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Morphological boundary forms by a novel inductive event mediated by Lunatic fringe and Notch during somitic segmentation

¹ Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0101, Japan
² Center for Developmental Biology, RIKEN, Japan

View larger version (60K): [in a new window]   Fig. 1. Definition of the levels of a forming boundary in the anterior PSM as seen in sagittal section of an E2 embryo under Nomarski optics. Anterior is at left. (A) The first feature of morphological segmentation is a gap within mesenchymal PSM, and this position is designated as the line 0. The site located one-somite length posterior to the line 0 is defined as the level –1. The border cells immediately anterior to the gap undergo epithelialization resulting in a solid boundary at the line +1. (B) Identification of the line 0 in living embryos was confirmed by the location of the condensed cell mass posterior to +1. The position of level +1 was judged by combining a landmark made by a GFP/COS implantation and histological criteria. GFP in a paraffin section was visualized by anti-GFP antibody (brown).

View larger version (69K): [in a new window]   Fig. 2. The prospective line –1 was already determined to make a fissure. (A,B) Transplantation of a piece of PSM containing level –1 into a non-segmented level (–1.5). The size of the graft largely corresponded to a prospective somite. Anterior and posterior halves of the graft were labeled with DiI (red) and DiO (green), respectively. On the operated side, a boundary (arrow) formed between the DiI- and DiO-labeled regions, and along this fissure epithelialized miniature somites were present (bracket). (C,D) In the control, an isotopic transplantation (–1.5 into –1.5) did not produce a boundary between DiI- and DiO-labeled areas. (B,D) Sagittal frozen sections of embryos treated similarly to A and C, respectively. Stained for F-actin with Phalloidin (white).

View larger version (70K): [in a new window]   Fig. 3. A fissure was induced to form by the posterior border cells. A group of cells taken from near –1 of a quail donor was inserted into the level –1.5 of a chicken host. This manipulation resulted in the ectopic formation of a fissure with supernumerary somites along it (brackets). Sixteen out of 34 specimens showed distribution of the quail cells confined to the posterior region of the ectopic border (A), whereas 18 embryos had the donor cells spanning the border (B). No embryo was observed where the graft was located only anteriorly to the ectopic fissure. A; A dorsal view under a dissecting microscope. (A',B,C) A horizontal section stained with QCPN antibody to visualize the quail cells (brown, arrowheads). Anterior at top. (C) Control isotopic graft (–1.5 into –1.5).

View larger version (107K): [in a new window]   Fig. 4. Expression patterns of Lfng (A), Delta1 (B) and Notch1 (C) mRNAs in the anterior PSM of E2 chicken embryos. The sharp anterior boundary of the Lfng-expressing area demarcates the prospective line –1. Red and black arrowheads indicate levels 0 and –1, respectively. (A-C) A dorsal view with anterior at top. (A'-C') A sagittal section of the same specimen shown in A-C with anterior at left.

View larger version (91K): [in a new window]   Fig. 5. In ovo DNA electroporation into PSM. (A) A diagram showing positions of a plus electrode (platinum) under the embryo, and a minus electrode (sharpened tungsten) near the DNA solution which was laid over the anterior primitive streak at HH stage 7-8. (B) 24 hours after the electroporation with pCAGGS-GFP, GFP signal was observed in the segmented somites and PSM. (C) A sagittal section of a GFP-electroporated embryo (anterior to the left). (D) An embryo co-electroporated with pCAGGS-Lfng and pCAGGS-GFP showing no gross disturbance in the somites. (E) Western blotting showing that pCAGGS-Lfng and pCAGGS-NotchE, used for the electroporation, gave rise to proteins of expected size when transfected into COS cells. Products of Lfng/FLAG (42 kDa, lane 1) and NotchE/Myc (83 kDa of an unprocessed form, and 70 kDa and 63 kDa fragments of processed forms translocating to the nucleus (Kopan et al., 1996), lane 3) were, respectively, detected by anti-FLAG and anti-Myc antibodies. Lanes 2 and 4 are controls for lanes 1 and 3, respectively.

View larger version (42K): [in a new window]   Fig. 6. A morphological boundary was induced to form at an interface between host and Lfng/Notch-electroporated donor tissues. (A) A diagram showing a manipulation to create a sharp border of Lfng activity. Lfng or Notch E DNA was electroporated into PSM of a donor embryo together with GFP, and subsequently a small piece was dissected from a donor PSM, and transplanted into a host. (B) An ectopic fissure was formed at the boundary between the donor (GFP-positive cells in the right panel; arrowhead) and host regions. (C) A confrontation between NotchE-positive and -negative cells gave a similar result to that in B, whereas Notch LNG did not show such effects (D). The left and right panels in B-D show the same specimen with the position of a fissure and electroporated donor-derived cells (green), respectively, indicated. (E) A sagittal section of the supernumerary somites resulting from a Lfng boundary. Anterior is at left. Epithelial cells visualized by phallodin stainin (red) were present anterior to the Lfng-producing cells (green), whereas control GFP-expressing cells did not affect the epithelialization (F). The diagrams on the right of E and F show the position of the boundaries more clearly.

View larger version (104K): [in a new window]   Fig. 7. Notch/Lfng acts directionally on the anteriorly located cells. (A) When Lfng-producing cells were located in the anterior half of a host somite, the interface (arrow) did not induce a fissure. (B) When a ‘strip’ of Lfng-producing cells was positioned in the middle of a host somite, only the anterior (arrowhead) but not the posterior (arrow) interface produced an ectopic fissure.

View larger version (68K): [in a new window]   Fig. 8. A Notch boundary did not induce a fissure in posterior PSM. A piece of NotchE/GFP-electroporated PSM was taken from level –1.5 and transplanted into posterior PSM near the level –4.5.

View larger version (94K): [in a new window]   Fig. 9. The AP polarity in the supernumerary somites. (A,A') When an ectopic fissure was induced to form by NotchE-producing cells (asterisk), the small somite located anterior to the fissure expressed Delta1 mRNA at its posterior margin (arrow). (B) NotchE-producing cells themselves showed an intense signal even when they were located outside the somite. (C,C') Notch LNG did not affect the AP polarity of the host somite. (D) When a large graft of level –1 was placed into level –1.5 (the same transplantation as Fig. 2A), the resulting miniature somites, after 5 hours of incubation, expressed Delta1 mRNA in the posterior margin of each somite. DiI was used to mark the grafted level along the AP axis of a host (pink in the control side). (E,F) QCPN staining of horizontal sections. Embryos with the same manipulation that were incubated for another 2 days formed supernumerary DRGs in the anterior part of each segment (E), whereas a control isotopic transplantation had no effect on DRG (F).

View larger version (38K): [in a new window]   Fig. 10. Summary and model for the action of Notch signaling. (A) The signal (arrows) emanating from the posterior border cells and acting on the anterior border cells is designated as the ‘segmenter’. See text for detailed explanation. Lfng mRNA normally located posterior to the line 0 is not shown to allow the molecular events at –1 to be clearly represented. (B) Two models proposing a mode of Notch action in somite boundary formation analogous to its action in Drosophila. See text for details.