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Hindbrain patterning: Krox20 couples segmentation and specification of regional identity

Octavian Voiculescu1, Emmanuel Taillebourg1, Cristina Pujades1,*, Chantal Kress2, Stephanie Buart1, Patrick Charnay1,{dagger} and Sylvie Schneider-Maunoury1

1 Laboratoire de Biologie Moléculaire du Développement, INSERM U368, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France
2 Laboratoire de Biologie du Développement, CNRS URA 1960, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
* Present address: Biologie Moléculaire et Cellulaire du Développement, UMR CNRS 7622, Université Pierre et Marie Curie, 9 Quai St. Bernard, 75252 Paris Cedex 05, France



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  		Fig. 1. Comparison of reporter gene expression in Krox20 heterozygous embryos. Reporter gene expression was assayed by X-gal staining in Krox20lacZ/+ (A,C,E,G) and Krox20Cre/+ R26R (B,D,F) embryos, and by AP staining in Krox20Cre/+ Z/AP embryos (H-J). Embryos shown in I,J were processed for Krox20 ISH (brown) after AP staining. (A-H) Hindbrains of whole-mounted embryos with rostral towards the left. (I,J) Flat-mounted hindbrains with rostral towards the top. From the 14-15 s stage onward, neural crest cells exiting r5 are positive for the reporter gene only in the Krox20lacZ/+ embryo (arrowhead in C). Derivatives of the r5 neural crest are labelled in the Krox20Cre/+ R26R embryo at 9.5 dpc in the third branchial arch (BA3) (arrowheads in F), and at 10.5 dpc in BA3 and in the superior ganglion of the glossopharyngeal nerve (IX) (arrow in H). At 10.5 dpc, in the Krox20lacZ/+ embryo (G), lacZ expression is detected in the boundary cap cells of the trigeminal (Vbc) and facial (VIIbc) nerves (arrow in G).

 


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  		Fig. 2. Fate mapping in Krox20-null embryos. Reporter gene expression was assayed by X-gal staining in Krox20lacZ/lacZ (A,C), Krox20lacZ/Cre R26R (B,D,F) and Krox20Cre/+ R26R (H) embryos and by AP staining in Krox20lacZ/Cre Z/AP (E) and Krox20Cre/+ Z/AP (G) embryos of the indicated stages. (A-E,G) Flat-mounted hindbrains with rostral towards the top; (F,H) Parasagittal sections of the hindbrain region with rostral towards the left. In B, expression from the Krox20lacZ allele and from the R26R reporter are superimposed in r5. In C, labelled neural crest (nc) cells that migrate rostrally and caudally to r5 have not been totally removed. In D, note that r3 blue cells are localised both rostral and caudal to the r2/r4 boundary (visible on the right side). In G, note that an unstained territory corresponding to r4 is present but is not observed on the picture because the rhombomeres are not perfectly perpendicular to the AP axis at this stage.

 


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  		Fig. 3. Cell death and proliferation in hindbrains of wild type and Krox20 mutant embryos. (A-C) Flat mounted hindbrains of 12 s wild type (A), Krox20lacZ/+ (B) and Krox20lacZ/lacZ (C) embryos stained with X-gal (blue) and processed for H3P immunohistochemistry (brown). (D,E) Coronal sections of 14 s Krox20lacZ/+ (D) and Krox20lacZ/lacZ (E) embryos whose mothers were injected with BrdU 1 hour before sacrifice. The sections were stained with X-gal (blue) and processed for BrdU immunohistochemistry (brown). (F-M) Flat mounted hindbrains of 8 s (F,G), 12 s (H,I), 15 s (J,K) and 9.5 dpc (L,M) Krox20lacZ/+ (F,H,J,L) and Krox20lacZ/lacZ (G,I,K,M) embryos stained for ß-galactosidase activity (blue), and for cell death by the TUNEL method (brown or purple). (N,O) Dissected hindbrains of 9.5 dpc wild-type (N) and Krox20lacZ/lacZ (O) embryos stained for cell death with Nile Blue Sulphate. Black arrowheads in F,H,I,J,K,M,O point to regions of intense cell death. ov, otic vesicle. Rostral is towards the top.

 


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  		Fig. 4. Modification of rhombomere identity of r3 cells in Krox20 mutant embryos. (A-C) Flat-mounted hindbrains of 8 s Krox20+/+ (A), Krox20lacZ/+ (B), or Krox20lacZ/lacZ (C) embryos carrying the AP transgene expressed in r2 (r2AP), stained for AP activity and processed for Hoxb1 ISH (both purple). (D) Hindbrain of an 8 s Krox20lacZ/lacZ whole embryo labelled with X-gal for 12 hours to show the presence of r3 cells at this stage. (E) r2-r3 region of the flat-mounted hindbrain of a Krox20+/+ r2AP embryo at the 10 s stage, processed for Krox20 protein immunochemistry (orange) and AP activity (purple). (F) The r2/r4 region of the flat-mounted hindbrain of an 8 s Krox20lacZ/lacZ r2AP embryo, stained for X-gal (blue) and AP activity (purple). The inset shows a high magnification of three cells, one of which is a double stained cell. (G) The r2/r4 region of the flat-mounted hindbrain of an 8 s Krox20lacZ/lacZ embryo stained with X-gal (blue) and processed for Hoxb1 ISH (brown). (H) r3/r5 region of the flat-mounted hindbrain of a 9.5 dpc Krox20Cre/lacZ R26R embryo stained with X-gal (blue) and processed for Hoxb1 ISH (purple). The arrowheads in F-H point to cells labelled by both markers. Rostral is towards the top.

