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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00609

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Msx1 is required for dorsal diencephalon patterning

Antoine Bach1,*, Yvan Lallemand1,*, Marie-Anne Nicola1, Casto Ramos1,{dagger}, Luc Mathis2, Mathilde Maufras1 and Benoît Robert1,{ddagger}

1 Unité de Génétique Moléculaire de la Morphogenèse, Institut Pasteur, URA 2578 du CNRS, 25 rue du Dr Roux, 75724 PARIS Cedex 15, France
2 Unité de Biologie Moléculaire du Développement, Institut Pasteur, URA 2578 du CNRS, 25 rue du Dr Roux, 75724 PARIS Cedex 15, France



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  		Fig. 1. Abnormalities of the pretectum in Msx1-/- embryos. (A,B) Sagittal sections at the level of the SCO of E15.5 wild-type (A) and homozygous mutant (B) embryos. Nuclei are stained in violet (Hematoxylin) and cytoplasm in pink (Eosin). In the homozygous mutant, the posterior commissure (B, arrowhead) exhibits an abnormal density of nuclei and a disorganization of fiber tracts, while the SCO is absent (arrow). (C) Comparison of Msx1 homozygous mutant with wild type at E11.5. Note the indentation at the caudal diencephalon level (white asterisk). In all panels, anterior is towards the right; Wt, wild type; -/-, Msx1-/- homozygous mutant.

 


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  		Fig. 5. Expression patterns of P1 and P2 markers in wild-type and homozygous mutant embryos at E14.5. In situ hybridization with a Pax7 (A,B), Pax6 (C,D), Lim1 (E,F) or Gbx2 (G,H) probe in wild-type (A,C,E,G) and in Msx1-/- homozygous (B,D,F,H) embryos. (A-F) Dorsal views. In Msx1-/- embryos, Pax6, Pax7 and Lim1 are expressed at the midline (B,D,F, black arrows), from which they are normally excluded (A,C,E). In addition, the dorsolateral domains of expression of these genes are reduced or absent in the entire P1 for Pax6 and in its anterior part for Lim1 and Pax7 (white arrowheads). Note that Pax7 and Lim1 are not downregulated in the caudal part of P1, at the level of the posterior commissure. Black asterisks indicate the position of the dorsal midline (A-F). (G,H) Lateral views. Gbx2 expression is normal in P2 and also in the lateral part of P1 in the Msx1-/- mutant (H). Note the dorsal depression at the level of P1 (H, white asterisk). The telencephalon has been removed to facilitate visualization of the entire diencephalon. In all panels, anterior is towards the right. m, mesencephalon; P1, prosomere 1; P2, prosomere 2; Wt, wild type; -/-, Msx1-/- homozygous mutant.

 


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  		Fig. 2. Interruption of the nlacZ expression domain in Msx1-/- embryos. (A,B) Whole-mount X-Gal stained brains of E14.5 homozygous mutant (A) and heterozygous (B) embryos show that the absence of a functional Msx1 leads to the loss of the Msx1-expressing domain in P1. (C,D) Dorsal views at a higher magnification of the P1 region of E12.5 homozygous mutant (C) and heterozygous (D) embryos after whole-mount X-Gal staining. Black arrows indicate the limits of P1 and white arrows the posterior commissure/SCO primordium region. In all panels, anterior is towards the right. m, mesencephalon; P1, prosomere 1; +/-, Msx1+/- heterozygote;; -/-, Msx1-/- homozygous mutant.

 


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  		Fig. 6. Wnt1 is downregulated before disappearance of the diencephalon midline in the Msx1-/- mutant. Embryos were stained for ß-galactosidase activity (blue staining) before in situ hybridization for Wnt1 (purple staining). At E9.5, some homozygous embryos (B) exhibit a reduced expression of Wnt1 when compared with heterozygotes (A), while the Msx1nlacZ domain is still present (arrowheads). (C,D) At E10.5, more embryos show this phenotype. This suggests that Msx1 is required for the initiation (E9.5) and maintenance (E10.5) of Wnt1 expression. Note that Wnt1 expression in the isthmus (A, i), which derives from the first phase of expression, is not affected in the mutant (B, i). In all panels, anterior is towards the right. d, diencephalon; i, isthmus; m, mesencephalon; +/-, Msx1+/- heterozygote;; -/-, Msx1-/- homozygous mutant.

