Planar cell polarity acts through septins to control collective cell movement and ciliogenesis

Abstract

The planar cell polarity (PCP) signaling pathway governs collective cell movements during vertebrate embryogenesis, and certain PCP proteins are also implicated in the assembly of cilia. The septins are cytoskeletal proteins controlling behaviors such as cell division and migration. Here, we identified control of septin localization by the PCP protein Fritz as a crucial control point for both collective cell movement and ciliogenesis in Xenopus embryos. We also linked mutations in human Fritz to Bardet-Biedl and Meckel-Gruber syndromes, a notable link given that other genes mutated in these syndromes also influence collective cell movement and ciliogenesis. These findings shed light on the mechanisms by which fundamental cellular machinery, such as the cytoskeleton, is regulated during embryonic development and human disease.

Fig. 1

Fritz and Septins control convergent extension. (A) Cell intercalation (black, white, and gray cells) drives CE, elongating the body axis and closing the blastopore (bp) during gastrulation.(B) Control embryo with closed blastopore (whitecircle). (C) Sibling Fritz morphants fail to close the blastopore (red).(D) Coinjection of GFP-Fritz mRNA rescued blastopore closure (blue). (E) Control embryo. (F) Sibling sept7 morphants fail to close the blastopore (red). (G) Sept7 mRNA rescues closure of blastopore (blue). (H) Quantification of blastopore closure (mean ± SEM; n values for each column, left to right, are as follows: 21, 20, 28, 18, 11, 8, 16, 12, and 18). (I) Still frame from movie of control Keller explant; green arrows indicate mediolaterally aligned cells, alignment of all cells is quantified in the inset diagram. (J) Xdd1, a PCP-specific dominant-negative Dishevelled, disrupts elongation and alignment (red arrows indicate misoriented cells). (K) Fritz morphant cells display defective elongation, but normal polarized alignment. (L) Sept2 morphant cells display defective elongation, but normal polarized alignment. (M) Control cells (movie 1); orange/yellow lines indicate positions of kymographs in m′ and m″. (N) Fritz morphant Keller explant; red arrows indicateintercellular spaces (movie 2). (O) Sept2 morphant Keller explant; red arrows indicate intercellular spaces (movie 3).

Fig. 2

Functional interaction between Fritz and septins. (A) Control embryo withclosed blastopore. (B) Sibling embryo treated with 100 μM FCF. (C) Sibling treated with 200μM FCF. (D) Sibling injected with a low dose of Fritz MO. (E) Sibling Fritz morphant treated with 100μM FCF. (F) Sibling Fritz morphant treated with 200 μM FCF. (G) Quantification of blastopore closure (mean ± SEM; n = nine embryos per column). (H) Sept2-GFP concentrated at the cortex (red in h′) of cells engaged in CE. (I) Cortical sept2-GFP concentration was reduced in Fritz morphants. (J) Sept2-GFP localization was quantified as the ratio of cortical versus cytoplasmic pixel intensities. (K)Cortical/cytoplasmic Sept2-GFP ratio was reduced significantly in Fritz morphants; the ratio of coexpressed membrane-RFP is unchanged (mean ± SEM; n = 123 cells for control, 150 for morphant).

Fig. 3

Fritz controls septin localization, ciliogenesis, and Hedgehog signaling.(A) Control tadpole (anterior view; arrowheads indicate eyes).(B) Sibling Fritz morphant (red arrowhead indicates single, medial eye). (C) Vax1 expression in control embryo.(D) Vax1 expression is lost in a Fritz morphant. (E) Sagittal view of Nkx2.2 expression in the spinal cord of a control embryo, black arrowheads. (F) Nkx2.2 expression is lost in Fritz morphant, red arrowheads. (G) A single multiciliated cell from the Xenopus epidermis. (H) Multiciliated cells in Fritz morphants display fewer and shorter cilia. (I) Sept7 in ring-like structures (inset) at the base of cilia in a confocal slice of a multiciliated cell. (J) Sept7 structures (green; j′) are highly ordered in a stack from a control multiciliated cell: cilia are visible in red. (K) Sept7 structures (green; k′) are disorganized in a Fritz morphant multiciliated cell: few cilia are visible in red. (j″) Z-projection reveals tight association of Sept7 with the apical surface (yellow line); intensity plot is shown at right (red line). (k″) Z-projection reveals loss of sept7 from the apical surface.

Fig. 4

Septins govern ciliogenesis and Hedgehog signaling in developing embryos.(A)Control Xenopus multiciliated cells (B) Cilia are shorter and fewer in number sept7 morphants (C) Sagittal view of the Hedgehog target gene Nkx2.2 in the spinal cord of a control embryo marked by black arrowheads. (D) Nkx2.2 expression is lost in sept2 and sept7 morphant embryos; red arrowheads. (E) Table of mutations in human Fritz (C2ORF86) in BBS and MKS patients. In only two cases (*, **) were mutations present in trans with mutations in a known BBS- or MKS-associated gene.