- Email: [email protected]
Covert Prepatterning of a Cell Division Wave
Developmental Cell
Previews Covert Prepatterning of a Cell Division Wave Michael Veeman1,* 1Division of Biology, Kansas State University, Manhattan, KS 66506, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.04.005
A directional wave of mitosis, progressing posterior to anterior across the epidermis, is important for neural tube closure in the invertebrate chordate Ciona intestinalis. In this issue of Developmental Cell, Ogura and Sasakura (2016) show that the patterning of this wave unexpectedly has complex origins in the previous cell cycle. Cell division is one of many cell behaviors under fine spatial and temporal control in developing embryos. Localized proliferation has an easily intuited importance, but there are also many cases in which cell division has to pause while complex tissue rearrangements are underway or in which the cell cycle is tightly coordinated with other morphogenetic cell behaviors (Duncan and Su, 2004). One example of this is seen in the simple embryos of the invertebrate ascidian chordate Ciona intestinalis. The early cleavages in Ciona are very rapid, giving rise to an embryo that undergoes the stereotypically chordate invagination and zippering of a neural plate into a hollow, dorsal neural tube only 7 hr after fertilization. Ogura, Sasakura, and colleagues previously showed that the 11th epidermal cell division occurs in a posterior-toanterior wave that matches the direction of neural tube zippering (Ogura et al., 2011). They found that this coincides with the insertion of a long G2 phase into the cell cycle and that experimental manipulations of the epidermal cell cycle can block neural tube closure. The posterior-to-anterior wave in the 11th cell cycle was thought to be the earliest sign of patterned cell division in the epidermal lineage. However, in a study published in this issue of Developmental Cell, Ogura and Sasakura (2016) make the remarkable observation that spatial variation in both G2- and S-phase length are already present in the 10th cell cycle but that S-phase length is longest in the anterior, whereas G2 length is longest in the posterior. The differences in G2- and S-phase lengths at this 10th cell cycle effectively cancel each other out to result in a cell cycle of uniform overall duration from anterior to posterior. It is then in the 11th cell cycle that G2-phase
length becomes uniformly long from posterior to anterior, while the spatial differences in S-phase length persist, thus giving rise to the distinct wave of division from posterior to anterior. The authors speculate that this striking compensatory mechanism for covertly prepatterning a subsequent overt wave of division might be important for ensuring a particularly rapid transition from synchronous to spatially patterned mitoses. The discovery by Ogura and Sasakura (2016) of this cryptic, compensated prepattern in cell-cycle timing hinged on a carefully selected fluorescent reporter: a GFP-tagged version of PCNA (Proliferating Cell Nuclear Antigen) that brightly labels replication foci and can thus distinguish cells in S phase from cells in G1 or G2. It also required the delicate live imaging of many replicate embryos coupled with extensive quantitative analysis. Ogura and Sasakura (2016) were aided in this by the unique attributes of the Ciona embryo, which has a stereotypically chordate body plan but is small and simple enough to be imaged in its entirety, with fine subcellular detail, in a single confocal stack. The identification of the cell-cycle compensation phenomenon discussed here provides an excellent new example of the compelling advantages of Ciona for quantitative approaches to chordate morphogenesis (reviewed by Veeman and Reeves, 2015). To identify the upstream cues controlling G2 length in the 10th and 11th epidermal cell cycles, Ogura and Sasakura (2016) take advantage of the strong tools for gene regulatory network analysis in Ciona (reviewed by Stolfi and Christiaen, 2012), including powerful community databases for identifying potential regulators and the ability to quickly test candidate enhancer regions in vivo by
