Spatiotemporal oscillations of Notch1, Dll1 and NICD are coordinated across the mouse PSM

Abstract

During somitogenesis, epithelial somites form from the pre-somitic mesoderm (PSM) in a periodic manner. This periodicity is regulated by a molecular oscillator, known as the 'segmentation clock', that is characterised by an oscillatory pattern of gene expression that sweeps the PSM in a caudal-rostral direction. Key components of the segmentation clock are intracellular components of the Notch, Wnt and FGF pathways, and it is widely accepted that intracellular negative-feedback loops regulate oscillatory gene expression. However, an open question in the field is how intracellular oscillations are coordinated, in the form of spatiotemporal waves of expression, across the PSM. In this study, we provide a potential mechanism for this process. We show at the mRNA level that the Notch1 receptor and Delta-like 1 (Dll1) ligand vary dynamically across the PSM of both chick and mouse. Remarkably, we also demonstrate similar dynamics at the protein level; hence, the pathway components that mediate intercellular coupling themselves exhibit oscillatory dynamics. Moreover, we quantify the dynamic expression patterns of Dll1 and Notch1, and show they are highly correlated with the expression patterns of two known clock components [Lfng mRNA and the activated form of the Notch receptor (cleaved Notch intracellular domain, NICD)]. Lastly, we show that Notch1 is a target of Notch signalling, whereas Dll1 is Wnt regulated. Regulation of Dll1 and Notch1 expression thus links the activity of Wnt and Notch, the two main signalling pathways driving the clock.

Keywords: Notch signalling; Oscillations; Somitogenesis.

Fig. 1.

Dll1 and Notch1 mRNA expression in mouse PSM. (A-F) In situ hybridisation of Dll1(i) (A-C) and Notch1(i) (D-F) in E10.5 PSM using intronic (i) RNA probes. (G-I) Fix-and-culture assay comparing the expression pattern of Dll1(i) (G) and Notch1(i) (H) with that of the clock gene Hes7 (I). (J-O) FISH of Dll1 (J-L) and Notch1 (M-O) mRNA in PSM using exonic (e) probes. (P-R) qRT-PCR for Dll1, Notch1, Hes7, β-actin and Gapdh in the caudal halves of individual E10.5 PSM explants following fix and culture. Data show the mean ± s.d. of technical replicates. (P,Q) Individual samples showing the fold change in mRNA concentration between fixed and cultured explants for Dll1, Notch1, Hes7 and β-actin once normalised to Gapdh. (R) Total variance of fold change in expression levels in fix:culture ratios of each gene measured by qRT-PCR across all 16 samples. a.u., arbitrary units. **P<0.001.

Fig. 2.

Pre-mRNA expression profile of Dll1 and Notch1 in the chick PSM. (A-E) In situ hybridisation of Dll1(i) and Notch1(i) in the PSM of HH8-HH13 embryos using intronic (i) RNA probes. Cyclical expression was confirmed by fix-and-culture analysis for Dll1(i) (B) and Notch1(i) (D). (E) Dll1(i) and Notch1(i) oscillate out of synchrony.

Fig. 3.

Comparison of Dll1 and Notch1 mRNA expression with that of clock genes. (A-E) Dll1(i) oscillates out of synchrony with Notch1(i) (A), Lfng(i) (B) and Hes7(i) (C). Dll1(i) oscillates in synchrony with Snail1 (D). Notch1(i) oscillates in synchrony with Lfng(i) (E). (F-H) Fold changes (normalised to Gapdh) between E10.5 caudal PSM fixed and cultured samples, as determined by using qRT-PCR, for Notch1 against Hes7 (F), β-actin against Hes7 (G) and Dll1 against Hes7 (H). a.u., arbitrary units.

Fig. 4.

