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Making the Clock Tick: Right Time, Right Pace
Developmental Cell
Previews Making the Clock Tick: Right Time, Right Pace Alexis Hubaud1 and Olivier Pourquie´1,* 1Institut de Ge ´ ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS UMR 7104, INSERM U964, Universite´ de Strasbourg, Illkirch F-67400, France *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2013.01.004
Two studies from Delaune et al. (2012) and Harima et al. (2012), published in Developmental Cell and Cell Reports, respectively, used elegant genetic and imaging techniques to shed new light on the role of the Notch pathway in regulating the pace and synchronization of the segmentation clock. The vertebrate body axis is organized in a serial repetition of segments, as evidenced, for instance, by the structure of the spine. This organization is established during embryogenesis by the sequential addition of structures called somites: at periodic intervals, a block of cells is pinched off from the paraxial mesoderm and forms a new segment (Pourquie´, 2011). A molecular oscillator known as the segmentation clock controls this process and dictates its periodicity (Oates et al., 2012). Each cell undergoes autonomous cyclic activation of the Notch, Wnt, and Fgf pathways, leading to periodic activation of a subset of their target genes (Pourquie´, 2011; Oates et al., 2012). At the tissue level, these oscillations are integrated first by local synchronization with the neighboring cells and then by an interaction with signaling gradients spanning the presomitic mesoderm. This results in kinematic gene-expression waves that are initiated in the posterior mesoderm and that travel and finally stop to form a new segment at the anterior end (Figure 1). How these oscillations are dynamically regulated and locally synchronized is not fully understood. Notch signaling plays an important role in this process: an autoregulatory feedback loop with the Hes/Her transcription factors has been proposed to act as a pacemaker in zebrafish and to a lesser extent in mice (Oates et al., 2012). Two recent papers have provided new insights into the role of the Notch pathway in vertebrate segmentation. In the first paper, published in a recent issue of Developmental Cell, Delaune et al. (2012) directly demonstrate the requirement for Notch to synchronize the oscillations of presomitic cells by using a zebrafish reporter with single-cell resolution. In the second study, recently published in
Cell Reports, Harima et al. (2012) confirm the role of the cyclic gene Hes7 in setting up the pace of the segmentation clock by engineering mice with accelerated Hes7 oscillations. The synchronization of cell oscillations by Notch signaling has been a longstanding hypothesis (Jiang et al., 2000; Horikawa et al., 2006). The original observation that interfering with the Notch pathway in zebrafish results in a saltand-pepper pattern of cyclic genes led to the suggestion that Notch is involved in synchronizing oscillations among neighboring cells (Jiang et al., 2000). However, direct evidence from live reporters at a single-cell resolution was lacking. To test this hypothesis, Delaune and colleagues (2012) created a fluorescent reporter comprising the regulatory sequences of the cyclic gene her1 and a fusion HER1-Venus for the segmentation clock. Despite the short period of oscillations in zebrafish, this tool accurately reported the clock activity and enabled live imaging at a single-cell resolution. Although reporters for the segmentation clock have already been developed by the Kageyama and Pourquie´ groups (Oates et al., 2012), this tool achieves single-cell resolution and constitutes a significant improvement in the field. Delaune and coauthors (2012) used mutants for the Notch pathway in combination with their reporter and computed the oscillation phases of single cells. In the absence of Notch signaling, presomitic cells continued to cycle as suggested by the salt-and-pepper expression pattern detected by in situ hybridization. Importantly, by calculating the phase shift between neighboring cells, they showed that without Notch signaling, the cells oscillated in an asynchronous manner. Therefore, the authors conclusively
demonstrated that the Notch pathway is required for the synchronization of cellular oscillators in zebrafish. Interestingly, this role of Notch signaling appears to be conserved in mice (Okubo et al., 2012). Previous reports suggested that Notchdependent synchronization was important to counteract the effect of noise due to stochasticity in gene expression or cell division (Horikawa et al., 2006). The resolution of this new reporter enabled Delaune and colleagues (2012) to address this question. By analyzing cell divisions and oscillations, they showed that mitosis mostly disrupted the synchrony between sibling cells and their neighbors. The authors observed a resynchronization of the sibling cells with their neighbors within two oscillation cycles. Whereas cell division was proposed to act as a source of noise in the system (Horikawa et al., 2006), their data show that sibling cells were significantly more synchronized with each other than with their neighbors during the oscillation cycle following their division. Surprisingly, this tighter synchrony was also observed in the Notch mutants, suggesting that the segregation of clock components is sufficient to control the oscillations of sibling cells. Moreover, cell division preferentially occurs when sibling cells and their neighbors are in the trough phase (‘‘off state’’) of the reporter oscillation. Thus, Delaune and colleagues (2012) proposed that a link between the cell cycle and the segmentation clock keeps the oscillations of sibling cells coupled despite mitosis. How the daughter cells remain synchronized without Notch signaling will provide new insights into the mechanism controlling these oscillations. The Notch pathway plays a crucial role not only in the synchronization of the clock, as shown by Delaune et al. (2012),
Developmental Cell 24, January 28, 2013 ª2013 Elsevier Inc. 115
Developmental Cell
Previews
Figure 1. Segmentation Clock in Vertebrates Hes/Her gene expression (in blue) oscillates in the posterior presomitic mesoderm with a similar period as somite formation. In the absence of Notch signaling in zebrafish, those oscillations become asynchronous, whereas partial removal of Hes7 introns in mice reduces their period.