electroporation. They find that G2 length in these cell cycles is under the control of the G2/M regulator Cdc25, which is initially expressed asymmetrically along the AP axis before being downregulated at the onset of neurulation. They show that Cdc25 is under transcriptional control by GATAb and AP-2like2, which are important ectodermal transcription factors that are themselves differentially expressed along the AP axis. Cdc25 has a long-established role in regulating mitotic entry in early embryos (Bouldin and Kimelman, 2014; Edgar et al., 1994), so it is not surprising that it controls G2 length in this context. What is more remarkable is the observation that S-phase length is also under fine spatial control. S-phase length increases dramatically post mid-blastula transition (MBT) in Drosophila, via global changes in cdk1 activity (Farrell et al., 2012), but it has not been widely considered as a potential mechanism for spatially regulating cell-cycle timing. Interestingly, one place where differential regulation of S-phase length has previously been seen is in the very early ascidian embryo, where the earliest cell-cycle asynchrony between animal and vegetal cells involves differential S-phase length downstream of b-catenin (Dumollard et al., 2013). The mechanism for the fine control of S-phase length in the epidermis is of great interest but has yet to be elucidated. Another important question for the future is the mechanism by which epidermal cell-cycle timing impacts neural tube closure. The authors find that if they induce a correctly spatially patterned but premature wave of mitosis, neural tube closure is perturbed. Ciona neural tube closure has recently been shown to involve the polarized accumulation of Developmental Cell 37, April 18, 2016 107
Developmental Cell
Previews F-actin in the zippering cells (Hashimoto et al., 2015). Ogura and Sasakura (2016) show this to be blocked when epidermal cell-cycle timing is manipulated, but the causal link between cell-cycle timing and F-actin accumulation remains unclear. One intriguing possibility is that the epidermis may be actively pushing dorsomedially to bring the neural folds together and that control of the epidermal cell cycle is essential for maintaining the balance of mechanical forces in the ectoderm. The small, simple Ciona embryo will be a powerful model to investigate the molecular details of how S-phase length is spatially patterned and the mechanistic details of how patterns of cell division in
the epidermis influence neural tube closure. The ability to easily image the full anterior-to-posterior length of a neurulating chordate embryo with subcellular detail made it more feasible to first detect this unusual cell-cycle compensation phenomenon in Ciona, but it will be interesting and important to determine whether similar mechanisms are used in other taxa.
Duncan, T., and Su, T.T. (2004). Curr. Biol. 14, R305–R307. Edgar, B.A., Lehman, D.A., and O’Farrell, P.H. (1994). Development 120, 3131–3143. Farrell, J.A., Shermoen, A.W., Yuan, K., and O’Farrell, P.H. (2012). Genes Dev. 26, 714–725. Hashimoto, H., Robin, F.B., Sherrard, K.M., and Munro, E.M. (2015). Dev. Cell 32, 241–255. Ogura, Y., and Sasakura, Y. (2016). Dev. Cell 37, this issue, 148–161.
REFERENCES
Ogura, Y., Sakaue-Sawano, A., Nakagawa, M., Satoh, N., Miyawaki, A., and Sasakura, Y. (2011). Development 138, 577–587.
Bouldin, C.M., and Kimelman, D. (2014). Cell Cycle 13, 2165–2171.
Stolfi, A., and Christiaen, L. (2012). Genetics 192, 55–66.
Dumollard, R., Hebras, C., Besnardeau, L., and McDougall, A. (2013). Dev. Biol. 384, 331–342.
Veeman, M., and Reeves, W. (2015). Genesis 53, 143–159.
