Bonnard C et al. (2012), Mutations in IRX5 impair craniofacial developme...

XB-ART-45233

Nat Genet 2012 May 13;446:709-13. doi: 10.1038/ng.2259.

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Mutations in IRX5 impair craniofacial development and germ cell migration via SDF1.

Bonnard C , Strobl AC , Shboul M , Lee H , Merriman B , Nelson SF , Ababneh OH , Uz E , Güran T , Kayserili H , Hamamy H , Reversade B .

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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.

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Species referenced: Xenopus laevis
Genes referenced: actb actl6a cxcl12 gata3 irx3 irx5 kcnd2 pgat slc12a3 trps1 twist1
GO keywords: neural crest cell migration [+]

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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.

Figure 4: Irx5 interacts with zinc-finger transcription factors Gata3 and Trps1 to regulate craniofacial morphogenesis. a–c) Irx5 (a), Trps1 (b) and Gata3 (c) were expressed in nested regions of developing X. laevis head at stage (st.) 33. (d) Irx5, Trps1 and Gata3 transcripts (pink) colocalized to ventral region of four branchial arches (1–4), otic vesicle (ov) and frontonasal process (fp). Irx5 and Trps1 (yellow) were coexpressed in most anterior three branchial arches (1–3). Irx5 and Gata3 mRNA (green) were seen in developing lens (le). (e) Irx5 coimmunoprecipitated with wild-type Trps1 (lane 2), and to a lesser extent with the variant18 (lane 4). IP, immunoprecipitation; IB, immunoblot. Irx5 N166K protein was pulled down by wild-type Trps1 (lane 3). (f) Gata3 coimmunoprecipitated with wild-type Irx5 (lane 2), and to a lesser extent with mutant Irx5 N166K (lane 3). (g,h) Trps1 promoted Irx5-dependent transactivation of Kcnd2 promoter luciferase construct. Conversely, Gata3 inhibited Irx5-dependent transactivation of Kcnd2 reporter. Data are mean ± s.d. *P < 0.05; **P < 0.005; one-tailed Student's t test). (i) Increasing amounts of Trps1 dose-dependently enhanced binding of Gata3 to Irx5 (lanes 2–5). (j) Increasing amounts of Trps1 potentiated inhibitory role of Gata3 on Irx5-mediated transactivation of Kcnd2 reporter. Data are mean ± s.d. *P < 0.05; **P < 0.005 (one-tailed Student's t test). (k) Schematic of IRX5 and IRX3 proteins, in complex with comodulators TRPS1 and GATA3, tuning transcription of target genes such as SDF1 for chemoattraction of migratory populations of NCCs and germ cells.

References [+] :

Bawle, Adult and two children with fetal methotrexate syndrome. 1998, Pubmed