but also in setting its periodicity. Indeed, Notch activates negative regulators, such as the Her/Hes factors in the presomitic mesoderm. Modeling has suggested that this negative-feedback loop can drive oscillations with appropriate timing of gene expression (Lewis, 2003). Notably, proper time delay between Notch induction and Notch repression leads to sustained oscillations. Such delay can originate from transcription, splicing, or translation of Her/Hes factors. This model has been strengthened by previous work by the Kageyama group, who increased the stability of Hes7 in mice or shortened the time required for its synthesis by removing all Hes7 introns (Takashima et al., 2011). These modifications of the time delay abolished Hes7 oscillations and strongly affected segmentation. However, it was argued that such drastic effects convey little information about the internal dynamics of the oscillator (Oates et al., 2012). Thus, to study the behavior of the
oscillator without breaking it, Harima and colleagues (2012) generated mice with a Hes7 transgene containing only its last intron. Whereas an intronless transgene led to major segmentation defects (Takashima et al., 2011), removal of two of the three introns caused a milder phenotype with an increased number of somites and rostral vertebrae and fusion of the caudal segments. To precisely show that the mice mutants had a more rapid segmentation clock, the authors used a reporter for Hes7 cycling and showed that the removal of introns leads to a slightly shorter period of Hes7 oscillations. These results from Harima et al. (2012) indicate that proper timing of Hes7 expression is essential in controlling the tempo of the segmentation clock. Unlike previous descriptions of mice and zebrafish mutants with a lengthened clock period (Oates et al., 2012), this study reports a mutant with accelerated pace of segmentation. It is therefore tantalizing to speculate that Hes7 consti-
116 Developmental Cell 24, January 28, 2013 ª2013 Elsevier Inc.
tutes the pacemaker of mouse segmentation. However, several lines of evidence suggest the existence of other players in mice (Pourquie´, 2011; Oates et al., 2012). For instance, mice overexpressing NICD have normal segmentation despite the apparent loss of Hes7 oscillations. In addition, it is unclear how the Hes7 module interacts with other cycling genes and with the gradients of morphogens such as Fgf, which initiates Hes7 oscillations. Further studies should address these questions, as well as the precise mechanism by which the clock conveys information to define a new somite. Indeed, recent experiments have elegantly shown that the phase gradient, not the period, of the clock is the key parameter in determining the segment size (Lauschke et al., 2013). Similar advances in imaging, manipulating, and modeling the signaling pathways are thus essential for further expanding our understanding of vertebrate segmentation.
REFERENCES Delaune, E.A., Franc¸ois, P., Shih, N.P., and Amacher, S.L. (2012). Dev. Cell 23, 995–1005. Harima, Y., Takashima, Y., Ueda, Y., Ohtsuka, T., and Kageyama, R. (2012). Cell Rep. Published online December 6, 2012. http://dx.doi.org/10.1016/j. celrep.2012.11.012. Horikawa, K., Ishimatsu, K., Yoshimoto, E., Kondo, S., and Takeda, H. (2006). Nature 441, 719–723. Jiang, Y.J., Aerne, B.L., Smithers, L., Haddon, C., Ish-Horowicz, D., and Lewis, J. (2000). Nature 408, 475–479. Lauschke, V.M., Tsiairis, C.D., Franc¸ois, P., and Aulehla, A. (2013). Nature 493, 101–105. Lewis, J. (2003). Curr. Biol. 13, 1398–1408. Oates, A.C., Morelli, L.G., and Ares, S. (2012). Development 139, 625–639. Okubo, Y., Sugawara, T., Abe-Koduka, N., Kanno, J., Kimura, A., and Saga, Y. (2012). Nat Commun 3, 1141. Pourquie´, O. (2011). Cell 145, 650–663. Takashima, Y., Ohtsuka, T., Gonza´lez, A., Miyachi, H., and Kageyama, R. (2011). Proc. Natl. Acad. Sci. USA 108, 3300–3305.