 


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  		Fig. 3. Expression of midline markers is affected in Msx1-/- mutant embryos. E11.5 wild-type (A,C) and mutant (B,D) embryos were whole-mount hybridized with a Bmp6 (A,B) or a Wnt1 (C,D) probe. Arrowheads in A,B indicate the Bmp6 expression domains in the telencephalic choroid plexus. (E,F) Transverse sections across P1 of E12.5 embryos hybridized with a Pax6 probe. In the mutant, the Pax6 expression domain extends over the midline (F, arrow) from which it is normally excluded (E, arrow)). In A-D, anterior is towards the right. m, mesencephalon; P1, prosomere 1; t, telencephalon; Wt, wild type; -/-, Msx1-/- homozygous mutant.

 


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  		Fig. 4. Cell death is reduced in the diencephalon midline of Msx1-/- mutant embryos. Dorsal views of the diencephalon regions from E11.5 wild-type (A) or Msx1-/- (B,C) embryos. (A,B) Nile Blue staining. Dead cells are abundant (>20) in the midline of wild-type (A) but undetectable in that of Msx1-/- mutant (B) embryos. (C) ß-Galactosidase staining of the same embryo as in B reveals that the diencephalon midline is still present. In all panels, anterior is towards the right. m, mesencephalon; P1, P1 prosomere; t, telencephalon; Wt, wild type; -/-, Msx1-/- homozygous mutant.

 


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  		Fig. 8. Msx genes are necessary for Wnt1 expression. (A,B) Comparison of Wnt1 expression patterns revealed by in situ hybridization between a wild-type (A) and a double homozygous (Msx1-/-/Msx2-/-) mutant embryo (B) at E10.5. Expression is absent in the diencephalon and observed only caudally in the mesencephalon. Note that the midline area of the anterior diencephalon and telencephalon appears abnormal (black arrow). (C,D) Dissected brains from E12.5 Msx1+/- (C) and Msx2+/- (D) embryos stained for ß-galactosidase. Msx1 and Msx2 are co-expressed in the mesencephalon, but the Msx2 expression level is very low in P1. Note that both genes are expressed in the pineal gland (arrowheads). Expression of both genes is prominent in the telencephalic choroid plexus. In all panels, anterior is towards the right. c, telencephalic choroid plexus, m, mesencephalon; pc, posterior commissure; P1, prosomere 1; t, telencephalon; Wt, wild-type.

 


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  		Fig. 7. Ectopic expression of Msx1 induces expression of Wnt1 in the chick brain and lateral ectoderm. (A-D) Dorsal views. Msx1 cDNA in pCIG was electroporated into one side of the diencephalon of chick embryos. Wnt1 is induced (A, arrowhead) in cells that have received pCIG-Msx1 DNA, visualized by GFP (B, arrowhead) that is expressed from the same plasmid. Broken lines delimit the domain in the neuroepithelium that has received plasmid DNA. (C,D) Another example of induction of Wnt1 by Msx1. The mesencephalic neuroepithelial domain where DNA has been transfected (D, arrowhead, green fluorescence) expresses Wnt1 (C, arrowhead). In A,C, the asterisk indicates the endogenous midline domain of Wnt1 expression. (E,F) Lateral view of a chick embryo in which the Msx1 cDNA has been electroporated in the lateral ectoderm, at the mesencephalon level. In E, Msx1 expression is monitored by in situ hybridization (red signal). After washing out the red signal, the same embryos was further processed for in situ hybridization with Wnt1 (F, purple signal). Note that Wnt1 expression is always observed in cells that express Msx1 at a higher level (arrows) and does not extend to neighboring cells. In E,F, anterior is towards the left. d, diencephalon; i, isthmus; m, mesencephalon; t, telencephalon.

 


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  		Fig. 9. A model for the induction and function of Msx genes in the dorsal midline of the diencephalon. (A) Initial induction of Msx expression in the neural folds is the consequence of a Bmp signal from the lateral ectoderm (Liem et al., 1995Go; Lee and Jessell, 1999Go). (B) After closure of the neural tube, Msx genes are expressed at the dorsal midline. They are necessary to induce the expression of Wnt1 in the dorsal midline and maintain the integrity of the dorsal midline cells which are in turn required to provide inductive signals to the dorsolateral domains, characterized by the expression of genes such as Pax3, Pax6, Pax7 and Lim1 in the posterior diencephalon.

 




© The Company of Biologists Ltd 2003