ+T548F, which introduces the GEF-inactivating mutation into the preceding strain (Fig. 3A).

Fig. 3.

Fig. 3.

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TIAM-1 N-terminal deletions lead to ectopic and unpolarized protrusions. (A) Domain maps of TIAM-1 and N-terminal deletions relevant to this study. tiam-1ΔN, truncated protein removing all N-terminal domains, leaving only the GEF domain; tiam-1ΔPDZ, truncated protein removing only the PDZ domain; tiam-1ΔPDZ+T548F, truncated protein removing the PDZ domain and containing the T548F point mutation (asterisk). The black line indicates the lesion in tiam-1(tm1556). (B) Compared to controls, tiam-1 N-terminal deletion mutants displayed ectopic filiform protrusions, which could be partially ameliorated by eliminating the GEF activity of TIAM-1 (tiam-1ΔPDZ+T548F). White arrowheads indicate protrusions. Scale bar: 5 µm. For analysis of orientation of filiform protrusions, see Fig. S1D. (C) Quantification of the number of protrusions generated during early, middle and late stages of dorsal intercalation in control and tiam-1 N-terminal deletion mutants. Error bars indicate s.e.m. *P<0.05; **P<0.01; ***P<0.001; one-way ANOVA with Tukey's post hoc test.

We first assessed the intercalation phenotypes of the various mutants using DIC microscopy. Mutants all showed significantly increased intercalation time (Fig. S3A). We next crossed the tiam-1 mutants with an actin reporter strain to visualize F-actin. We could not detect a statistically significant difference in the base-to-tip length of the main medial protrusion in various tiam-1 loss-of-function backgrounds (Fig. S3B). Although dorsal cells in all mutants eventually produced a main, medially directed lamellipodium, mutant embryos that produced an N-terminally truncated form of TIAM-1 generated significantly more filiform protrusions compared to those in control embryos, specifically during middle to late intercalation, suggesting an inhibitory effect of the N-terminal domains on filiform protrusions at later stages (Fig. 3B, quantified in Fig. 3C). Compared with tiam-1T548F, tiam-1ΔPDZ+T548F mutants generated significantly more filiform protrusions during mid-intercalation (Fig. S3C), suggesting that the TIAM-1 N-terminus contributes to filiform protrusive activity via a previously unknown mechanism that is partly independent of the GEF domain. The N terminal domains of TIAM-1 are also required for normal medial polarization of filiform protrusions; tiam-1 ΔPDZ and ΔN deletion mutants showed significantly reduced polarization compared to that in controls (Fig. S3D). This mispolarization depended in part on TIAM-1 GEF activity, as loss of GEF activity or the GEF domain entirely reverted protrusions to a more normal distribution compared to that in controls (tiam-1ΔPDZ+T548F and tiam-1null were not significantly different from controls; Fig. S3D).

The netrin pathway component UNC-5 is a potential upstream regulator of protrusive activity during dorsal intercalation

TIAM-1 has been implicated in netrin-based signaling in growth cones through its association with the netrin receptor, UNC-40 (DCC) (Chan et al., 1996; Demarco et al., 2012; Fearon et al., 1990). We hypothesized that netrin signaling components might similarly act upstream of TIAM-1 during dorsal intercalation. During neurite outgrowth, the netrin pathway can mediate both attraction and repulsion. In the classic case of C. elegans growth cone guidance, attractive signaling acts via the extracellular ligand UNC-6 (netrin) and UNC-40. Repulsion often requires the UNC-5 receptor in neurons (Mahadik and Lundquist, 2023; Merz and Culotti, 2000; Norris and Lundquist, 2011; Norris et al., 2014) and other cell types (Su et al., 2000; Ziel and Sherwood, 2010), but classic experiments indicate that unc-5 can also act independently of unc-40 (Hedgecock et al., 1990; reviewed in Yam and Charron, 2020). We therefore examined the effects of loss-of-function mutations for unc-6, unc-40 and unc-5 on intercalation time. Although loss of function of all three loci resulted in a statistically significant increase in intercalation time, the most pronounced effect resulted from loss of unc-5 function (Fig. 4A). unc-5 mutants also occasionally displayed mildly aberrant dorsal cell shapes in some embryos.

