Krox-20 patterns the hindbrain through both cell-autonomous and non cell-autonomous mechanisms

Abstract

The Krox-20 gene encodes a zinc finger transcription factor, which has been shown previously, by targeted inactivation in the mouse, to be required for the development of rhombomeres (r) 3 and 5 in the segmented embryonic hindbrain. In the present work, Krox-20 was expressed ectopically in the developing chick hindbrain by use of electroporation. We demonstrate that Krox-20 expression is sufficient to confer odd-numbered rhombomere characteristics to r2, r4, and r6 cells, presumably in a cell-autonomous manner. Therefore, Krox-20, appears as the major determinant of odd-numbered identity within the hindbrain. In addition, we provide evidence for the existence of a non cell-autonomous autoactivation mechanism allowing recruitment of Krox-20-positive cells from even-numbered territories by neighboring Krox-20-expressing cells. On the basis of these observations, we propose that Krox-20 regulates multiple, intertwined steps in segmental patterning: Initial activation of Krox-20 in a few cells leads to the segregation, homogenization, and possibly expansion of territories to which Krox-20 in addition confers an odd-numbered identity.

Figure 1

Ectopic Krox-20 expression leads to EphA4 induction and follistatin and Hoxb1 repression. Flat-mounted hindbrains (A–E,H–J) or whole mounts (F,G) from chick embryos electroporated with LacZ (A), wild-type (B–G), or R409W mutant (H–J) mouse Krox-20 expressing plasmids between stages HH8 and HH10 (A–E,H–J) or between stages HH10 and HH11 (F,G). The embryos were collected 24 h after electroporation (18 h for D and 16 h for F and G) and the expression of the indicated markers was analyzed by X-Gal staining (A), immunochemistry (B,C,F–H) or in situ hybridization (D,E,I,J). (A) Analysis of β-galactosidase distribution after electroporation with a LacZ expression plasmid. (B) Analysis of Krox-20 expression with an antibody that recognizes both mouse and chicken proteins after electroporation with a Krox-20 expression plasmid. Note that ectopic Krox-20 is present in isolated cells with a distribution similar to that of β-galactosidase (A). (C) EphA4 ectopic expression is detected in large patches of cells in r2 and r4. (D) follistatin expression is severely down-regulated upon Krox-20 ectopic expression in the hindbrain, including r5, where endogenous Krox-20 is also present. The patchy appearance of the follistatin-positive domain on the control side is normal at this stage. (E) Hoxb1 is repressed following ectopic Krox-20 expression. Large patches of Hoxb1-negative cells are observed within the entire domain of normal expression, including r4 and r7. (F,G) EphA4 activation in rhombomere 2 (arrowheads) is not due to cell migration from r3 because, in (G), the embryo was cut immediately after electroporation at the level of prospective rhombomere 2 and the two parts were kept separated. (H,I,J) Ectopic expression of the Krox-20 mutant allele (R409W) does not induce EphA4 (H), nor does it repress follistatin (I) or Hoxb1 (J). In I, the red staining corresponds to in situ hybridization with a mouse-specific Krox-20 probe, revealing the transfected cells. Electroporated side is on the left.

Figure 2

Time-course of the effects of Krox-20 ectopic expression on Hoxb1 and EphA4 expression. Flat-mounted hindbrains from embryos electroporated with Krox-20, incubated for the indicated period of time and hybridized in situ with Hoxb1 (purple) and EphA4 (red) probes. The developmental stages of the harvested embryos are indicated (bottomright of A–E, and I–K). (F–H,L–N) Higher magnifications of the embryos shown in C–E and I–K, respectively. At early stages, EphA4 expression fills all Hoxb1-negative patches in r4, but not in the caudal domain of Hoxb1 expression. Later, EphA4 is down-regulated in basal Hoxb1-negative patches in r4 (white arrowheads in H and M). Note the formation of a thin unstained boundary at the interface between adjacent rhombomeres as well as between EphA4-positive and Hoxb1-positive domains within r4 (black arrowheads in G and N). The apparent overlap in EphA4 and Hoxb1 labeling in some areas in M is due to cytoplasmic overlap of different cells. Electroporated side is on the left.

Figure 3

Krox-20 misexpression does not affect mafB/kr, Hoxa2, Hoxa3, and Hoxb3 expression. Flat mounts of hindbrains of embryos electroporated with Krox-20, incubated for 24 h (18 h in A and B) and hybridized with the indicated probes. (A) EphA4-positive patches (red) in r2 and r4 do not express mafB/kr (purple) whereas those in r6 maintain this expression. (B) MafB/kr expression (red) is not affected by Krox-20 expression. In particular, the Hoxb1-negative domains in r4 and r7 do not activate mafB/kr. (C,D) Krox-20 ectopic expression does not lead to significant modifications in the patterns of Hoxa3 or Hoxb3 expression. (E) Hoxa2 expression (purple) is also maintained in r4 in the patches negative for Hoxb1 (red). Electroporated side is on the left.

