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A role for iro1 and iro7 in the establishment of an anteroposterior compartment of the ectoderm adjacent to the midbrain-hindbrain boundary

Motoyuki Itoh, Tetsuhiro Kudoh, Michael Dedekian, Cheol-Hee Kim and Ajay B. Chitnis*

Laboratory of Molecular Genetics, NICHD/NIH, Bethesda, MD 20892, USA



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  		Fig. 1. Sequence alignment of zebrafish and mouse Iroquois proteins. (A) Schematic structure of Iro1 and Iro7. HD, homeodomain; IRO, Iro box. (B) Alignment of zebrafish Iro1, Iro3, Iro5 and Iro7 (ziro1, 3, 5 and 7), and mouse Irx1-Irx6 (mIrx1-Irx6) in part of the N-terminal domain and the homeodomain (blue shaded box in A); broken line represents the homeodomain. (C) Alignment of zebrafish and mouse Iro genes in the IRO box domain (green shaded box in A). Brackets in the right margin show orthologs and paralogs with most similarity (B,C). (D) Percentage similarity of amino acids between mouse Irx (MUS IRX1-IRX6) and zebrafish Iro (ZEF Iro1, Iro3, Iro5 and Iro7) proteins in the region shown in B. Each mouse Irx from the IrxA cluster is shown together with its paralog from the IrxB cluster.

 


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  		Fig. 2. Expression patterns of iro1 and iro7 at early embryonic stages. (A-I) iro1 expression. (J-R) iro7 expression. (A-C,J-L) Dorsal view of iro1 and iro7 expression at blastula and gastrula stages. Anterior is towards the top. iro1 and iro7 show similar patterns of expression until the late gastrula stage. (D-G,M-P) Dorsal view of iro1 and iro7 expression at the tailbud stage (TB) and early segmental stages (2s, 2 somites; 5s, 5 somites), anterior towards the left. (G,P) Viewed from side. Expression of iro1 and iro7 begins to diverge by the end of the gastrula stage. At 24 hours post fertilization (24h) (H,I,Q,R), expression of iro1 is broad, while iro7 expression is much more restricted (H,Q, dorsal view; I,R, side view).

 


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  		Fig. 3. Double in situ hybridization defining the domains of iro1 and iro7 expression. Expression patterns of iro1 (A-D,I-L) and iro7 (E-H,M-P). (A-H) Double in situ hybridization with otx2 (A,E), with pax2.1 (B,F), with gbx1 (C,G), and with hoxb1b (D,H) at 80% epiboly. (I,M) At the tailbud stage, iro1 and iro7 are expressed in partially overlapping patterns with ngn1. (J,N) neural crest cells marked by fkd6 expression are within a domain where iro1 and iro7 are expressed. (K,O) By early somitogenesis, expression of iro1 and iro7 is excluded from the MHB region marked by pax2.1. (L,P) iro1 and iro7 are expressed in different locations in the hindbrain: iro1 is expressed in rhombomere 1 and caudally from rhombomere 5, while iro7 is expressed in rhombomeres 3 and 4. (A-P) Anterior towards the top, dorsal view.

 


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  		Fig. 4. Rostral expansion of trigeminal ganglia, the MHB domain, and the premigratory neural crest cells correlates with expansion of iro1 and iro7 in headless (hdl) mutants. (A,B) Expression of iro1 (A) and iro7 (B) is expanded rostrally in hdl mutants at the tailbud stage. (C-E) The trigeminal ganglia (C), MHB domain (D) and neural crest (E), marked respectively by ngn1 expression at the one-somite stage (C), pax2.1 at the three-somite stage (D) and fkd6 at the tailbud stage (E), are expanded rostrally in hdl mutants. Expression of krox20 (krx20) in the hindbrain is unaffected in hdl (D). Red arrowheads indicate anterior limit of expression of each gene in wild-type embryos for comparison.

 


