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Evidence for medial/lateral specification and positional information within the presomitic mesoderm

Catarina Freitas^1,*, Sofia Rodrigues^1,*, Jean-Baptiste Charrier², Marie-Aimée Teillet² and Isabel Palmeirim^1,

¹ Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal
² Institut d’Embryologie Cellulaire et Moléculaire du Centre National de la Recherche Scientifique et du Collège de France, 49 bis Avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex, France
^* These two authors contributed equally to this work

View larger version (46K): [in a new window]   Fig. 1. Variability of hairy1, hairy2 and lunatic fringe expression patterns is evident at the level of sinus rhomboidalis. In situ hybridisation with the hairy1 (A-C), hairy2 (D-F) and lunatic fringe (G-I) antisense mRNA probes, showing the variety of expression patterns observed at the level of PSM and sinus rhomboidalis, in six-somite stage embryos. (Left) Electron scanning micrograph of the sinus rhomboidalis of a six-somite stage embryo (dorsal view). The lines define the AP levels of the transverse sections. (A1-I1) Cross-sections at the level of the posterior limit of the median pit. (A2-I2) Cross sections 80-100 µm caudal to the posterior limit of the median pit. (A3-I3) Cross-sections 160-180 µm caudal to the posterior limit of the median pit. (A4-I4) Cross-sections 240-260 µm caudal to the posterior limit of the median pit. We consider the median pit to be the region that comprises the pit itself and the slope surrounding it. A transverse section at the same level, in two different embryos hybridised with the same probe, evidences distinct patterns of expression (e.g. A2-C2). Transverse sections at adjacent levels also evidence distinct patterns of expression, within the same embryo (e.g. G2-G3). PM-PSM, prospective medial–presomitic mesoderm. Rostral is towards the top. Scale bar: 250 µm in whole-mount embryos; 110 µm in cross sections.

View larger version (91K): [in a new window]   Fig. 2. Double whole-mount in situ hybridisation shows that at the level of sinus rhomboidalis the expression pattern of the cycling genes is not coincident. (A-C) Double in situ hybridisation in six-somite stage embryos with hairy1 (blue staining) and hairy2 (red staining) RNA probes. The expression domains of these genes are distinct, as evidenced by the caudal extension of the hairy2 domain in relation to hairy1. (D-F) Double in situ hybridisation with hairy1 (blue staining) and lunatic fringe (red staining) RNA probes. At the level of sinus rhomboidalis, we observe that, in some embryos, the expression pattern of lunatic fringe is different from hairy1, extending more posteriorly (see D). (G-I) Double in situ hybridisation with lunatic fringe (blue staining) and hairy2 (red staining) RNA probes. The expression pattern of hairy2 protrudes more caudally than that of lunatic fringe (see G-I). At the level of PSM, the expression domains of the three genes coincide at its anterior border, although at its caudal limit they are not completely coincident. Rostral is towards the top. Scale bar: 300 µm

View larger version (108K): [in a new window]   Fig. 3. Quail/chick chimaera fate map of deep mesenchymal tissue extending 100-150 µm posterior to the median pit. (A) Schematic diagram illustrating the localisation of transplanted quail tissue into the six-somite chick embryo. (B) Cross-sections at the level of rostral PSM and (C) epithelial somite, of chimaeras incubated for 24 hours, showing the localisation of the quail donor cells in the medial part of these tissues. Note that a cross section in the more rostral PSM evidences the Wolffian duct. Scale bar: 70 µm

View larger version (64K): [in a new window]   Fig. 4. Somites are not formed in the absence of medial PSM cells. (A,E) The type of PSM ablation performed on 15 to 20 somite stage embryos. (B) Dorsal view of an embryo cultured for 9 hours after lateral PSM ablation. The black arrowheads indicate somites formed during the in vitro culture period. The same number of somites is formed both in operated and control sides, although in the former the size of the somites appears smaller. (C) Lateral PSM ablated embryo hybridised with Delta-1. Somites formed in culture express Delta-1 in its normal pattern of expression. (D) Lateral PSM ablated embryo hybridised with Tbx6L present a normal pattern of expression. (F) Dorsal view of an embryo cultured for 9 hours, whose medial PSM has been removed. The brackets indicate the operated side where no somites have been formed in the absence of medial PSM tissue. (G) Medial PSM ablated embryo hybridised with Delta-1 reveals that the remaining lateral PSM does not express the Delta-1 gene and that it is restricted to a more caudal domain, when compared with the control PSM. (H) Medial PSM ablated embryo hybridised with Tbx6L clearly shows that lateral PSM tissue remains in the operated side (arrowhead). Scale bars: 200 µm in B-F; 300 µm in C,D,G,H.

View larger version (49K): [in a new window]   Fig. 5. Explant culture experiment: control experiment. (A) Two types of explants were generated, one comprising the lateral PSM (left explant) and the other containing the corresponding medial PSM, the axial structures and the control PSM (right explant). In order to ensure that the PSM tissue was being subdivided into equivalent medial and lateral halves, some explants were randomly taken, immediately fixed and hybridised either with probes for Delta-1 (B) or with BMP-4 (C). These control experiments show that the Delta-1 domain of expression is longitudinally subdivided into two halves. In the BMP-4 hybridised explant, a thin domain negative to this gene is present corresponding to the lateral PSM tissue. Scale bar: 250 µm.

View larger version (90K): [in a new window]   Fig. 6. Explant culture experiment: molecular segmentation markers are not expressed in the absence of medial PSM. Dorsal view of medial (right) and lateral (left) PSM explants, generated as described in Fig. 5 and subsequently hybridised with probes for Delta-1 (A), Notch-1 (B), paraxis (C), hairy1 (D), hairy2 (E), lunatic fringe (F), BMP-4 (G), Noggin (H) and Tbx6L (I) antisense RNA probes. The expression of molecular segmentation markers (Delta-1, Notch-1 and paraxis) as well as cycling genes (hairy1, hairy2 and lunatic fringe) is affected in the isolated lateral PSM cells. Nevertheless, the usual expression patterns of Tbx6L, BMP-4 and Noggin are maintained. Scale bar: 300 µm.

View larger version (65K): [in a new window]   Fig. 7. A mediolateral asynchrony originates oblique stripes in the PSM. (A-C) Dorsal view of PSM/newly formed somites of six-somite stage embryos hybridised with hairy1. Rostral is towards the top. The schematics depict the PSM stripe anterior progression. The transition between two horizontal stripes originates an oblique stripe in the PSM: the transition between a posterior to an anterior stripe implies the upregulation of the gene in the lateral domain of the anterior stripe concomitant with the downregulation in the lateral part of the posterior stripe. In a second phase, cells in the medial domain follow the same progression in their expression. Scale bar: 100 µm.