Evolutionarily ancient association of the FoxJ1 transcription factor with the motile ciliogenic program

Abstract

It is generally believed that the last eukaryotic common ancestor (LECA) was a unicellular organism with motile cilia. In the vertebrates, the winged-helix transcription factor FoxJ1 functions as the master regulator of motile cilia biogenesis. Despite the antiquity of cilia, their highly conserved structure, and their mechanism of motility, the evolution of the transcriptional program controlling ciliogenesis has remained incompletely understood. In particular, it is presently not known how the generation of motile cilia is programmed outside of the vertebrates, and whether and to what extent the FoxJ1-dependent regulation is conserved. We have performed a survey of numerous eukaryotic genomes and discovered that genes homologous to foxJ1 are restricted only to organisms belonging to the unikont lineage. Using a mis-expression assay, we then obtained evidence of a conserved ability of FoxJ1 proteins from a number of diverse phyletic groups to activate the expression of a host of motile ciliary genes in zebrafish embryos. Conversely, we found that inactivation of a foxJ1 gene in Schmidtea mediterranea, a platyhelminth (flatworm) that utilizes motile cilia for locomotion, led to a profound disruption in the differentiation of motile cilia. Together, all of these findings provide the first evolutionary perspective into the transcriptional control of motile ciliogenesis and allow us to propose a conserved FoxJ1-regulated mechanism for motile cilia biogenesis back to the origin of the metazoans.

Figure 1. FoxJ1 from T. adhaerens and S. purpuratus are nuclear localized and can regulate the expression of ciliary genes.

Anti-myc antibodies were used to detect Placozoa (A) and sea urchin (B) FoxJ1 (red, white arrow). Nuclei were stained with DAPI (blue). (C) Expression of dynein intermediate chain in the spinal cord (long arrow) and pronephric (kidney) duct (short arrow) of a wild-type zebrafish embryo. The wdr78 and efhc1 genes are expressed in a similar pattern in wild-type embryos (see Figure 2A and data not shown). Ectopic expression of dynein intermediate chain in embryos ectopically expressing placozoan (D) and sea urchin (E) FoxJ1, respectively. Ectopic expression of wdr78 in embryos ectopically expressing placozoan (F) and sea urchin (G) FoxJ1, respectively. Ectopic expression of efhc1 in embryos ectopically expressing placozoan (H) and sea urchin (I) FoxJ1, respectively. Mis-expression of the different ciliary genes in D–I is indicated by the arrows. Embryos depicted are at 20 hpf, oriented anterior to the left, dorsal to the top.

Figure 2. Zebrafish FoxJ2 and FoxJ3 are unable to induce the expression of ciliary genes.

(A) Expression of efhc1 in the spinal cord (long arrow) and pronephric (kidney) duct (short arrow) of a wild-type zebrafish embryo, and in embryos ectopically expressing zebrafish FoxJ2 (B) and FoxJ3 (C), respectively. (D) Expression of spag6 in the spinal cord (long arrow) and pronephric (kidney) duct (short arrow) of a wild-type zebrafish embryo, and in embryos ectopically expressing zebrafish FoxJ2 (E) and FoxJ3 (F), respectively. Embryos depicted are at 20 hpf, oriented anterior to the left, dorsal to the top.

Figure 3. S. mediterranea foxJ1 genes are expressed in ciliated tissues.

Sense control (A) and expression pattern of Smed-foxJ1-4 depicted in dorsal (B) and ventral view (E). Sense control (C) and expression pattern of Smed-ift172 shown in dorsal (D) and ventral view (F). Expression pattern of Smed-foxJ1-1 (G) and expression pattern of Smed-foxJ1-2 (H). Arrows in B, G and H denote expression in the dorsal stripe of presumptive ciliated sensory cells. Scale bars: 300 µm for A–F and 200 µm for G–H.

Figure 4. S. mediterranea foxJ1-4 is required for the differentiation of motile cilia.

(A–C) Control (A), ift172(RNAi) (B) and foxJ1-4(RNAi) worms (C) 14 days after the last RNAi feeding. Worms shown in panels B and C display tissue edema. Scale bar: 1 mm. (D–F) Anti-α-tubulin staining (green) of the multi-ciliated ventral epithelium in control (D), ift172(RNAi) (E) and foxJ1-4(RNAi) worms (F), respectively. The even spacing of nuclei (magenta) characteristic of the ventral epithelium demonstrates epithelial integrity in E and F. Images are single optical sections. Scale bar: 20 µm. (G) foxJ1-4 expression is unaffected by control RNAi (dsred). (H) foxJ1-1 expression is not altered in a foxJ1-4(RNAi) worm. (I) foxJ1-4 expression is substantially reduced in a foxJ1-4(RNAi) worm. Scale bar: 200 µm.