Functional interactions between Fat family cadherins in tissue morphogenesis and planar polarity

Abstract

The atypical cadherin fat (ft) was originally discovered as a tumor suppressor in Drosophila and later shown to regulate a form of tissue patterning known as planar polarity. In mammals, four ft homologs have been identified (Fat1-4). Recently, we demonstrated that Fat4 plays a role in vertebrate planar polarity. Fat4 has the highest homology to ft, whereas other Fat family members are homologous to the second ft-like gene, ft2. Genetic studies in flies and mice imply significant functional differences between the two groups of Fat cadherins. Here, we demonstrate that Fat family proteins act both synergistically and antagonistically to influence multiple aspects of tissue morphogenesis. We find that Fat1 and Fat4 cooperate during mouse development to control renal tubular elongation, cochlear extension, cranial neural tube formation and patterning of outer hair cells in the cochlea. Similarly, Fat3 and Fat4 synergize to drive vertebral arch fusion at the dorsal midline during caudal vertebra morphogenesis. We provide evidence that these effects depend on conserved interactions with planar polarity signaling components. In flies, the transcriptional co-repressor Atrophin (Atro) physically interacts with Ft and acts as a component of Fat signaling for planar polarity. We find that the mammalian orthologs of atro, Atn1 and Atn2l, modulate Fat4 activity during vertebral arch fusion and renal tubular elongation, respectively. Moreover, Fat4 morphogenetic defects are enhanced by mutations in Vangl2, a 'core' planar cell polarity gene. These studies highlight the wide range and complexity of Fat activities and suggest that a Fat-Atrophin interaction is a conserved element of planar polarity signaling.

Fig. 1.

Loss of Fat4 alters the morphology of kidney, cochlea and caudal vertebra. (A-B′) H&E-stained sections of postnatal day (P) 0 wild-type (A) and Fat4^–/– (B) mouse kidney. Dilated tubular lumens are outlined in light blue in the wild-type (A′) and Fat4^–/– (B′) kidney. Percentages of luminal space relative to total volume of wild-type and Fat4^–/– kidneys are reported in A′ and B′. Rc, renal cortex; Rm, renal medulla. (C-D′) Phalloidin-stained P0 cochlear epithelia from wild-type (C) and Fat4^–/– (D) inner ear. Lengths of wild-type and Fat4^–/– cochlea (white spiral lines) are indicated in C′ and D′. The sensory epithelium was divided into four domains (apex, mid-apical, mid-basal and basal turn) for quantitative analysis, as defined by the white dashed lines in C. (E-F′) H&E-stained transverse sections of a P0 wild-type (E) and Fat4^–/– (F) lumbar vertebra. Vertebrae, composed of vertebral body (Vb) and vertebral arches (Va), are highlighted in light blue in wild type (E′) and Fat4^–/– mutant (F′). Ratios of mediolateral axis (horizontal line) to dorsoventral axis (vertical line) in wild-type and Fat4^–/– animals are reported in E′ (1.42) and F′ (1.49). Dorsal is up and lateral is to the right and left, here and in all subsequent figures. (G-L′) Immunofluorescence with antibodies against the kinocilium marker acetylated α-tubulin (green) and phalloidin staining of actin (red) on P0 wild-type (G,H,I) and Fat4^–/– (J,K,L) cochlear sensory epithelia. Fluorescent images of mid-apical, mid-basal and basal turn are shown in the top, middle and bottom panels. The apex was not analyzed owing to low fidelity of hair cell patterning (Brooker et al., 2006). Outer hair cells (OHCs) are labeled by row number (1-3) in white (G′-L′), with discontinuous ectopic OHC4s (4) in yellow (J′,K′). Scale bars: 500 μm.

Fig. 2.