Notch and Wnt inhibition reveals differing regulation of Dll1(i) and Notch1(i) expression. (A-J) In situ hybridisation was performed on mouse or chick explants cultured with (+) or without (−) LY411575 (A-F), pyrvinium pamoate (G,H) or XAV939 (I,J). (K,L) Western blot analysis of Dll1 protein from pooled PSM samples following 4 h exposure to pyrvinium pamoate (K), or analysis of Notch1 and NICD proteins following 4 h exposure to LY411575 (L).

Fig. 5.

Oscillations of Dll1 and Notch1 proteins in mouse PSM. (A-F) Following FISH to detect Lfng(i) in one half of a set of E10.5 tails, immunohistochemistry was performed on the contralateral half of the explants to detect Dll1 (A-C) or Notch1 (D-F) protein. (G-H′) Double immunohistochemistry on sections of individual E10.5 tails to detect PSM expression of NICD and Dll1 (G,H), or NICD and Notch1 (G′,H′) (n=15). The dotted lines demarcate the positions of the most recently formed somite(s), outer edges of the PSM, adjacent neural tissue (C,E) or hind gut (H). (I,J) Western blot analysis for Dll1 and Notch1 or α-tubulin on the caudal half of individual PSM explants following fix and culture.

Fig. 6.

Quantification of spatiotemporal dynamics of Dll1 and Notch1 protein expression. (A) An example of an intensity plot depicting axial variation in signal intensity across the PSM. Data plotted from two explant pairs showing Lfng pre-mRNA (black broken line) in one explant compared with Notch1 protein (red) in the contralateral half explant, and Lfng pre-mRNA (black unbroken line) in a half explant from a second tail compared with Dll1 protein (green) in the contralateral half explant of the second tail. Measured intensities (y axis) are plotted against axial position [x axis; rostral (‘A’) to right and caudal (‘P’) to left]. (B-H) Kymographs show spatial distribution of Notch1, Dll1 and NICD, and of Lfng(i) across numerous PSMs. (B,C) NICD (B) and Dll1 (C) expression in PSM sections; (D,E) Lfng(i) (D) and Dll1 (E) in contralateral explant halves; (F,G) Lfng(i) (F) and Notch1 (G) in contralateral explant halves. a.u., arbitrary units.

Fig. 7.

Quantification of pulsatile Dll1 and Notch1 protein expression in the caudal PSM of mouse tails. (A) Overlay of kymographs from Fig. 6D [Lfng(i)], Fig. 6E (Dll1) and Fig. 6G (Notch1) reveals their spatiotemporal expression during one oscillation cycle. (B) The periodic extension of the data shown in A highlights the oscillatory nature of the dynamics. (C) The periodic extension of Fig. 6B and E depicts regular oscillations and activity waves of both NICD and Dll1 from caudal to rostral that are out of phase with each other. (D) Kymograph to show the intensity of Notch1 alone within the magnified region from B. (E) The average signal intensity for Notch1, Dll1, activated NICD and Lfng in caudal regions of the periodically extended kymographs is plotted as a function of time. Note that parentheses in the key denote the corresponding paired sample. Hence, for instance, Dll1 appears twice, as it is paired with both NICD and Lfng(i). a.u. arbitrary units. (F) A proposed model. Pulses of Notch1 protein followed closely by Dll1 originate in the caudal PSM, which initiates a sequence of events detected as spatially separated events along the PSM, depicted as coloured bands of expression. The red boxed region that is magnified to the right contains coloured boxes to show the details of events in each colour band in the PSM. Blue, Notch is translated and translocated to cell membranes; green, Notch 1 and Dll1 are translated and translocate to cell membranes, leading to transactivation; orange, transactivation leads to cleavage and release of NICD, which translocates to the nucleus and activates downstream transcription of Notch target genes (including Notch1, Lfng and Hes7), while Dll1 and/or extracellular Notch1 are endocytosed to be recycled or degraded. Phase 1, 2 and 3 refer to well-established phases of Lfng/NICD expression domains.