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Onset of the segmentation clock in the chick embryo: evidence for oscillations in the somite precursors in the primitive streak

Caroline Jouve^*, Tadahiro Iimura and Olivier Pourquie

Laboratoire de génétique et de physiologie du développement (LGPD), Developmental Biology Institute of Marseille (IBDM), CNRS-INSERM-Université de la méditerranée-AP de Marseille, Campus de Luminy, Case 907, 13288 Marseille Cedex 09, France
^* Present address: Trophos SA, Bat CCIMP-Case 931, Campus de Luminy, 13009 Marseille, France

View larger version (64K): [in a new window]   Fig. 1. Onset of c-hairy2 and lunatic fringe RNA expression in paraxial mesoderm precursors. (A-G) Expression of c-hairy2; (H-N) expression of lunatic fringe. (O,P) Transverse sections from the embryo seen in D. The arrow indicates the level of Hensen’s node. (A,H) Stage HH3: expression of c-hairy2 and lunatic fringe is not detected in the presumptive territory of the paraxial mesoderm in the epiblast lateral to the primitive streak (asterisks). Transient expression of lunatic fringe (H,I) is detected in the neural plate (np). (B,I) Stage HH3+: expression is observed in the caudal primitive streak. (C-E,J-L). In progressively older embryos, expression of the c-hairy2 (C-E) and lunatic fringe (J-L) mRNA appears as a chevron, which sweeps caudorostrally along the primitive streak. (F,M) At stage HH4, the chevron of c-hairy2 and lunatic fringe expression ends its rostral progression. Its rostral tip coincides with the axial prechordal mesoderm, which maintains c-hairy2 (F,G) but not lunatic fringe (M,N) expression. (F,G,M,N) Subsequently a second chevron of c-hairy2 and lunatic fringe expression is initiated in the mid-primitive streak. This chevron also undergoes an anterior progression that terminates at the level of Hensen’s node. (O) The centre of the chevron appears to include the primitive streak (ps), and the immediately adjacent cells of the epiblast (e) and already ingressed mesoderm (m), while the wings of the chevrons are comprised solely of epiblast cells (P). (Q) Fate map of the precursors of the head and somitic mesoderm in the streak, adapted from Psychoyos and Stern (Psychoyos and Stern, 1996), compared with the expression domains of c-hairy2 or lunatic fringe in the primitive streak (R). The two chevron-like waves (W1, W2) sweep across the rostral primitive streak, which at these stages contains the precursors of the head and somitic mesoderm. EM, extra-embryonic mesoderm; GC, germinal crescent; IM, intermediate mesoderm; LP, lateral plate.

View larger version (57K): [in a new window]   Fig. 2. (A-H) Evidence for periodic waves of lunatic fringe expression before formation of the first pair of somites. Existence of a dynamic wave of expression of lunatic fringe in the paraxial mesoderm was demonstrated by performing half-embryo cultures. (A-H) Embryos from stage HH3+ to stage HH6 were bisected along the midline, and one half was fixed immediately (left half in each panel), while the other half (right half in each panel) was cultured for various time. Three phases were distinguished on the basis of the AP location of the streak expression domain. These phases were termed E (early), when the domain was located at its most caudal position in the streak, and M (intermediate) and L (late), when it became located progressively more rostral. Asterisks indicate the position of the Hensen’s node. (A) Stage HH3+ half embryo (left) showing caudal expression of lunatic fringe (1E, early phase of first chevron). After 3 hours in culture (right explant), expression has reached the mid-streak level (1M, intermediate phase of the first chevron). (B) Stage HH4– half embryo (left) with chevron-like expression of lunatic fringe located at the mid-streak level (1M). After 1 hour in culture, the chevron migrated rostrally and reached Hensen’s node level (right explant, 1L; late phase of first chevron). (C) Stage HH 4– half embryo (left, 1M). After 3 hours in culture, two chevrons of lunatic fringe expression are observed at the level of the Hensen’s node, indicating a late phase of second chevron migration (2L) (right explant). (D) Stage HH4 half embryo showing an early phase of second chevron migration (2E, left). After 1 hour in culture, the second chevron has reached the level of the Hensen’s node (right, 2L). (E) Stage HH4+ half embryo showing a late stage of the second chevron migration (left, 2L). After 1 hour in culture, the expression domain of lunatic fringe now appears as a stripe at the level of the node (right, 3L: late phase of third wave). Note the expression in the rostral primitive streak adjacent to the Hensen’s node. (F) Stage HH4+ half embryo (left, 2M). When cultured for 3 hours, the streak expression domain is separated from the Hensen’s node by a negative domain. Expression of lunatic fringe now appears as an intermediate stage of the fourth wave (4M: intermediate phase of fourth wave). Note the non-expressing domain between the streak-positive domain and the Hensen’s node. (G) Stage HH5 half embryo showing the stripe lateral to the Hensen’s node characteristic of the late stage (left, 3L). After a 1 hour culture period, the expression now corresponds to the intermediate stage of the fourth wave (right, 4M). (H) Stage HH6 half embryo showing the caudal expression domain in the primitive streak and the lateral mesodermal domain characteristic of the early stage of the sixth wave (6E, left). After 1 hour in culture, expression appears as a stripe adjacent to the Hensen’s node, indicating a late stage of the 6th wave (6L, right). (I) Schematic representation of the kinetics of the early waves of lunatic fringe expression based on the results of half-embryo cultures. Each arrow corresponds to a half embryo-culture in which the length of the arrow indicates the culture period of the right half (a small unit in the scale indicates 30 minutes). lunatic fringe expression pattern before and after culture are indicated on both sides of the arrows.

