The chick somitogenesis oscillator is arrested before all paraxial mesoderm is segmented into somites

Abstract

Background: Somitogenesis is the earliest sign of segmentation in the developing vertebrate embryo. This process starts very early, soon after gastrulation has initiated and proceeds in an anterior-to-posterior direction during body axis elongation. It is widely accepted that somitogenesis is controlled by a molecular oscillator with the same periodicity as somite formation. This periodic mechanism is repeated a specific number of times until the embryo acquires a defined specie-specific final number of somites at the end of the process of axis elongation. This final number of somites varies widely between vertebrate species. How termination of the process of somitogenesis is determined is still unknown.

Results: Here we show that during development there is an imbalance between the speed of somite formation and growth of the presomitic mesoderm (PSM)/tail bud. This decrease in the PSM size of the chick embryo is not due to an acceleration of the speed of somite formation because it remains constant until the last stages of somitogenesis, when it slows down. When the chick embryo reaches its final number of somites at stage HH 24-25 there is still some remaining unsegmented PSM in which expression of components of the somitogenesis oscillator is no longer dynamic. Finally, we identify a change in expression of retinoic acid regulating factors in the tail bud at late stages of somitogenesis, such that in the chick embryo there is a pronounced onset of Raldh2 expression while in the mouse embryo the expression of the RA inhibitor Cyp26A1 is downregulated.

Conclusions: Our results show that the chick somitogenesis oscillator is arrested before all paraxial mesoderm is segmented into somites. In addition, endogenous retinoic acid is probably also involved in the termination of the process of segmentation, and in tail growth in general.

Figure 1

Regression of the PSM domain in the chick tail bud. Lateral view of whole-mount chick tail buds at (A, F, K) HH stage 20, (B, G, L) stage 22, (C, H, M) stage 24, (D, I, N) stage 26 and (E, J, O) stage 27 analysed by in situ hybridisation with (A-J) c-Tbx6 and (K-O) c-Dact2/c-MyoD showing the position of the PSM or the first somite, respectively. (F-J) Sections of tail buds stained with the c-Tbx6 probe; the borders of the PSM domain (p) and the last somite (s) are demarcated. (P) Bar chart showing the size of the PSM (green bars) and the size of the last somite (red bars) at HH stages 10-25. (Q) Bar chart showing the PSM/last somite ratio during the same window of development calculated separately for each embryo and then the average ratio was calculated for each stage. Error bars represent standard deviation.

Figure 2

Time course of somite number during chick somitogenesis. Extension of the somite formation period. (A) Graph plotting increasing time intervals along the x axis and increasing number of somites and the corresponding HH stage of chick development along the y axis. The red line is a theoretical line showing the progression if the speed of somitogenesis were to remain constant at 90 minutes throughout development. Each blue dot corresponds to an individual sample/embryo. The blue line is a best fit line. Dotted green line demarcates the average final number of somites. (B-G) Fix and culture analysis using the half embryo system using the caudal region of a chick embryo. Left half samples were immediately fixed whereas right half samples were cultured in vitro for different periods of time and then the expression of c-Lfng was analysed in the two halves by in situ hybridisation. (B) HH stage 20 and (C) stage 23 samples cultured for 90 minutes. (D-G) HH stage 23 samples were cultured for (D) 60 minutes, (E) 90 minutes, (F) 120 minutes and (G) 150 minutes.

Figure 3

Components of the Notch-based somitogenesis oscillator are downregulated in the chick tail bud at HH stage 25. Lateral views of chick embryos of (A-B) HH stage 22, (C, E, K, M, O, Q) stage 24 and (D, F, L, N, P, R) stage 25, and (G-J) frontal views of chick embryos of HH stage 26 analysed by in situ hybridisation to show the expression of (A-D) c-Lfng, (E-J) c-Tbx6, (K, L) c-Meso1, (M, N) c-Nrarp, (O, P) c-Delta1 and (Q, R) c-Notch1. From HH stage 24 the expression of components of the somitogenesis oscillator becomes restricted to the rostral region of the PSM and then at HH stage 25 their expression is downregulated.

Figure 4

Onset of c-Raldh2 expression in the chick tail bud from HH stage 21. Lateral view of whole-mount chick tail buds at (A, F, K) HH stage 20, (B, G, L) stage 21, (C, H, M) stage 22, (D, I, N) stage 23 and (E, J, O) stage 24 analysed by in situ hybridisation with (A-E) c-Wnt3a, (F-J) c-Raldh2 and (K-O) c-Cyp26A1 to show their expression in the tail bud region. Red arrowheads show that from HH stage 20 to stage 21 there is a severe downregulation of c-Wnt3a simultaneous to an onset of c-Raldh2 in the tail bud. Lateral view of mouse tail buds at (P, T, X) E10.5, (Q, U, Y) E11.5, (R, V, Z) E12.5 and (S, W, AA) E13.5 analysed by in situ hybridisation using (P-S) m-Wnt3a, (T-W)m-Raldh2, and (X-AA)m-Cyp26A1 probes to show their expression in the tail bud region. Red arrowheads show that from E10.5 to E13.5 there is a severe downregulation of m-Wnt3a simultaneous to an onset of m-Raldh2 in the tail bud and a downregulation of m-Cyp26A1.

Figure 5

Wnt3a diminution and RA activity in the chick tail bud. RA detection using F9-1.8 cells cultured in (A) RA-free media, (B) with 1 nM RA or co-cultured with (C) somitic tissue, (D) tail bud tissue from a HH stage 20, (E) HH stage 22 and (F) HH stage 26. (G) Schematic representation. From HH stage 21 there is an onset of c-Raldh2 in the proximity of the precursor cells located in the chick tail bud. This local RA activity is likely to negatively affect the expression of c-Wnt3a, which subsequently affects the growth of the precursor population and the speed of somite formation, and might also impinge on the observed loss of Notch activity associated with the somitogenesis oscillator. (H-J) Dorsal view of chick embryo analysed by in situ hybridisation using a c-Fgf8 probe. (H) control, (I) exposure to DMSO and (J) 1.5 mM RA soaked beads. (K) Scheme of experiment with RA soaked beads. Red circle shows the position of the DMSO/RA soaked bead in the tail bud; yellow circle represents the DMSO reference bead used as a means of measuring PSM size before and after treatment. (L-M) 1.5-10 mM RA soaked beads in the tail bud (L) slow down the growth of the PSM and (M) reduces the number of somites formed in a defined culture period. Blue bars denote DMSO treated samples and green bars denote RA treated samples. Error bars represent standard deviation. Abbreviations: som (somites) and tb (tail bud).