Publication information 7LWOH Ca2+ coding and decoding strategies for the specification of neural and renal precursor cells during development $XWKRU V Moreau, Marc; Neant, Isabelle; Webb, Sarah E.; Miller, Andrew L.; Riou, Jean-Francois; Leclerc, Catherine 6RXUFH Cell Calcium , v. 59, March 2016, p. 75-83 9HUVLRQ Pre-published version '2, https://doi.org/10.1016/j.ceca.2015.12.003 3XEOLVKHU Elsevier Copyright information © 2016 Elsevier Notice This version is available at HKUST Institutional Repository via http://hdl.handle.net/1783.1/78363 If it is the author’s pre-published version, changes introduced as a result of publishing processes such as copy-editing and formatting may not be reflected in this document. For a definitive version of this work, please refer to the published version. http://repository.ust.hk/ir/ This is the Pre-Published Version Moreau et al, review in Cell Calcium 2015 Titre : Ca2+ coding and decoding strategies for the specification of neural and renal progenitor cells during development. Authors : Marc Moreau, Isabelle Néant, Sarah E. Webb, Andrew L. Miller, Jean-François Riou, and Catherine Leclerc Corresponding author: Catherine Leclerc Introduction Calcium (Ca2+) signalling has long been reported to play a role in the early development of vertebrate embryos [1-3]. During embryogenesis, a rise in intracellular Ca2+ is known to be a widespread trigger for directing stem cells towards a specific tissue fate, but the precise Ca2+ signalling mechanisms involved in achieving these pleiotropic effects are still poorly understood. In this review, we compare the Ca2+ signalling pathways that are involved in regulating neural determination, which is the first step for both neural development, (neurogenesis) and kidney development (nephrogenesis). Figure 1 is a schematic representation of the Ca2+ signals that are known to be generated in the amphibian Xenopus laevis in the dorsal ectoderm during the process of neural induction, and in the dorso-lateral mesoderm during the specification of the embryonic kidney. Early neurogenesis / neural induction It is important to bear in mind that although neural induction is considered to be similar (from the point of view of the morphological events and signalling pathways involved), for most if not all vertebrates, in reality only a few species have been studied in great detail. Indeed, much of the research on neural induction has been conducted with amphibian embryos. Traditionally, Xenopus sp. has been used as an animal model for investigating the inductive events and cellular mechanisms that occur during organogenesis because the embryos are easy to obtain in high numbers and are relatively large in size. Thus Xenopus embryos are an especially easy model system to use when performing microinjection and microsurgery. Neural induction occurs during gastrulation and the neural tissues, like the epidermal tissues, are derived from the ectoderm. During gastrulation in vertebrates, the ectodermal 1 This is the Pre-Published Version Moreau et al, review in Cell Calcium 2015 cells give rise to epidermal progenitors on the ventral side of the embryo and to neural progenitors in the dorsal side. This binary choice of cell fate during neural induction is controlled by complex mechanisms that involve both positive effectors (such as fibroblast growth factors, FGFs) and negative effectors (such as bone morphogenetic proteins, BMPs; Wingless/Int proteins, Wnts and Nodal) [4-6]. One of the key regulatory mechanisms involved in the conversion of ectoderm into neuroectoderm is the inhibition of the BMP pathway by noggin, chordin, and follistatin, which are factors secreted by the dorsal mesoderm. Following neural induction, the neuroectoderm develops into the neural plate, which consists of undifferentiated dividing neuroepithelial cells, and then later in development these cells exit the cell cycle and differentiate into neurons and glial cells. For the purposes of this article, however, we are limiting the topic of our review to that of neural induction, as this is the stage that specifically involves embryonic stem cells. With a combination of the bioluminescent Ca2+ reporter, aequorin, and a custom- designed luminescence imaging microscope, it has previously been shown that during 2+ 2+ gastrulation in amphibian embryos, a gradual elevation of intracellular Ca ([Ca ]i) and a series of superimposed rapid Ca2+ transients are generated exclusively in the dorsal ectoderm cells (the tissue where neural induction takes place). On the other hand, neither the Ca2+ elevation nor the distinct Ca2+ transients are generated in the ventral ectoderm cells, which are at the origin of the epidermis [7, 8]. The onset of this Ca2+ signalling activity occurs at the blastula stage, long