Spinal Cords Built to Scale Benjamin L. Allen1,* 1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.04.007
An outstanding question in embryogenesis is how different-sized animals maintain similarly proportioned body plans. In this issue of Developmental Cell, Uygur et al. (2016) tackle this issue in the context of neural tube patterning, discovering that differential sensitivity to the Hedgehog pathway may provide one answer to this fundamental issue. One of the fundamental questions in developmental biology is: what regulates size? Cell size, organ size, and embryo size can all be dynamically regulated both within and across species; however, the mechanisms that control these processes remain largely unexplored, particularly in the context of the whole embryo, where the maintenance of similarly proportioned body plans in the face of significant differences in organism size is referred to as scaling. Recent studies have addressed scaling in the context of early, BMP-dependent dorsoventral patterning in the developing Xenopus embryo (Inomata et al., 2013), as well as via the Bicoid morphogen gradient in Drosophila embryos (Wu et al., 2015). This question has also been addressed during dorsoventral patterning of the vertebrate neural tube, where recent work suggested that altered differentia108 Developmental Cell 37, April 18, 2016
tion in neural cell progenitors is essential for regulating scaling in this tissue (Kicheva et al., 2014). Despite these studies, the fundamental mechanisms that control scaling in related organisms of different sizes remain unclear. In this issue of Developmental Cell, Uygur et al. (2016) investigate the role of the Hedgehog signaling pathway in scaling of the neural tube across different avian species, specifically comparing spinal cord patterning in zebra finch and chicken embryos, two species of significantly different embryonic and adult size. The authors initially analyzed the expression of three transcription factors (NKX2.2, OLIG2, and NKX6.1) that play essential roles in mediating cell fate specification in the neural tube and are themselves direct transcriptional targets of the HH pathway (Oosterveen et al., 2012; Peterson et al., 2012). Strikingly, patterning occurs faster
and across a smaller tissue field in the zebra finch embryo than in the chicken embryo. One might expect that the faster patterning in the zebra finch embryos could be due to relatively higher levels of HH ligand production; however, Sonic HH (SHH) levels are actually lower in the zebra finch neural tube compared to the chicken neural tube. These data suggested that zebra finch neuroepithelial cells are more sensitive to SHH levels than their chicken counterparts. To test this hypothesis, the authors obtained transgenic lines of both species that ubiquitously express GFP. From these animals, the authors created chimeric neural tubes, transplanting GFP-positive donor tissue into GFP-negative host tissue within neural tubes of embryos from the opposite species. Consistent with their hypothesis, the zebra finch neuroepithelial cells are more sensitive to endogenous SHH ligand than
Previews Covert Prepatterning of a Cell Division Wave Michael Veeman1,* 1Division of Biology, Kansas State University, Manhattan, KS 66506, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.04.005
A directional wave of mitosis, progressing posterior to anterior across the epidermis, is important for neural tube closure in the invertebrate chordate Ciona intestinalis. In this issue of Developmental Cell, Ogura and Sasakura (2016) show that the patterning of this wave unexpectedly has complex origins in the previous cell cycle. Cell division is one of many cell behaviors under fine spatial and temporal control in developing embryos. Localized proliferation has an easily intuited importance, but there are also many cases in which cell division has to pause while complex tissue rearrangements are underway or in which the cell cycle is tightly coordinated with other morphogenetic cell behaviors (Duncan and Su, 2004). One example of this is seen in the simple embryos of the invertebrate ascidian chordate Ciona intestinalis. The early cleavages in Ciona are very rapid, giving rise to an embryo that undergoes the stereotypically chordate invagination and zippering of a neural plate into a hollow, dorsal neural tube only 7 hr after fertilization. Ogura, Sasakura, and colleagues previously showed that the 11th epidermal cell division occurs in a posterior-toanterior wave that matches the direction of neural tube zippering (Ogura et al., 2011). They found that this coincides with the insertion of a long G2 phase into the cell cycle and that experimental manipulations of the epidermal cell cycle can block neural tube closure. The posterior-to-anterior wave in the 11th cell cycle was thought to be the earliest sign of patterned cell division in the epidermal lineage. However, in a study published in this issue of Developmental Cell, Ogura and Sasakura (2016) make the remarkable observation that spatial variation in both G2- and S-phase length are already present in the 10th cell cycle but that S-phase length is longest in the anterior, whereas G2 length is longest in the posterior. The differences in G2- and S-phase lengths at this 10th cell cycle effectively cancel each other out to result in a cell cycle of uniform overall duration from anterior to posterior. It is then in the 11th cell cycle that G2-phase