 


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  		Fig. 5. Modification of rhombomere identity of r5 cells in Krox-20 homozygous embryos. (A,B) Flat-mounted hindbrains of wild-type (A) and Krox20lacZ/lacZ (B) embryos at the 8 s stage processed for ISH with probes for Hoxb1 and Cdh6 probes (both purple). (C,D) Flat-mounted hindbrains of 13 s Krox20lacZ/lacZ embryos stained with X-gal (C) or stained with X-gal and processed for Mafb/kr ISH (D). (E,F) Flat-mounted hindbrains of wild-type (E) and Krox20lacZ/lacZ (F) 20 s embryos processed for Mafb/kr ISH. Rostral is towards the top.

 


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  		Fig. 6. Analysis of cell identity and cell mingling in embryonic chimaeras. All the pictures show flat-mounted hindbrains. (A) Combined X-gal staining and NeoR ISH performed on a 15 s wild-type/Krox20lacZ/lacZ chimaeric embryo. The white arrowheads point to groups of blue cells in even-numbered rhombomeres. (B) Combined X-gal staining and NeoR ISH performed on a 18 s wild-type/Krox20lacZ/+ chimaeric embryo. (C) X-gal staining of a 14 s chimaeric embryo between wild-type and Epha4/lacZ transgenic lines. (D) Epha4 ISH performed on a 15 s wild-type embryo. (E,F) Combined X-gal staining and Epha4 ISH performed on 15 s (E) and 12 s (F) wild-type/Krox20lacZ/lacZ chimaeric embryos. (G) Combined X-gal staining and NeoR ISH performed on a 9.5 dpc wild-type/Krox20lacZ/Cre R26R chimaeric embryo. (H) Combined X-gal staining and double Krox20 (orange) and Hoxb1 (purple-brown) ISH performed on a 14 s wild-type/Krox20lacZ/lacZ chimaeric embryo. (I) Combined X-gal staining and Hoxb1 ISH performed on a 12 s wild-type/Krox20lacZ/lacZ chimaeric embryo.

 


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  		Fig. 7. Schematic model for hindbrain segmentation in wild-type and Krox20–/– embryos. (A) Wild-type situation. At the 1-5 s stages, Krox20 is activated in a few scattered cells in prospective r3, and in a more coherent group of cells in prospective r5 (light blue circles). Hoxb1 (green) and Mafb/kr (Kr) (orange), and the Hoxa2 (r2) (yellow) enhancer are also activated. Additional cells are subsequently recruited to express Krox20, probably by a non cell-autonomous autoactivation process (light blue arrows). At the 8-10 s stage, prospective r3 and r5 now express Krox20 homogeneously, and continue to expand by cell recruitment at the expense of r2/r4 (for r3) and of r6 (for r5). In addition, an autoregulatory loop (curved arrows) leads to enhancement of Krox20 expression, presumably in a cell-autonomous manner. Krox20 expression in these cells leads to acquisition of r3/r5 molecular identity (Epha4, Hoxa2-r3/r5 enhancer activation) and repression of even-numbered rhombomere molecular identity (e.g. Hoxb1, Hoxa2-r2 enhancer repression). At the 12 s stage, r3 and r5 express Krox20 at high levels (dark blue) and have acquired r3/r5 identity, including cell mingling properties. This leads to the sorting of even- and odd-numbered rhombomere cells, and sharpening of gene expression limits. At the 20-25 s stage, boundaries are morphologically conspicuous. (B) In Krox20-null mutants, at the 1-5 s stages, early activation of Krox20 occurs normally (light blue circles). However, the Krox20-expressing territories do not expand, because of defective cell recruitment based on non cell-autonomous autoregulation. At the 8-10 s stage, Krox20-expressing cells are still scattered. They have acquired even-numbered rhombomere identity and are incorporated into adjacent even-numbered territories, namely r2 and r4 for cells that should belong to r3, and r6 for cells that should give rise to r5. At the 12 s stage, Krox20 gene expression is not maintained in these cells, owing to impaired cell autonomous autoregulation. Finally, at the 20-25 s stage, cell death in the even-numbered rhombomeres leads to a significant size reduction of the hindbrain.

 

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© The Company of Biologists Ltd 2001