before the start of gastrulation (i.e., before mesoderm invagination). These observations have been confirmed in Xenopus [9] and have also been demonstrated in the chick [10] suggesting that neural induction starts before gastrulation. In Xenopus embryos, the spontaneous Ca2+ transients are initially localized in the most anterior part of the dorsal ectoderm and the accumulation pattern of these Ca2+ transients over time correlates with the formation of the prospective neuroectoderm. In addition, as gastrulation proceeds, the Ca2+ transients increase both in number and intensity, so that they reach a peak activity by mid- gastrulation, a stage where neural determination is thought to have occurred [8]. Therefore, the Ca2+ transients observed in the dorsal ectoderm constitute the first directly visualized events linked to neural induction (Figure 1A, B). The animal cap model 2 This is the Pre-Published Version Moreau et al, review in Cell Calcium 2015 In blastula stage Xenopus embryos, the animal cap constitutes the region around the animal pole, which is destined to form the ectoderm during normal development. This tissue retains it pluripotent nature and it can be easily dissected and maintained in simple culture medium. Upon exposure to specific inducers, however, the animal cap can differentiate into neural, mesodermal, or endodermal tissues. In this way, therefore, the cells of the animal cap have been described as being equivalent to mammalian embryonic stem cells [11]. Animal caps cultured in vitro have been shown to reproduce the in vivo induction of amphibian tissues and they are therefore considered to be a very useful tool for investigating the differentiation mechanisms that occur in normal embryonic development. Indeed, experiments with animal caps have demonstrated that in the amphibian embryo, Ca2+ is a necessary and sufficient to convert ectoderm into neuroectoderm [8]. In addition, animal caps have been induced to differentiate into neurons and glial cells following short term culture in the presence of caffeine, which triggers a rise of intracellular Ca2+ [12]. This ex vivo assay has also been used to further characterize the Ca2+ signalling pathways that lead to the expression of neural specific genes. Implication of calcium channels. The ability of ectoderm cells to be induced to differentiate into neural tissue (called neural competence) [13, 14], is acquired shortly before gastrulation and is then lost during the late gastrula stages. In amphibians, neural competence is associated with the expression of 2+ functional CaV1.2 channels (i.e., L-type voltage-dependent Ca channels) in the plasma membrane [15, 16]. The highest density of CaV1.2 channels is reached at mid-gastrula, when neural competence of the ectoderm is optimal. There is subsequently a decrease in the density of CaV1.2 channels, which occurs simultaneously with the normal loss of competence, at the end of gastrulation. This temporal pattern of CaV1.2 channel expression correlates with the dynamic pattern of Ca2+ transients. When the function of CaV1.2 channels was inhibited by treatment with specific antagonists during gastrulation, there was a concomitant inhibition in the generation of Ca2+ transients and a decrease in the resting level of intracellular Ca2+, which suggests that the Ca2+ transients might be generated via the activation of CaV1.2 channels [7, 8]. In addition, the inhibition of these Ca2+ transients induced a downregulation of at least two of the early neural genes (i.e., zic3 and geminin) as well as severe abnormalities in the anterior nervous system. 3 This is the Pre-Published Version Moreau et al, review in Cell Calcium 2015 The most apparent defects were observed in the head including a reduction in the size or total absence of eyes, as well as a lack of melanophores [8, 17]. Thus, in the naïve ectoderm cells 2+ of Xenopus, an influx of Ca through CaV1.2 channels is likely to be the main component of 2+ the signalling pathway that induces the changes in [Ca ]i observed both in vivo and ex vivo during neural induction [18]. However, It should be noted that members of the TRP (transient receptor potential) channel family, particularly TRPC1, are also likely to be involved [19]. Acquisition of neural fate in amphibians therefore appears to require the expression of functional CaV1.2 channels in the ectoderm. This finding, however, prompted several new 2+ questions: (1) Given that CaV1.2 channels are voltage-operated Ca channels, what is the mechanism by which these channels are specifically activated in the dorsal ectoderm during the process of neural induction? (2) What are the genes activated downstream of the Ca2+ signals? (3) Are the Ca2+ signals that are involved in neural induction, conserved among