length becomes uniformly long from posterior to anterior, while the spatial differences in S-phase length persist, thus giving rise to the distinct wave of division from posterior to anterior. The authors speculate that this striking compensatory mechanism for covertly prepatterning a subsequent overt wave of division might be important for ensuring a particularly rapid transition from synchronous to spatially patterned mitoses. The discovery by Ogura and Sasakura (2016) of this cryptic, compensated prepattern in cell-cycle timing hinged on a carefully selected fluorescent reporter: a GFP-tagged version of PCNA (Proliferating Cell Nuclear Antigen) that brightly labels replication foci and can thus distinguish cells in S phase from cells in G1 or G2. It also required the delicate live imaging of many replicate embryos coupled with extensive quantitative analysis. Ogura and Sasakura (2016) were aided in this by the unique attributes of the Ciona embryo, which has a stereotypically chordate body plan but is small and simple enough to be imaged in its entirety, with fine subcellular detail, in a single confocal stack. The identification of the cell-cycle compensation phenomenon discussed here provides an excellent new example of the compelling advantages of Ciona for quantitative approaches to chordate morphogenesis (reviewed by Veeman and Reeves, 2015). To identify the upstream cues controlling G2 length in the 10th and 11th epidermal cell cycles, Ogura and Sasakura (2016) take advantage of the strong tools for gene regulatory network analysis in Ciona (reviewed by Stolfi and Christiaen, 2012), including powerful community databases for identifying potential regulators and the ability to quickly test candidate enhancer regions in vivo by
electroporation. They find that G2 length in these cell cycles is under the control of the G2/M regulator Cdc25, which is initially expressed asymmetrically along the AP axis before being downregulated at the onset of neurulation. They show that Cdc25 is under transcriptional control by GATAb and AP-2like2, which are important ectodermal transcription factors that are themselves differentially expressed along the AP axis. Cdc25 has a long-established role in regulating mitotic entry in early embryos (Bouldin and Kimelman, 2014; Edgar et al., 1994), so it is not surprising that it controls G2 length in this context. What is more remarkable is the observation that S-phase length is also under fine spatial control. S-phase length increases dramatically post mid-blastula transition (MBT) in Drosophila, via global changes in cdk1 activity (Farrell et al., 2012), but it has not been widely considered as a potential mechanism for spatially regulating cell-cycle timing. Interestingly, one place where differential regulation of S-phase length has previously been seen is in the very early ascidian embryo, where the earliest cell-cycle asynchrony between animal and vegetal cells involves differential S-phase length downstream of b-catenin (Dumollard et al., 2013). The mechanism for the fine control of S-phase length in the epidermis is of great interest but has yet to be elucidated. Another important question for the future is the mechanism by which epidermal cell-cycle timing impacts neural tube closure. The authors find that if they induce a correctly spatially patterned but premature wave of mitosis, neural tube closure is perturbed. Ciona neural tube closure has recently been shown to involve the polarized accumulation of Developmental Cell 37, April 18, 2016 107
Developmental Cell
Previews F-actin in the zippering cells (Hashimoto et al., 2015). Ogura and Sasakura (2016) show this to be blocked when epidermal cell-cycle timing is manipulated, but the causal link between cell-cycle timing and F-actin accumulation remains unclear. One intriguing possibility is that the epidermis may be actively pushing dorsomedially to bring the neural folds together and that control of the epidermal cell cycle is essential for maintaining the balance of mechanical forces in the ectoderm. The small, simple Ciona embryo will be a powerful model to investigate the molecular details of how S-phase length is spatially patterned and the mechanistic details of how patterns of cell division in
the epidermis influence neural tube closure. The ability to easily image the full anterior-to-posterior length of a neurulating chordate embryo with subcellular detail made it more feasible to first detect this unusual cell-cycle compensation phenomenon in Ciona, but it will be interesting and important to determine whether similar mechanisms are used in other taxa.