Vangl2 cooperatively interacts with Fat4 in kidney, cochlea and caudal vertebra. (A-D) H&E-stained longitudinal sections of P0 wild-type (A), Vangl2^Lp/+ (B), Fat4^–/– (C) and Fat4^–/–;Vangl2^Lp/+ (D) mouse kidney. (E-H) Phalloidin-stained cochlear epithelia of P0 wild type (E) and Vangl2^Lp/+ (F), Fat4^–/– (G) and Fat4^–/–;Vangl2^Lp/+ (H) mutants. (I-L) H&E-stained transverse sections of P0 wild-type (I), Vangl2^Lp/+ (J), Fat4^–/– (K) and Fat4^–/–;Vangl2^Lp/+ (L) lumbar vertebra. (M-O′) Immunofluorescence on Fat4^–/–;Vangl2^Lp/+ cochlear sensory epithelium as in Fig. 1G-L′. (P-R) Morphometric analyses of renal tubular dilation (P), cochlear shortening (Q) and vertebral arch broadening (R). Statistical analysis was by two-sample t-test assuming equal variances. Error bars indicate s.d. (S) Frequency plots of gain or loss of OHC patches in the OOC. Phenotypic severity was assessed by the frequency of emergence of ectopic OHC4s (red plots in left panels) or loss of OHC3s (green plots in right panels). Average frequency and s.d. are shown by horizontal and vertical black bars adjacent to the plot, respectively. Statistics was performed by analysis of variance (ANOVA). Scale bars: 500 μm.

Fig. 3.

Fat1 synergizes with Fat4 in kidney and cochlea. (A-D) H&E-stained longitudinal sections of P0 wild-type (A), Fat1^–/+ (B), Fat4^–/– (C) and Fat1^–/+;Fat4^–/– (D) mouse kidney. (E-H) Phalloidin-stained cochlear epithelia of P0 wild type (E) and Fat1^–/+ (F), Fat4^–/– (G) and Fat1^–/+;Fat4^–/– (H) mutants. (I-L) H&E-stained transverse sections of P0 wild-type (I), Fat1^–/+ (J), Fat4^–/– (K) and Fat1^–/+;Fat4^–/– (L) lumbar vertebra. (M-O′) Immunofluorescence on Fat1^–/+;Fat4^–/– cochlear sensory epithelium as in Fig. 1G-L′. (P-R) Morphometric analyses of renal tubular dilation (P), cochlear shortening (Q) and vertebral arch broadening (R). Statistics performed as in Fig. 2P-R. (S) Frequency plots of gain or loss of OHC patches in the OOC. Phenotypic severities were defined and presented as in Fig. 2S. Scale bars: 500 μm.

Fig. 4.

Fat1 synergistically interacts with Fat4 and Fjx1 in the cranial neural tube. (A,B) The number of double mutants with cranial neural tube defects. E18.5 mouse embryos were obtained from Fat1^–/+;Fat4^–/+ (A) or Fat1^–/+;Fjx1^–/+ (B) intercrosses. (C-I) Appearance of Fat1^–/–;Fat4^–/– (C-E), Fat1^–/– (F) or Fat1^–/–;Fjx1^–/– (G-I) E18.5 embryos. (J,L) H&E-stained longitudinal sections of E18.5 Fat1^–/–;Fat4^–/– (J) and Fat1^–/–;Fjx1^–/– (L) kidneys. (K,M) H&E-stained transverse sections of E18.5 Fat1^–/–;Fat4^–/– (K) and Fat1^–/–;Fjx1^–/– (M) lumbar neural tube and vertebra. (N,O) H&E-stained longitudinal sections through wild-type (N) and Fat1^–/–;Fat4^–/– (O) E18.5 heads. Ears are boxed (black dashed lines). Scale bars: 500 μm.

Fig. 5.

Fat3 synergizes with Fat4 in caudal vertebra, but antagonizes Fat4 in kidney and cochlea. (A-D) H&E-stained longitudinal sections of P0 wild-type (A), Fat3^–/– (B), Fat3^–/+;Fat4^–/– (C) and Fat3^–/–;Fat4^–/– (D) mouse kidney. (E-H) H&E-stained transverse sections of P0 wild-type (E), Fat3^–/– (F), Fat3^–/+;Fat4^–/– (G) and Fat3^–/–;Fat4^–/– (H) lumbar vertebra. (I-L) Phalloidin-stained cochlear epithelia of P0 wild-type (I), Fat3^–/– (J), Fat4^–/– (K) and Fat3^–/–;Fat4^–/– (L) inner ear. (M,M′) Immunofluorescence on Fat3^–/– cochlear sensory epithelium as in Fig. 1G-L′. (N-P) Morphometric analyses of renal tubular dilation (N), cochlear shortening (O) and vertebral arch broadening (P). Statistics performed as in Fig. 2P-R. (Q) Frequency plots of gain or loss of OHC patches in the OOC. Phenotypic severities defined and presented as in Fig. 2S. Scale bars: 500 μm.

