Using homozygosity mapping and locus resequencing, we found that alterations in the homeodomain of the IRX5 transcription factor cause a recessive congenital disorder affecting face, brain, blood, heart, bone and gonad development. We found through in vivo modeling in Xenopus laevis embryos that Irx5 modulates the migration of progenitor cell populations in branchial arches and gonads by repressing Sdf1. We further found that transcriptional control by Irx5 is modulated by direct protein-protein interaction with two GATA zinc-finger proteins, GATA3 and TRPS1; disruptions of these proteins also cause craniofacial dysmorphisms. Our findings suggest that IRX proteins integrate combinatorial transcriptional inputs to regulate key signaling molecules involved in the ontogeny of multiple organs during embryogenesis and homeostasis.
PubMed ID: 22581230
Article link: Nat Genet.
Genes referenced: actl6a gata3 irx3 irx5 kcnd2 slc12a3 trps1 twist1
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|Figure 3: Irx5 orchestrates migration of cranial NCCs and primordial germ cells by repressing Sdf1 expression. (a–e) Cranial NCC migration patterns visualized by Twist1 expression in X. laevis embryos. Lateral view of head region of stage 26 embryos, anterior to the left. (a) Expression of Twist1 demarcated NCCs in four branchial arches (1–4; n = 36/36). Dashed red line outlines eye vesicle and dashed white line outlines first branchial arch. Scale bar, 0.25 mm. (b) Irx5 morpholino (MO)-injected embryos specifically lacked NCCs in first branchial arch (n = 23/38). Red arrowhead, ectopic NCC migration over eye vesicles. (c) Injection of wild-type mouse Irx5 DNA in Irx5 morphant embryos rescued NCC migration to first branchial arch (n = 30/40). (d) Injection of DNA encoding mouse Irx5 A150P in Irx5 morphant embryos did not rescue NCC migration to first branchial arch (n = 27/48). (e) Injection of DNA encoding mouse Irx5 N166K partially rescued NCC migration in Irx5 morphant embryos (n = 23/48). (f) Irx5 A150P protein was not detected in embryos injected with DNA encoding mouse Irx5 A150P compared to embryos injected with DNA encoding wild-type mouse Irx5. α-actin, loading control. Quantitative RT-PCR results show comparable transcription for each Irx5 DNA construct injected. Actb, positive control. (g,h) At stage 25, Sdf1 was markedly overexpressed in head region of Irx5 morphants relative to control embryos (n = 18/18). Scale bar, 0.25 mm. (i) Injection of a human SDF1 promoter reporter showed greater basal luciferase activity in Irx5-depleted embryos. Overexpression of mouse Irx5 in MS5 cells repressed transactivation of SDF1 reporter. Data are mean ± s.d. *P < 0.05 (one-tailed Student's t test). (j) Twist1 expression demarcated NCCs in four branchial arches (1–4; n = 70/70). Dashed red line, eye vesicle. Scale bar, 0.25 mm. (k) Sdf1 morpholino–injected embryos did not show overt NCC migration defects (n = 46/46). (l) Irx5 morpholino–injected embryos lacked NCCs in first branchial arch (n = 37/58). Dashed black line, absence of first branchial arch. (m) Reduction of Sdf1 by injection of Sdf1 morpholino rescued NCC migration to first branchial arch of Irx5 morphants (n = 42/58). (n) Defective migration of primordial germ cells marked by Xpat was seen in Irx5 morphant embryos (n = 33/40). Scale bar, 0.5 mm.|