Previews Making the Clock Tick: Right Time, Right Pace Alexis Hubaud1 and Olivier Pourquie´1,* 1Institut de Ge ´ ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS UMR 7104, INSERM U964, Universite´ de Strasbourg, Illkirch F-67400, France *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2013.01.004
Two studies from Delaune et al. (2012) and Harima et al. (2012), published in Developmental Cell and Cell Reports, respectively, used elegant genetic and imaging techniques to shed new light on the role of the Notch pathway in regulating the pace and synchronization of the segmentation clock. The vertebrate body axis is organized in a serial repetition of segments, as evidenced, for instance, by the structure of the spine. This organization is established during embryogenesis by the sequential addition of structures called somites: at periodic intervals, a block of cells is pinched off from the paraxial mesoderm and forms a new segment (Pourquie´, 2011). A molecular oscillator known as the segmentation clock controls this process and dictates its periodicity (Oates et al., 2012). Each cell undergoes autonomous cyclic activation of the Notch, Wnt, and Fgf pathways, leading to periodic activation of a subset of their target genes (Pourquie´, 2011; Oates et al., 2012). At the tissue level, these oscillations are integrated first by local synchronization with the neighboring cells and then by an interaction with signaling gradients spanning the presomitic mesoderm. This results in kinematic gene-expression waves that are initiated in the posterior mesoderm and that travel and finally stop to form a new segment at the anterior end (Figure 1). How these oscillations are dynamically regulated and locally synchronized is not fully understood. Notch signaling plays an important role in this process: an autoregulatory feedback loop with the Hes/Her transcription factors has been proposed to act as a pacemaker in zebrafish and to a lesser extent in mice (Oates et al., 2012). Two recent papers have provided new insights into the role of the Notch pathway in vertebrate segmentation. In the first paper, published in a recent issue of Developmental Cell, Delaune et al. (2012) directly demonstrate the requirement for Notch to synchronize the oscillations of presomitic cells by using a zebrafish reporter with single-cell resolution. In the second study, recently published in
Cell Reports, Harima et al. (2012) confirm the role of the cyclic gene Hes7 in setting up the pace of the segmentation clock by engineering mice with accelerated Hes7 oscillations. The synchronization of cell oscillations by Notch signaling has been a longstanding hypothesis (Jiang et al., 2000; Horikawa et al., 2006). The original observation that interfering with the Notch pathway in zebrafish results in a saltand-pepper pattern of cyclic genes led to the suggestion that Notch is involved in synchronizing oscillations among neighboring cells (Jiang et al., 2000). However, direct evidence from live reporters at a single-cell resolution was lacking. To test this hypothesis, Delaune and colleagues (2012) created a fluorescent reporter comprising the regulatory sequences of the cyclic gene her1 and a fusion HER1-Venus for the segmentation clock. Despite the short period of oscillations in zebrafish, this tool accurately reported the clock activity and enabled live imaging at a single-cell resolution. Although reporters for the segmentation clock have already been developed by the Kageyama and Pourquie´ groups (Oates et al., 2012), this tool achieves single-cell resolution and constitutes a significant improvement in the field. Delaune and coauthors (2012) used mutants for the Notch pathway in combination with their reporter and computed the oscillation phases of single cells. In the absence of Notch signaling, presomitic cells continued to cycle as suggested by the salt-and-pepper expression pattern detected by in situ hybridization. Importantly, by calculating the phase shift between neighboring cells, they showed that without Notch signaling, the cells oscillated in an asynchronous manner. Therefore, the authors conclusively
demonstrated that the Notch pathway is required for the synchronization of cellular oscillators in zebrafish. Interestingly, this role of Notch signaling appears to be conserved in mice (Okubo et al., 2012). Previous reports suggested that Notchdependent synchronization was important to counteract the effect of noise due to stochasticity in gene expression or cell division (Horikawa et al., 2006). The resolution of this new reporter enabled Delaune and colleagues (2012) to address this question. By analyzing cell divisions and oscillations, they showed that mitosis mostly disrupted the synchrony between sibling cells and their neighbors. The authors observed a resynchronization of the sibling cells with their neighbors within two oscillation cycles. Whereas cell division was proposed to act as a source of noise in the system (Horikawa et al., 2006), their data show that sibling cells were significantly more synchronized with each other than with their neighbors during the oscillation cycle following their division. Surprisingly, this tighter synchrony was also observed in the Notch mutants, suggesting that the segregation of clock components is sufficient to control the oscillations of sibling cells. Moreover, cell division preferentially occurs when sibling cells and their neighbors are in the trough phase (‘‘off state’’) of the reporter oscillation. Thus, Delaune and colleagues (2012) proposed that a link between the cell cycle and the segmentation clock keeps the oscillations of sibling cells coupled despite mitosis. How the daughter cells remain synchronized without Notch signaling will provide new insights into the mechanism controlling these oscillations. The Notch pathway plays a crucial role not only in the synchronization of the clock, as shown by Delaune et al. (2012),
Developmental Cell 24, January 28, 2013 ª2013 Elsevier Inc. 115
Developmental Cell
Previews
Figure 1. Segmentation Clock in Vertebrates Hes/Her gene expression (in blue) oscillates in the posterior presomitic mesoderm with a similar period as somite formation. In the absence of Notch signaling in zebrafish, those oscillations become asynchronous, whereas partial removal of Hes7 introns in mice reduces their period.