View larger version (132K): [in a new window]   Fig. 3. Waves of lunatic fringe expression before formation of the first somite. (A-H) lunatic fringe expression sequence in developing embryos from stage HH4+ to stage HH7. After stage HH4+, lunatic fringe becomes expressed in a sequence resembling that observed in 15-20 somite stage embryos (McGrew et al., 1998). (A) Third wave: expression first appears in a caudal domain of the rostral primitive streak and laterally in two adjacent domains in the newly ingressed mesoderm. Remnants of the first two chevrons can been seen rostral and caudal to Hensen’s node (white arrow). (B) Slightly later, the expression domain has moved anteriorwards and appears as two broad stripes lateral and then anterior to Hensen’s node. Subsequently, three similar waves of expression of lunatic fringe (C-F) are observed before the formation of the first somite (G,H). The red arrow indicates the level of the caudal boundary of the first somite. White arrows indicate the level of Hensen’s node.

View larger version (98K): [in a new window]   Fig. 4. Somitic progenitors in the primitive streak undergo pulses of c-hairy2 expression. Right and left panels show two stage 4+ embryos hybridised in wholemount with the c-hairy2 probe. The right embryo is slightly older than the left one, as is seen by the slightly extended length of axial mesoderm. The left embryo corresponds to the end of the third wave, while the right embryo marks the beginning of the fourth wave (see Fig. 6 and Fig. 3B,C). (A-E) Transverse cryosections of the left embryo at the levels shown on the wholemount. Mesodermal expression is observed in the prechordal mesoderm and notochord and in the overlying neural midline (A and not shown). At the level of Hensen’s node, the expression is detected only in the paraxial mesoderm precursors, in two stripes lateral to the node (B), which probably correspond to the prospective first somite. The other major site of expression is the epiblast (C,D) and the caudal mesoderm fated to give rise to extra-embryonic mesoderm (E). (F-J) Transverse sections of the right embryo. In contrast to the left embryo, no expression is detected in the mesoderm that flanks Hensen’s node, while Hensen’s node itself expresses the c-hairy2 message (G). In this embryo, the only expression detected in the paraxial mesoderm precursors includes the primitive streak and the adjacent mesoderm in a territory located caudal to Hensen’s node (I). As in the younger embryo, strong expression of c-hairy2 is also detected in the ventral neural plate and underlying axial mesoderm (F), in the epiblast (H-J) and in the mesoderm ingressing at the level of the caudal primitive streak (J). At this stage, expression of c-hairy2 message in the paraxial mesoderm is detected as a broad caudal domain in the newly ingressed presomitic mesoderm. This data demonstrates that pulses of expression occur in the territory containing the precursors of the paraxial mesoderm, i.e. the rostral primitive streak (compare D with I). hn, Hensen’s node; pc, prechordal mesoderm; ps, primitive streak.

View larger version (40K): [in a new window]   Fig. 5. Dynamic expression of lunatic fringe is independent of propagatory signals spreading anteriorwards along the primitive streak. (A) The experiment testing for signal propagation along the primitive streak. Embryos were divided sagittally, and in one of the two halves the caudal part was removed and fixed immediately (C). Both halves, the entire (D) and the truncated one (B), were incubated for the same period of time. The positions of the cuts are marked by the broken red lines. The black chevron on the primitive streak corresponds to the expression pattern of lunatic fringe in the embryo shown in (B-D) prior to culture. (E) The expression pattern of lunatic fringe, showing the two chevrons of expression (in black) in the embryo shown in (B-D) after culture. The same expression pattern is observed in ablated and unoperated halves, even after extended culture (B-D). Therefore, progression of lunatic fringe expression along the primitive streak does not rely on a posteriorly derived signal spreading through the primitive streak. hn, Hensen’s node; pc, prechordal mesoderm.

View larger version (17K): [in a new window]   Fig. 6. Onset of cyclic gene expression in the paraxial mesoderm in relation to head and somitic mesoderm development. The top row shows a diagrammatic representation of primitive streak elongation and regression, and the forming axial and paraxial mesoderm of embryos between stage HH3+ and HH7. Head mesoderm (pink) is produced by the primitive streak between stage HH4– and 5 and it then undergoes a striking anteriorwards elongation when the primitive streak regresses. Presomitic mesoderm (light-green domain) starts to be formed from stage HH4–/5 and its production progresses together with primitive streak regression movements. Formation of somites (dark green) starts from stage 6 onwards. Bottom row shows the waves of cyclic gene expression prior to somite formation. The expression domains of c-hairy2 (or lunatic fringe) in the paraxial mesoderm and its precursors in the streak are presented in colour, each wave being represented by a different colour. W1 and W2 correspond to the first two chevrons described in Fig. 1. Subsequently, waves of expression sweep across the rostral primitive streak and the newly formed PSM. Altogether, a dynamic expression sequence in the paraxial mesoderm and its precursors is reiterated five times before the formation of the first morphological somite. The black arrow marks the position of the first somite precursors.

View larger version (24K): [in a new window]   Fig. 7. The fate of the paraxial mesoderm with respect to head segmentation. As shown on the left, only two pulses of cyclic gene expression are detected for the whole mesodermal territory anterior to the first somite. The prechordal mesoderm shares with the paraxial mesoderm the ability to give rise to skeletal muscle derivatives. Moreover, the prechordal mesoderm precursors experience the first chevron of cyclic genes in the streak and they subsequently maintain this expression. We propose that the first pulse of cyclic gene expression (W1) marks the production of the prechordal territory, while the second one (W2) marks the production of the whole paraxial head mesoderm.