Figure 4

Non cell-autonomous effects on EphA4 and Hoxb1 expression. Flat-mounted hindbrains from embryos electroporated with mouse Krox-20 and revealed by in situ hybridization (A,B,E–G) or immunochemistry (C–E) with the indicated probes or antibodies. (A) Double in situ hybridization performed with an EphA4 probe (red) and a mouse-specific Krox-20 probe (purple) which labels only electroporated cells. Note that the mouse Krox-20 probe labels isolated cells within the EphA4-positive patches. (B) Double in situ hybridization performed on an embryo harvested 24 h after electroporation with an EphA4 probe (red) and a Krox-20 probe (purple), which hybridizes with both the endogenous chicken and the exogenous mouse mRNAs. Note the complete overlap between both labelings (brown color, compare with red/orange staining in A), except in r1, where only Krox-20 is detected (red arrowhead). (C,D) Double immunochemistry performed on embryos harvested 14 h (C) and 20 h (D) following electroporation with antibodies directed against EphA4 (red) and Krox-20 (purple). The latter antibody recognizes both the chick and mouse Krox-20. Note again that all EphA4-positive territories express Krox-20. (E) Hoxb1 in situ hybridization (purple) combined with anti-Myc immunochemistry (brown) on an embryo electroporated with the Myc-tagged Krox-20. (F,G) Double in situ hybridization performed on embryos harvested at stages HH14− and HH14+ respectively with Hoxb1 (red) and Krox-20 (purple) probes. The Krox-20 probe recognizes both chick and mouse mRNAs. Note the presence of white patches (Hoxb1– and Krox-20-negative) within basal r4 (black arrowhead in G). Electroporated side is on the left.

Figure 5

Expression of exogenous Krox-20 leads to non-autonomous activation of endogenous Krox-20. Flat-mounted hindbrains from embryos electroporated with mouse Krox-20 (A–D) or a construct (pAdRSVβgalKrox20) directing co-transcription of LacZ and mouse Krox-20 (E–H). They were analyzed by in situ hybridization with chicken-specific (purple) and mouse-specific (red, AD) Krox-20 probes, and Bluo-Gal staining (dark blue, E–H). (A,B) HH13 embryos harvested 18 h after the electroporation. (Red arrowheads) Patches of endogenous Krox-20 expression in r1. (C) HH15 embryo harvested 24 h after the electroporation. (D) Higher magnification view of the embryo shown in C. While mouse Krox-20 is expressed in a punctuate manner along the neural tube, chicken Krox-20 is ectopically activated in cell patches restricted to the r1–r7 region. The extent of chicken Krox-20 expression appears to broaden at later stages, and positive patches are observed in absence of close mouse Krox-20-expressing cells (black arrowheads in D). (E,F) HH13+ embryos harvested 18 h after electroporation. (G,H) Higher magnification views of the embryos shown in E and F. Chicken Krox-20 is expressed in Bluo-Gal-negative cells (examples are indicated by green arrowheads). Electroporated side is on the left.

Figure 6

Early neurogenesis is affected by ectopic Krox-20 expression. Flat-mounted hindbrains from control (A,C) and Krox-20 electroporated (B,D) embryos. (A,B) Immunochemistry with an antibody directed against neurofilaments (3A10), which reveals cell bodies and growing axons of differentiated neurons. The control embryo in A was electroporated with a mutant Krox-20 allele (R409W). In B, neurogenesis is impaired and delayed on the electroporated side. (C,D) Double labeling with antibodies directed against neurofilaments and EphA4. The embryo in C was not electroporated. In the Krox-20 electroporated case (D), the density of neurofilament staining is lower in EphA4-positive patches in r4 and r6 and in contrast reinforced in their vicinity (arrowheads). Electroporated side is on the left.

Figure 7

A model for chick hindbrain patterning. (a) Time-course of Krox-20 and Hoxb1 expression in the developing chick hindbrain. (A–F) Flat mounts of embryos at the indicated somite stages of development, subjected to double in situ hybridization with chicken Krox-20 (purple) and Hoxb1 (red) probes. (b) Schematic representation of the development of Krox-20 and Hoxb1 expression and of putative genetic interactions. For a detailed description, see Discussion. Hoxa1/Hoxb1- and Krox-20-expressing territories are represented in orange and purple-grey, respectively. (Light purple-grey) High level expression of Nab1 and Nab2 within the Krox-20-positive territories. (Black arrows and bars) Established (solid stem) or putative (dashed stem) cell-autonomous induction and repression/inhibition, respectively. (Purple-grey arrowheads) Different putative signals involved in Krox-20 activation and originating from unidentified tissues (dashed stem), Krox-20-expressing cells (purple-grey stem) and prospective r4 (orange stem). Somite stages are indicated on the left.