Duncan, T., and Su, T.T. (2004). Curr. Biol. 14, R305–R307. Edgar, B.A., Lehman, D.A., and O’Farrell, P.H. (1994). Development 120, 3131–3143. Farrell, J.A., Shermoen, A.W., Yuan, K., and O’Farrell, P.H. (2012). Genes Dev. 26, 714–725. Hashimoto, H., Robin, F.B., Sherrard, K.M., and Munro, E.M. (2015). Dev. Cell 32, 241–255. Ogura, Y., and Sasakura, Y. (2016). Dev. Cell 37, this issue, 148–161.
REFERENCES
Ogura, Y., Sakaue-Sawano, A., Nakagawa, M., Satoh, N., Miyawaki, A., and Sasakura, Y. (2011). Development 138, 577–587.
Bouldin, C.M., and Kimelman, D. (2014). Cell Cycle 13, 2165–2171.
Stolfi, A., and Christiaen, L. (2012). Genetics 192, 55–66.
Dumollard, R., Hebras, C., Besnardeau, L., and McDougall, A. (2013). Dev. Biol. 384, 331–342.
Veeman, M., and Reeves, W. (2015). Genesis 53, 143–159.
Spinal Cords Built to Scale Benjamin L. Allen1,* 1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.04.007
An outstanding question in embryogenesis is how different-sized animals maintain similarly proportioned body plans. In this issue of Developmental Cell, Uygur et al. (2016) tackle this issue in the context of neural tube patterning, discovering that differential sensitivity to the Hedgehog pathway may provide one answer to this fundamental issue. One of the fundamental questions in developmental biology is: what regulates size? Cell size, organ size, and embryo size can all be dynamically regulated both within and across species; however, the mechanisms that control these processes remain largely unexplored, particularly in the context of the whole embryo, where the maintenance of similarly proportioned body plans in the face of significant differences in organism size is referred to as scaling. Recent studies have addressed scaling in the context of early, BMP-dependent dorsoventral patterning in the developing Xenopus embryo (Inomata et al., 2013), as well as via the Bicoid morphogen gradient in Drosophila embryos (Wu et al., 2015). This question has also been addressed during dorsoventral patterning of the vertebrate neural tube, where recent work suggested that altered differentia108 Developmental Cell 37, April 18, 2016
tion in neural cell progenitors is essential for regulating scaling in this tissue (Kicheva et al., 2014). Despite these studies, the fundamental mechanisms that control scaling in related organisms of different sizes remain unclear. In this issue of Developmental Cell, Uygur et al. (2016) investigate the role of the Hedgehog signaling pathway in scaling of the neural tube across different avian species, specifically comparing spinal cord patterning in zebra finch and chicken embryos, two species of significantly different embryonic and adult size. The authors initially analyzed the expression of three transcription factors (NKX2.2, OLIG2, and NKX6.1) that play essential roles in mediating cell fate specification in the neural tube and are themselves direct transcriptional targets of the HH pathway (Oosterveen et al., 2012; Peterson et al., 2012). Strikingly, patterning occurs faster
and across a smaller tissue field in the zebra finch embryo than in the chicken embryo. One might expect that the faster patterning in the zebra finch embryos could be due to relatively higher levels of HH ligand production; however, Sonic HH (SHH) levels are actually lower in the zebra finch neural tube compared to the chicken neural tube. These data suggested that zebra finch neuroepithelial cells are more sensitive to SHH levels than their chicken counterparts. To test this hypothesis, the authors obtained transgenic lines of both species that ubiquitously express GFP. From these animals, the authors created chimeric neural tubes, transplanting GFP-positive donor tissue into GFP-negative host tissue within neural tubes of embryos from the opposite species. Consistent with their hypothesis, the zebra finch neuroepithelial cells are more sensitive to endogenous SHH ligand than