Fig. 4.

Fig. 4.

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UNC-5 depletion leads to ectopic protrusive activity. (A) Mutants in netrin pathway components unc-5, unc-6 and unc-40 displayed delayed completion of dorsal intercalation. Box plots show the 25–75th percentiles, whiskers show the minimum and maximum, and the median is marked with a line. ns, no significant difference; ***P<0.001; unpaired two-tailed Student's t-test. (B) UNC-5 became peripherally enriched as dorsal intercalation progressed. Error bars indicate s.e.m. ****P<0.0001; unpaired two-tailed Student's t-test. (C) Expression pattern of UNC-5::2×mNG during dorsal intercalation. Yellow arrowheads indicate lateral boundaries of intercalating dorsal cells. Scale bar: 10 µm. (D) Dorsal epidermal cells in a nonsense mutant [unc-5(e152)] exhibit ectopic filiform protrusions during late intercalation that are partially suppressed via tiam-1 knockdown. White arrowheads indicate protrusions. Scale bar: 5 µm. (E) Quantification of the number of protrusions generated during early, middle and late stages of dorsal intercalation in wildtype and unc-5(e152) mutants. Error bars indicate s.e.m. ns, no significant difference; **P<0.01; ***P<0.001; one-way ANOVA with Tukey's post hoc test. (F) Protrusions showed reduced medial polarization in unc-5(e152) mutants, which could be partially rescued by RNAi knockdown of tiam-1. Rose plots of the angle of protrusion relative to the cell centroid at the late stage of dorsal intercalation. Annotated P-values were calculated using the Mardia–Watson–Wheeler test. P-values below the plots compare mutants with wildtype; the P-value above the black line compares unc-5(e152) with unc-5(e152);tiam-1(RNAi).

We next examined localization of UNC-6, UNC-40 and UNC-5 using available knock-in strains (Jayadev et al., 2022). UNC-6 was expressed predominantly ventrally, without detectable signal in the epidermis near or shortly after the time dorsal intercalation occurs (Fig. S4A). UNC-40 was expressed at low levels in dorsal epidermal cells throughout dorsal intercalation, with stronger expression in ventral neuroblasts, including prominent foci (Fig. S4B). In contrast, UNC-5 showed strong expression only in the dorsal epidermis (Fig. S4C). UNC-5 was initially visible in cytosolic granules, which we assume are secretory vesicles. As dorsal intercalation progressed, UNC-5 accumulated at the lateral membranes of dorsal epidermal cells. Strikingly, UNC-5 showed an increased accumulation at the lateral edges of dorsal cells (Fig. 4B,C). The localization pattern of UNC-5 and effects on intercalation time following loss of unc-5 function make unc-5 a promising upstream candidate involved in the previously unreported inhibitory regulatory function of TIAM-1, and so we focused on it for further analysis.

Our previous work had shown that dorsal epidermal cells quickly become polarized at the onset of dorsal intercalation (Walck-Shannon et al., 2015). As the presence of UNC-5 is often associated with growth cone repulsion (Keleman and Dickson, 2001; Mahadik and Lundquist, 2023; Norris and Lundquist, 2011), the localization of UNC-5 in dorsal epidermal cells suggests a simple model: UNC-5 inhibits filiform protrusive activity at membrane surfaces where it is expressed, biasing protrusion towards the leading edge. Consistent with this model, dorsal epidermal cells in embryos homozygous for a nonsense allele of unc-5(e152) exhibited ectopic filiform protrusions (Fig. 4D, quantified in Fig. 4E) that largely lacked polarity (Fig. 4F). In contrast, unc-6(RNAi) and unc-40(RNAi) embryos showed no defects in protrusion formation (Fig. S4D), despite loss of detectable mNG signal (Fig. S4E). Several previous studies uncovered UNC-40-independent functions for UNC-5, based on comparison of the severity of defects between unc-5 and unc-40 mutants (Killeen et al., 2002; MacNeil et al., 2009; Mahadik and Lundquist, 2023). These results, combined with our analysis, suggest that UNC-5 likely has an UNC-40-independent function in inhibiting protrusions during dorsal intercalation.

