Dynamic Delta-like1 expression in presomitic mesoderm cells during somite segmentation

Highlights

• New fluorescent reporter mice enable live-imaging of Dll1 protein expression.

• Dll1 oscillation propagates from the posterior to anterior presomitic mesoderm.

• Dll1 expression oscillates on the surface of presomitic mesoderm cells.

• The Hes7 response to Dll1 becomes slower in the anterior PSM.

Abstract

During somite segmentation, the expression of clock genes such as Hes7 oscillates synchronously in the presomitic mesoderm (PSM). This synchronous oscillation slows down in the anterior PSM, leading to wave-like propagating patterns from the posterior to anterior PSM. Such dynamic expression depends on Notch signaling and is critical for somite formation. However, it remains to be determined how slowing oscillations in the anterior PSM are controlled, and whether the expression of the Notch ligand Delta-like1 (Dll1) oscillates on the surface of individual PSM cells, as postulated to be responsible for synchronous oscillation. Here, by using Dll1 fluorescent reporter mice, we performed live-imaging of Dll1 expression in PSM cells and found the oscillatory expression of Dll1 protein on the cell surface regions. Furthermore, a comparison of live-imaging of Dll1 and Hes7 oscillations revealed that the delay from Dll1 peaks to Hes7 peaks increased in the anterior PSM, suggesting that the Hes7 response to Dll1 becomes slower in the anterior PSM. These results raise the possibility that Dll1 protein oscillations on the cell surface regulate synchronous Hes7 oscillations, and that the slower response of Hes7 to Dll1 leads to slower oscillations in the anterior PSM.

Introduction

Cell-cell interactions play an important role in coordinating gene expression during tissue morphogenesis. One such example is the somite segmentation process, in which a bilateral pair of somites is generated periodically by segmentation of the anterior regions of the presomitic mesoderm (PSM). This periodic event is controlled by the segmentation clock genes, such as the transcriptional repressors Hes1 and Hes7, whose expression oscillates synchronously between neighboring cells (Hubaud and Pourquié, 2014; Kageyama et al., 2012; Oates et al., 2012). This synchronous oscillatory gene expression slows in the anterior PSM, thereby leading to wave-like propagating patterns from the posterior to anterior PSM. This dynamic expression is critical for somite formation, as dampening and/or desynchronization of the oscillations leads to segmentation defects, causing the fusion of somite-derived tissues such as the vertebrae and ribs (Takashima et al., 2011; Niwa et al., 2011). When PSM cells are dissociated, oscillatory gene expression becomes unstable and desynchronized, suggesting that cell-cell interactions play an important role in synchronized oscillations (Maroto et al., 2005; Masamizu et al., 2006).

Notch signaling regulates such cell-cell interactions; the Notch ligand Delta-like1 (Dll1) activates Notch signaling in neighboring cells, in which Hes1 and Hes7 are induced (Artavanis-Tsakonas et al., 1999; Jiang et al., 2000; Horikawa et al., 2006; Riedel-Kruse et al., 2007; Mara et al., 2007; Özbudak and Lewis, 2008; Kageyama et al., 2012). Hes1 and Hes7 expression oscillates with a periodicity of ~2 h by negative feedback in mouse PSM cells (Hirata et al., 2002; Bessho et al., 2003), and these oscillations lead to the oscillatory expression of Dll1 (Shimojo et al., 2016). Optogenetic induction of Dll1 oscillations can entrain Hes1 oscillations in neighboring cells (Isomura et al., 2017), suggesting that Dll1 oscillations regulate synchronous oscillations in the PSM. Indeed, when dissociated PSM cells are re-aggregated, they exhibit synchronous oscillations in a Notch signaling-dependent manner (Tsiairis and Aulehla, 2016). Furthermore, when Dll1 oscillations are dampened, Hes7 oscillations are also dampened, resulting in severe somite fusion (Shimojo et al., 2016). All of these data suggest that Dll1 oscillation-dependent cell-cell interactions are essential for coordinated gene expression during somite segmentation.

Despite the above findings, the dynamics of Dll1 expression in the PSM are still obscure. Particularly, it has been difficult to analyze Dll1 protein dynamics, because Dll1 protein oscillations exhibit amplitudes that are too small to be detected by immunostaining (Okubo et al., 2012), but they have been reliably detected by time-lapse imaging (Shimojo et al., 2016). Furthermore, Dll1 protein, as a ligand, should function on the cell surface, but the oscillatory expression of Dll1 protein in the PSM has only been shown at the tissue level (Bone et al., 2014; Shimojo et al., 2016). Dll1 protein is present not only on the cell surface but also in cytoplasmic vesicles including the Golgi apparatus (Geffers et al., 2007); therefore, the total expression levels of Dll1 protein in the PSM do not reflect the functional Dll1 expression levels. We previously examined Dll1 expression dynamics by using Dll1 reporter mice, in which luciferase cDNA was inserted into the Dll1 gene so that Dll1-luciferase fusion protein was expressed from the endogenous locus. Although we confirmed Dll1 protein oscillations in the PSM (Shimojo et al., 2016), it was technically difficult to examine its expression at the cellular level, because luciferase-dependent luminescence imaging exhibits only a low spatial resolution.

Here, to overcome this problem, we used a new Dll1 reporter mouse line (Dll1-Venus-T2A-mCherry mice), in which Venus fluorescent protein cDNA was inserted into the Dll1 gene so that a Dll1-Venus fusion protein was expressed from the endogenous Dll1 locus (Tateya et al., 2019). We performed time-lapse imaging using these mice to examine Dll1 protein expression dynamics at the cellular level and the mechanism of how oscillatory gene expression slows in the anterior PSM.