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  		Fig. 5. The role of iro1 and iro7 in neurogenesis. (A-H) A knock-down of iro1 and iro7 with morpholino antisense oligos MO1 and MO7 reveals that iro7 is essential for expression of the ngn1 in trigeminal ganglia. Expression of ngn1 in trigeminal ganglia (black arrowhead) is similar in uninjected (A) and MO1-injected embryos (B). Injection of MO7 leads to a loss of ngn1 expression in the trigeminal ganglia (C, open arrowhead). The knockdown of iro7 by MO7-injected at the one-cell stage is rescued on one side by co-injection with {Delta}Niro7 mRNA (D). ngn1 expression in the trigeminal is not lost (red arrowhead) on the side injected with {Delta}Niro7 and lacZ mRNA, while it is reduced on the side MO7 was injected without {Delta}Niro7 RNA (open arrowhead). The distribution of co-injected mRNA is visualized with a blue X-gal reaction product. (E-G) Examination of embryos at 24 hpf with an acetylated {alpha}-tubulin antibody reveals trigeminal neurons (black arrowhead) in uninjected and MO1-injected embryos but not in MO7-injected embryos (G, open arrowhead). (H,I) Expression of ngn1 in trigeminal ganglia in hdl mutants (H, black arrowhead) is expanded to the anterior but its expression is lost in MO7-injected hdl mutants (I, open arrowhead). (J) Antisense morpholinos specifically block iro1 and iro7 translation. Radiolabeled proteins, Iro1 and Iro7, were synthesized simultaneously in vitro in the presence of an increasing log molar ratio (101-104) of morpholinos, iro1 (MO1) or iro7 (MO7), and were run out on a SDS-PAGE gel. Lane 1: control, no morpholino. Increasing amounts of MO1 (lane 2-5) and MO7 (lane 6-9) lead to a specific reduction in the synthesis of Iro1 and Iro7 protein, respectively. (K) Structure of two artificial constructs; top, wild-type iro7; middle, VP16-iro7HD, the homeodomain of iro7 was fused to the activator region of VP16 herpes simplex virus (blue box); bottom, En-iro7HD, the homeodomain of iro7 was fused to the Drosophila Engrailed repressor region (red box). Purple box represents acidic region; green represents the Iroquois box. (L-O) Ectopic expression of iro1 and iro7 mRNA induces relatively broad ngn1 expression in the ventral ectoderm (arrowheads in L,M). En-iro7HD mRNA induces ngn1 expression in a salt-and-pepper pattern in the ventral ectoderm (N), while VP16-iro7HD mRNA is more effective at inducing broad ngn1 expression within the neural plate (O). (L-O) Anterior is towards the left, side view. Broken lines show the boundary between neural plate and ventral ectoderm. Embryos are at the three-somite stage. Distribution of ectopic mRNA is marked by red salmon-Gal staining to detect co-injected nuclear ß-galactosidase activity. (P-S) Expression of gata2 is reduced in iro1 (Q), iro7 (R), En-iro7HD (S) mRNA injected embryos when compared with uninjected control embryos (P). Embryos are at tailbud stage and viewed from ventral side.

 


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  		Fig. 6. iro1 and iro7 act together as repressors in neural crest formation. A combination of both morpholinos (MO1+7) causes a strong reduction of fkd6 expression at an early somite stage (D) compared with either single morpholino MO1 (B), MO7 (C) or uninjected control embryos. (E) fkd6 expression is recovered (red arrowhead) by {Delta}Niro1 and {Delta}Niro7 mRNA co-injection in MO1+MO7-injected embryos. (F, G) Expression of fkd6 in hdl mutants (F) is expanded to the anterior but its expression is lost in MO1 and MO7 co-injected hdl mutants (G). Injection of iro1 (H) and iro7 (I) mRNA does not induce ectopic fkd6 expression; however, En-iro7HD mRNA induces a little ectopic fkd6 expression in the anterior neuroectoderm (J, arrowhead). By contrast, injection of VP16-iro7HD mRNA represses endogenous expression of fkd6 (K). The distribution of injected mRNA is visualized with red salmon-Gal staining. All embryos are viewed from dorsal side and anterior is towards the left.

 


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  		Fig. 7. iro1 and iro7 act together as repressors in formation of the MHB domain, while ectopic iro1 or iro7 induces the ectopic expression of MHB genes. (A-E) The effects of the iro1 (MO1) and iro7 (MO7) morpholinos on expression of MHB markers eng2 (A), pax2.1 (B), fgf8 (C), wnt1 (D) at 24 hpf and pax2.1 (E) at tailbud stage. Red arrows show reduction of MHB markers. (F) Expression of pax2.1 in hdl mutants (left) is expanded to the anterior but its expression is reduced in MO1 and MO7 co-injected hdl mutants (right). (G) Expression of pax2.1 is recovered (red arrowhead) by {Delta}Niro1 and {Delta}Niro7 mRNA co-injection in double morpholino-injected embryos. (H-J) Embryos injected with iro1 mRNA or En-iro7HD mRNA showed ectopic expression of pax2.1 and fgf8, but not wnt1 (F,H), while iro7 mRNA induces fgf8 and reduces wnt1 (G). The distribution of injected mRNA is marked by red staining. (K-M) VP16-iro7HD mRNA injected embryos show loss of pax2.1 expression (K) at tailbud stage and the isthmus at the MHB region (L, arrowhead) and loss of fgf8 expression (M) at 24 hpf. Anterior is towards the left (A-M). Embryos are viewed from the left side (A-D,M) or dorsal (E-L).

 

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© The Company of Biologists Ltd 2002