Considering the similarity of defects shown in unc-5(e152) and tiam-1 N-terminal mutants (ectopic filiform protrusions and loss of medially polarized protrusions at late stages of intercalation) we next assessed whether there was any connection between UNC-5 and TIAM-1 function during dorsal intercalation. RNAi knockdown of tiam-1 in unc-5(e152) mutants rescued the ectopic protrusions observed in unc-5(e152) mutants alone such that their number and distribution were statistically indistinguishable from those of controls (Fig. 4E,F), suggesting that UNC-5 is involved in regulating Rac-dependent filiform protrusions during dorsal intercalation in part via TIAM-1.

The cortical localization of UNC-5 to lateral membranes in dorsal epidermal cells is intriguing and presumably results from contact-mediated or other signaling events. Mechanisms of UNC-5 localization have not been extensively investigated. One study implicates UNC-51 (a serine-threonine kinase) and the RUN domain protein UNC-14 in normal localization of UNC-5 in neurons (Ogura and Goshima, 2006). These two proteins are involved in autophagy in other contexts, suggesting that these proteins regulate trafficking of UNC-5 to the membrane. Whether such trafficking is spatially polarized is not clear and is an interesting area for future study.

In summary, our results indicate that TIAM-1 acts as a Rac-GEF in promoting protrusive activity during dorsal intercalation. Its N-terminus also appears to exert an inhibitory effect on filiform protrusions independent of its GEF activity, specifically during middle to late stages of intercalation. Finally, UNC-5 might act upstream of TIAM-1 during dorsal intercalation independent of UNC-6 and UNC-40, establishing a previously unreported role for this highly conserved receptor in regulating filiform protrusions during directed cell rearrangement.

MATERIALS AND METHODS

Strains

Caenorhabditis elegans strains were maintained on standard nematode growth medium plates seeded with OP50 Escherichia coli at 20°C (Brenner, 1974). Bristol N2 was used as the wild type. Details of strains used in this study can be found in Table S1.

CRISPR/Cas9 genome editing

All novel mutant alleles with the ‘jc##’ designation were generated via plasmid-based CRISPR/Cas9 editing (Dickinson et al., 2015) using repair templates cloned by SapTrap cloning (Schwartz and Jorgensen, 2016). Both deletion mutations (syb4155 and syb4244 alleles) were generated by SunyBiotech (Fujian, China). Guides, homology arm primers and single-stranded repair templates for all CRISPR/Cas9 editing can be found in Table S2.

RNA interference

Feeding RNAi was performed as described by transferring L4 worms to plates seeded with E. coli expressing double-stranded (ds)RNA from the relevant target gene or L4440 (control) vectors overnight (Walston et al., 2004). The following day, embryos were dissected from RNAi-treated hermaphrodites and mounted for imaging.

Injection RNAi was performed as described previously (Walston et al., 2004). dsRNA was generated using the T7 Megascript kit (Invitrogen). dsRNA was injected at a concentration of 2 μg/µl in nuclease-free water. L4 worms were injected and aged overnight before embryos were dissected from mature adults for imaging. The templates for C11D9.1 (tiam-1), F55C7.7 (unc-73), T19B4.7 (unc-40), F41C6.1 (unc-6) and control RNAi were obtained from the Ahringer feeding library (Kamath et al., 2003).

DIC imaging

Four-dimensional DIC movies were collected on either a Nikon Optiphot-2 microscope connected to a QiCAM camera (QImaging) or an Olympus BX50 microscope connected to a Scion CFW-1512M camera using Micro-Manager software (v1.4) (Edelstein et al., 2010, 2014). All embryos were mounted on 10% agar pads in M9 solution as previously described (Raich et al., 1999).