Fig. 6.

Fjx1 does not interact with Fat4 in kidney, cochlea or caudal vertebra. (A-D) H&E-stained longitudinal sections of P0 wild-type (A), Fjx1^–/– (B), Fat4^–/– (C) and Fat4^–/–;Fjx1^–/– (D) mouse kidney. (E-H) Phalloidin-stained cochlear epithelia of P0 wild-type (E), Fjx1^–/– (F), Fat4^–/– (G) and Fat4^–/–;Fjx1^–/– (H) inner ear. (I-L) H&E-stained transverse sections of P0 wild-type (I), Fjx1^–/– (J), Fat4^–/– (K) and Fat4^–/–;Fjx1^–/– (L) lumbar vertebra. (M-O′) Immunofluorescence on Fat4^–/–;Fjx1^–/– cochlear sensory epithelium as in Fig. 1G-L′. (P-R) Morphometric analyses of renal tubular dilation (P), cochlear shortening (Q) and vertebral arch broadening (R). Statistics performed as in Fig. 2P-R. (S) Frequency plots of gain or loss of OHC patches in the OOC. Phenotypic severities defined and presented as in Fig. 2S. Scale bars: 500 μm.

Fig. 7.

Atn1 antagonizes Fat4 in caudal vertebra. (A-D) H&E-stained longitudinal sections of a P0 wild-type (A), Atn1^–/– (B), Fat4^–/– (C) and Atn1^–/+;Fat4^–/– (D) mouse kidney. (E-H) Phalloidin-stained cochlear epithelia of P0 wild-type (E), Atn1^–/– (F), Fat4^–/– (G) and Atn1^–/–;Fat4^–/– (H) inner ear. (I-L) H&E-stained transverse sections of P0 wild-type (I), Atn1^–/– (J), Atn1^–/+;Fat4^–/– (K) and Atn1^–/–;Fat4^–/– (L) lumbar vertebra. (M,M′) Immunofluorescence on Atn1^–/– cochlear sensory epithelium as in Fig. 1G-L′. (N-P) Morphometric analyses of renal tubular dilation (N), cochlear shortening (O) and vertebral arch broadening (P). Statistics performed as in Fig. 2P-R. (Q) Frequency plots of gain or loss of OHC patches in the OOC. Phenotypic severities defined and presented as in Fig. 2S. Scale bars: 500 μm.

Fig. 8.

Atn2l cooperates with Fat4 in kidney and cochlea. (A-D) H&E-stained longitudinal sections of P0 wild-type (A), Atn2l^om/+ (B), Fat4^–/– (C) and Atn2l^om/+;Fat4^–/– (D) mouse kidney. (E-H) Phalloidin-stained cochlear epithelia of P0 wild-type (E), Atn2l^om/+ (F), Fat4^–/– (G) and Atn2l^om/+;Fat4^–/– (H) inner ears. (I-L) H&E-stained transverse sections of P0 wild-type (I), Atn2l^om/+ (J), Fat4^–/– (K) and Atn2l^om/+;Fat4^–/– (L) lumbar vertebra. (M-O′) Immunofluorescence on Atn2l^om/+;Fat4^–/– cochlear sensory epithelium as in Fig. 1G-L′. (P-R) Morphometric analyses of renal tubular dilation (P), cochlear shortening (Q) and vertebral arch broadening (R). Statistics performed as in Fig. 2P-R. (S) Frequency plots of gain or loss of OHC patches in the OOC. Phenotypic severities were defined and presented as in Fig. 2S. Scale bars: 500 μm.