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

             face development

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.

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

Bawle, Adult and two children with fetal methotrexate syndrome. 1998, Pubmed
Cavodeassi, The Iroquois family of genes: from body building to neural patterning. 2001, Pubmed , Xenbase
Chang, Identification of a submicroscopic 3.2 Mb chromosomal 16q12.2-13 deletion in a child with short stature, mild developmental delay, and craniofacial anomalies, by high-density oligonucleotide array-a recognizable syndrome. 2010, Pubmed
Cheng, The Iroquois homeobox gene, Irx5, is required for retinal cone bipolar cell development. 2005, Pubmed
Chi, Homeodomain revisited: a lesson from disease-causing mutations. 2005, Pubmed
Costantini, The homeodomain transcription factor Irx5 establishes the mouse cardiac ventricular repolarization gradient. 2005, Pubmed
Day, Celsius: a community resource for Affymetrix microarray data. 2007, Pubmed
French, Acquired variation outweighs inherited variation in whole genome analysis of methotrexate polyglutamate accumulation in leukemia. 2009, Pubmed
Gans, Neural crest and the origin of vertebrates: a new head. 1983, Pubmed
García-Moruja, Functional characterization of SDF-1 proximal promoter. 2005, Pubmed
Grigorieva, Gata3-deficient mice develop parathyroid abnormalities due to dysregulation of the parathyroid-specific transcription factor Gcm2. 2010, Pubmed
Hall, The neural crest and neural crest cells: discovery and significance for theories of embryonic organization. 2008, Pubmed
Hamamy, Severe hypertelorism, midface prominence, prominent/simple ears, severe myopia, borderline intelligence, and bone fragility in two brothers: new syndrome? 2007, Pubmed
He, Interaction between transcription factors Iroquois proteins 4 and 5 controls cardiac potassium channel Kv4.2 gene transcription. 2009, Pubmed
Kayserili, ALX4 dysfunction disrupts craniofacial and epidermal development. 2009, Pubmed
Le Douarin, Role of the neural crest in face and brain development. 2007, Pubmed
Lee, Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing. 2009, Pubmed
Malik, Transcriptional repression and developmental functions of the atypical vertebrate GATA protein TRPS1. 2001, Pubmed , Xenbase
Momeni, Mutations in a new gene, encoding a zinc-finger protein, cause tricho-rhino-phalangeal syndrome type I. 2000, Pubmed
O'Connor, SeqWare Query Engine: storing and searching sequence data in the cloud. 2010, Pubmed
Reversade, Mutations in PYCR1 cause cutis laxa with progeroid features. 2009, Pubmed , Xenbase
Rodríguez-Seguel, The Xenopus Irx genes are essential for neural patterning and define the border between prethalamus and thalamus through mutual antagonism with the anterior repressors Fezf and Arx. 2009, Pubmed , Xenbase
Rohmann, Mutations in different components of FGF signaling in LADD syndrome. 2006, Pubmed
Staton, miRNA regulation of Sdf1 chemokine signaling provides genetic robustness to germ cell migration. 2011, Pubmed
Takeuchi, Analysis of SDF-1/CXCR4 signaling in primordial germ cell migration and survival or differentiation in Xenopus laevis. 2010, Pubmed , Xenbase
Theveneau, Collective chemotaxis requires contact-dependent cell polarity. 2010, Pubmed , Xenbase
Tian, Loss of CHSY1, a secreted FRINGE enzyme, causes syndromic brachydactyly in humans via increased NOTCH signaling. 2010, Pubmed
Van Esch, GATA3 haplo-insufficiency causes human HDR syndrome. 2000, Pubmed
Wilkie, Genetics of craniofacial development and malformation. 2001, Pubmed