Confocal imaging

Embryos were dissected from adult hermaphrodites and mounted onto 10% agar pads in M9 solution and imaged. For fluorescence imaging of actin reporter lines, a Dragonfly 500 spinning disc confocal microscope (Andor), mounted on a Leica DMi8 microscope equipped with a Zyla camera and controlled by Fusion software (Andor), was used to collect images with 0.3 μm slices with a 100×/1.3 NA oil immersion Leica objective at 20°C. Movies were acquired either over a period of 30 min at 20 s intervals or over a period of 60 min at 1 min intervals. For imaging of unc-6(RNAi) and unc-40(RNAi), the same system was used; images of mNG knock-in strains were collected at 90 s intervals for 60 min using a 63× glycerol immersion Leica objective at 20°C. Focal planes totaling 6 µm in depth were acquired and projected to assess knockdown. For imaging of the tiam-1 transcriptional reporter line, the same system was used, except that an Andor iXon EMCCD camera was used for acquisition to increase sensitivity. Movies were acquired over a period of 120 min at 3 min intervals.

Quantification of dorsal intercalation time

Quantification of intercalation time was performed from DIC movies of embryos mounted on agar pads. For this study, we defined intercalation time as the time between terminal epidermal divisions (specifically, the divisions of Cpaaa and Cpaap) and when cells met contralateral seam cells. At least 20 embryos from at least eight mounts were analyzed per genotype.

Quantification of protrusions

Protrusions were quantified from maximum-intensity projections of 13 z-stacks of F-actin reporter expression as described above. Quantification was performed essentially as described previously (Walck-Shannon et al., 2015). In order to avoid bias in assuming which protrusions were functionally important for migration, protrusion number was obtained by counting aggregations of F-actin reporter signal extending at least 0.2 µm from the cell body. Angles were obtained by comparing the protrusion extension directions relative to the anterior–posterior axis of the embryo in ImageJ. At least ten cells from at least five embryos were analyzed per genotype. We designated cells as ‘early’ if they were in the process of polarizing into a wedge shape, ‘mid’ if the cell tips were ≤2 µm past the dorsal midline, and ‘late’ if the cell tips were ≤2 µm from the contralateral seam cells. The main medial protrusion length in mid-intercalation-stage embryos was determined from actin reporter movies by manually drawing a line selection from the base of the protrusion to its tip using Fiji (https://fiji.sc).

Statistical analysis

Data from control and experimental groups were compared using one-way ANOVA with Tukey’s post hoc testing to assess significance between individual groups using Prism (GraphPad). Protrusion angle-related data were compared using the Mardia–Watson–Wheeler test in R using the Circular package (https://CRAN.R-project.org/package=circular).

Supplementary Material

joces-137-261509_review_history.pdf (303.9KB, pdf)
Supplementary information
joces-137-261509-s1.pdf (4.1MB, pdf)
DOI: 10.1242/joces.261509_sup1

Acknowledgements

The strain tiam-1T548F was provided by the Bülow laboratory. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440).

Footnotes

Author contributions

Conceptualization: Y.Z., J.H.; Methodology: Y.Z.; Validation: Z.T., M.T.; Formal analysis: Y.Z., Z.T.; Investigation: Y.Z., Z.T., M.T.; Resources: J.H.; Writing - original draft: Y.Z.; Writing - review & editing: Y.Z., Z.T., J.H.; Visualization: Y.Z., Z.T.; Supervision: J.H.; Project administration: J.H.; Funding acquisition: J.H.

Funding

This work was supported by grants R01GM127687 and R35GM145312 from the National Institutes of Health awarded to J.H. Deposited in PMC for release after 12 months.

Data availability

All relevant data can be found within the article and its supplementary information.

Peer review history

The peer review history is available online at https://journals.biologists.com/jcs/lookup/doi/10.1242/jcs.261509.reviewer-comments.pdf

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DOI: 10.1242/joces.261509_sup1

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