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Requirement for downregulation of kreisler during late patterning of the hindbrain

Thomas Theil^*, Linda Ariza-McNaughton, Miguel Manzanares, Jim Brodie, Robb Krumlauf and David G. Wilkinson^¶

Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK
^* Present address: Developmental and Molecular Biology of Animals, Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
Present address: The Sanger Centre, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK
Present address: Instituto Cajal, CSIC, Av. Doctor Arce 37, 28002 Madrid, Spain
Present address: Stowers Institute for Medical Research, 1000 East 50th, Kansas City, Missouri 64110, USA

View larger version (67K): [in a new window]   Fig. 1. Ectopic expression of kreisler in the hindbrain of transgenic mice. (A) Construct for the generation of transgenic mice. An EphA4 r3/r5 enhancer in combination with a ß-globin minimal promoter (ßG) is used to drive expression of a kreisler mini gene in the hindbrain. (B-D) In situ hybridisation analysis of wild-type and transgenic embryos with a kreisler probe. (B) In wild-type E9.0 embryos, kreisler transcripts can be detected in r5/r6, in neural crest cells migrating to the third branchial arch and in the roof plate of the neural tube (arrow). (C) Dorsal view of an age-matched transgenic kreislerr3/r5 littermate showing additional kreisler expression in r3 and elevated expression levels in r5. (D) Side view of the same embryo shown in C revealing a flexure of the neural tube at the level of r3.

View larger version (75K): [in a new window]   Fig. 2. Segmental expression in the hindbrain has not been affected in kreislerr3/r5 mice. Wild-type (A-C) and kreislerr3/r5 embryos (D-F) were analysed for the expression of several marker genes. Whole-mount in situ hybridisation for Hoxb1 revealed its expression in r4 of both wild type (A) and kreislerr3/r5 embryos (D). Expression of an alkaline phosphatase reporter gene under the control of an r2 enhancer remains confined to r2 in wild-type (B) and transgenic embryos (E). Transcripts of the neuregulin gene can be detected in the dorsal part of the even-numbered rhombomeres and of r1 (C). This pattern of expression is maintained in kreislerr3/r5 mice (F); the weaker signal is due to the staining having been carried out for less time.

View larger version (67K): [in a new window]   Fig. 3. Expression analysis of r5 specific marker genes. Dorsal view of wild type (A-C; G-J) and kreislerr3/r5 embryos (D-F; K-N). (A,D) 16s embryos hybridised with Fgf3 antisense RNA showing weak ectopic activation in r3 of kreislerr3/r5 embryos (D). (B,E) Fgf3 expression in E10.0 embryos. Ectopic transcripts can be detected in r3 and prolonged expression occurs in r5. (C,F) E10.0 embryos analysed for Krox20 expression. By this time point Krox20 expression is completely downregulated in r3 of wild-type embryos, but remains in kreislerr3/r5 embryos. (G,K) E9.25 embryos hybridised with Hoxa3 antisense RNA. In wild-type mice, Hoxa3 is expressed up to the r4/r5 boundary (G), kreislerr3/r5 embryos show an additional expression domain in r3 (K). (H,L) In situ hybridisation analysis using an Hoxb3 probe. In kreislerr3/r5 mutants, Hoxb3 is ectopically activated in r3. (I,M) Activity of a lacZ reporter gene under the control of a Hoxa3 regulatory element is confined to r5 and r6 in wild-type embryos, whereas kreislerr3/r5 embryos show additional activation in r3. (J,N) Similarly, activity of a Hoxb3 r5 enhancer is ectopically activated in r3 in kreislerr3/r5 embryos.

View larger version (96K): [in a new window]   Fig. 4. Disruption of fbm neuronal migration in kreislerr3/r5 embryos. The expression pattern of neuronal markers and location of fbm neurones is shown in dorsal views and sections of embryos after in situ hybridisation or ß-galactosidase staining of lacZ reporter line. (A-D) Dorsal views of Isl1 expression in wild-type embryos at E11.5 (A) and kreislerr3/r5 embryos at E11.5 (B,C) and E12.5 (D). (E,F) Ret expression detected in coronal (E) and longitudinal (F) sections through hindbrain of E12.5 embryos. (G-I) Hoxb1/lacZ reporter gene expression in wild-type (G) and kreislerr3/r5 (H, dorsal view; I, sagittal longitudinal section) embryos at E12.5. (J-L): Dorsal views of Phox2b expression in wild-type (J) and kreislerr3/r5 (K,L) embryos at E11.5. The arrows in E,F,I point to the ectopic motor nucleus that protrudes into the hindbrain ventricle.

View larger version (119K): [in a new window]   Fig. 5. Disruption of onset of Isl1 and Phox2b expression in neurones. (A,B) The expression of Isl1 was analysed in wild type (A) and kreislerr3/r5 (B) E10.5 embryos. (C,D) The expression of Phox2b was analysed in wild-type (C) and kreislerr3/r5 (D) E10.5 embryos. Expression of both markers was found to be reduced in r3 and r5 of kreislerr3/r5, indicating that there is a decrease in the number of neurones expressing these markers that have initiated differentiation.

View larger version (89K): [in a new window]   Fig. 6. Disruption of neuronal differentiation in kreislerr3/r5 embryos. (A-D) The differentiation of neurones revealed by immunodetection of ß-tubulin is shown in dorsal views (A-C) and a lateral view (D) of E12.5 wild type (A) and kreislerr3/r5 (B-D) embryos. (C) A higher magnification view of the ectopic nucleus seen in B; axons are seen extending dorsolaterally along the cleft at the r4/r5 boundary. (E-H) ß-galactosidase staining of r3/r5 lacZ reporter line shown in dorsal view at E11.5 (E,F) and parasagittal longitudinal section at E12.5 (G,H). v, ventricle; m, mantle region of neural tube. Arrows in B,D indicate the clefts that form at r3 and r5 in kreislerr3/r5 embryos.