RhoA GEF Mcf2lb regulates rosette integrity during collective cell migration

ABSTRACT Multicellular rosettes are transient epithelial structures that serve as important cellular intermediates in the formation of diverse organs. Using the zebrafish posterior lateral line primordium (pLLP) as a model system, we investigated the role of the RhoA GEF Mcf2lb in rosette morphogenesis. The pLLP is a group of ∼150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA-sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This resulted in an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are properly polarized. In contrast, RhoA activity, as well as signaling components downstream of RhoA, Rock2a and non-muscle Myosin II, were diminished apically. Thus, Mcf2lb-dependent RhoA activation maintains the integrity of epithelial rosettes.

(A) Heatmap illustrating pLLP module scores for GO terms that are associated with various processes involving actin.(B) Regulation of actin cytoskeleton KEGG pathway map shows presence (red star) of molecules that are associated with of this process in our scRNA-seq data set.

Fig. S2. Expression of genes that regulate actin dynamics in pLLP clusters. (A-C) Using
our scRNA-seq data set, we performed GO term enrichment analysis to reveal expression of actin-binding genes (A), Rho GTPases, GAPS, and GEFs (B), and actin polymerization genes (C) among the three pLLP specific clusters.Note enhancement of expression of actin-binding genes and actin polymerization genes in the follower population compared to the leaders and proliferating cells.

Fig. S7. Subclustering of pLLP cells at different resolutions.
A resolution sweep from 0.1 to 1 at 0.1 increments identified resolution 0.3 as optimally maximizing silhouette width.However, we noted that at this resolution, follower cells (cluster 0 in A) separated into two clusters that have extremely similar gene signatures (cluster 2 and 3 in B): compare gene signature for clusters 2 and 3 (D).This gene signature also contained 9 out of 10 genes from cluster 0 (C).

Fig. S1 .
Fig. S1.GO term and KEGG analysis of the scRNA-seq data set for genes that regulate actin dynamics.

Fig. S5 .
Fig. S5.Hair cells are properly specified in the pLL of mcf2lb mutant embryos.(A -D) In situ hybridization of the hair cell markers atoh1a (A, B) and deltaA (C, D) in WT and mcf2lb mutant pLLs at 48 hpf.Dotted lines indicate NM. (E, F) 3D reconstruction of hair cells in NM L5 in WT and (F) mcf2lb mutants at 3dpf.(G) Quantification of hair cell volume in WT (n = 24 hair cells from 12 NMs) and mcf2lb mutants (n = 20 hair cells from 10 NMs).(H) Quantification of apical width of hair cells in WT (n = 21 hair cells from 10NMs) and mcf2lb mutants (n = 20 hair cells from 10NMs).(I) Quantification of basal width of hair cells in WT (n = 21 hair cells from 10NMs) and mcf2lb mutants (n = 20 hair cells from 10NMs).** = p < 0.01 (G, I unpaired two tailed t-test; H Mann -Whitney U test) Error bars are SD.A-D: Scale bars = 5 μm.E, F: Scale bars = 5 μm.

Fig. S6 .
Fig. S6.Microlumen integrity is compromised in the mcf2lb mutant pLLP and differentiating NMs.LexOP-secGFP plasmid was injected into TgBAC(Cxcr4b:LePR; LexOP:memRFP) transgenic fish.LexPR was induced for 6 hours (29-35 hpf) to induce expression of secGFP (green) and memRFP (magenta).(A, B) secGFP expression in the trailing rosette of WT and mcf2lb mutant pLLPs.Solid ovals denote presumptive microlumen and dotted line marks the trailing rosette.(A', B') Higher magnification of the rosettes outlined in panels A and B. (C, D) secGFP expression in deposited NM2 of WT and mcf2lb mutant embryos (presumptive microlumen is outlined).(E) Quantification of fluorescence intensity of secGFP in the microlumen normalized to the fluorescence intensity of cells expressing secGFP that contribute to the rosette centers of trailing rosettes and deposited NM2 in WT (n = 7 microlumen from 7 embryos) and mcf2lb mutants (n = 6 microlumen from 5 embryos).***p < 0.001 (unpaired two tailed t-test).Error bars are SD.Scale bars in panels A, B= 10 μm and in panels A', B', C, C', D, and D' = 5 μm.

Table S1 .
Key Resources Table