but also in setting its periodicity. Indeed, Notch activates negative regulators, such as the Her/Hes factors in the presomitic mesoderm. Modeling has suggested that this negative-feedback loop can drive oscillations with appropriate timing of gene expression (Lewis, 2003). Notably, proper time delay between Notch induction and Notch repression leads to sustained oscillations. Such delay can originate from transcription, splicing, or translation of Her/Hes factors. This model has been strengthened by previous work by the Kageyama group, who increased the stability of Hes7 in mice or shortened the time required for its synthesis by removing all Hes7 introns (Takashima et al., 2011). These modifications of the time delay abolished Hes7 oscillations and strongly affected segmentation. However, it was argued that such drastic effects convey little information about the internal dynamics of the oscillator (Oates et al., 2012). Thus, to study the behavior of the
oscillator without breaking it, Harima and colleagues (2012) generated mice with a Hes7 transgene containing only its last intron. Whereas an intronless transgene led to major segmentation defects (Takashima et al., 2011), removal of two of the three introns caused a milder phenotype with an increased number of somites and rostral vertebrae and fusion of the caudal segments. To precisely show that the mice mutants had a more rapid segmentation clock, the authors used a reporter for Hes7 cycling and showed that the removal of introns leads to a slightly shorter period of Hes7 oscillations. These results from Harima et al. (2012) indicate that proper timing of Hes7 expression is essential in controlling the tempo of the segmentation clock. Unlike previous descriptions of mice and zebrafish mutants with a lengthened clock period (Oates et al., 2012), this study reports a mutant with accelerated pace of segmentation. It is therefore tantalizing to speculate that Hes7 consti-
116 Developmental Cell 24, January 28, 2013 ª2013 Elsevier Inc.
tutes the pacemaker of mouse segmentation. However, several lines of evidence suggest the existence of other players in mice (Pourquie´, 2011; Oates et al., 2012). For instance, mice overexpressing NICD have normal segmentation despite the apparent loss of Hes7 oscillations. In addition, it is unclear how the Hes7 module interacts with other cycling genes and with the gradients of morphogens such as Fgf, which initiates Hes7 oscillations. Further studies should address these questions, as well as the precise mechanism by which the clock conveys information to define a new somite. Indeed, recent experiments have elegantly shown that the phase gradient, not the period, of the clock is the key parameter in determining the segment size (Lauschke et al., 2013). Similar advances in imaging, manipulating, and modeling the signaling pathways are thus essential for further expanding our understanding of vertebrate segmentation.
REFERENCES Delaune, E.A., Franc¸ois, P., Shih, N.P., and Amacher, S.L. (2012). Dev. Cell 23, 995–1005. Harima, Y., Takashima, Y., Ueda, Y., Ohtsuka, T., and Kageyama, R. (2012). Cell Rep. Published online December 6, 2012. http://dx.doi.org/10.1016/j. celrep.2012.11.012. Horikawa, K., Ishimatsu, K., Yoshimoto, E., Kondo, S., and Takeda, H. (2006). Nature 441, 719–723. Jiang, Y.J., Aerne, B.L., Smithers, L., Haddon, C., Ish-Horowicz, D., and Lewis, J. (2000). Nature 408, 475–479. Lauschke, V.M., Tsiairis, C.D., Franc¸ois, P., and Aulehla, A. (2013). Nature 493, 101–105. Lewis, J. (2003). Curr. Biol. 13, 1398–1408. Oates, A.C., Morelli, L.G., and Ares, S. (2012). Development 139, 625–639. Okubo, Y., Sugawara, T., Abe-Koduka, N., Kanno, J., Kimura, A., and Saga, Y. (2012). Nat Commun 3, 1141. Pourquie´, O. (2011). Cell 145, 650–663. Takashima, Y., Ohtsuka, T., Gonza´lez, A., Miyachi, H., and Kageyama, R. (2011). Proc. Natl. Acad. Sci. USA 108, 3300–3305.