A Gene Regulatory Network Balances Neural and Mesoderm Specification during Vertebrate Trunk Development

摘要

class="head no_bottom_margin" id="sec1title">IntroductionCell fate decisions in developing tissues are made by gene regulatory networks comprising transcription factors and intercellular signals. These networks determine the rate of self-renewal and differentiation to ensure the balanced generation of different cell types and the production of well-proportioned tissues (, ). The formation of the vertebrate trunk, which extends progressively during embryogenesis, is one example. Successively more posterior neural and paraxial presomitic mesodermal (PSM) cells of the trunk are generated from a bipotential population of cells (), termed neuromesodermal progenitors (NMPs) at the posterior end of the embryo. Proliferation of NMPs fuels the elongation of axial tissues (, , , , , ). Hence, the rate at which NMPs are generated, self-renew, and differentiate must be carefully regulated in order to balance the production of different trunk tissues and to prevent the premature or delayed depletion of NMPs that will affect the length of the embryo.NMPs reside in the node-streak border (NSB), caudal lateral epiblast (CLE) and the chordoneural hinge (CNH) of elongating embryos (, , , , ). These regions express Wnt and FGF ligands (). Interfering with either signal results in the depletion of NMPs and the premature truncation of the body axis (href="#bib86" rid="bib86" class=" bibr popnode">Takada et al., 1994, href="#bib101" rid="bib101" class=" bibr popnode">Wilson et al., 2009, href="#bib108" rid="bib108" class=" bibr popnode">Yoshikawa et al., 1997, href="#bib97" rid="bib97" class=" bibr popnode">van de Ven et al., 2011). Both Wnt and FGF signaling are implicated in posteriorizing cells by inducing the Cdx transcription factors (TFs) that promote the expression of more posterior Hox genes (href="#bib98" rid="bib98" class=" bibr popnode">van den Akker et al., 2002, href="#bib63" rid="bib63" class=" bibr popnode">Nordstrom et al., 2006, href="#bib97" rid="bib97" class=" bibr popnode">van de Ven et al., 2011, href="#bib55" rid="bib55" class=" bibr popnode">Mazzoni et al., 2013, href="#bib59" rid="bib59" class=" bibr popnode">Neijts et al., 2016, href="#bib4" rid="bib4" class=" bibr popnode">Amin et al., 2016). Moreover, Wnt signaling is required for the differentiation of NMPs to mesodermal tissue (href="#bib53" rid="bib53" class=" bibr popnode">Martin and Kimelman, 2012, href="#bib29" rid="bib29" class=" bibr popnode">Garriock et al., 2015) and the loss of Wnt3a results in a depletion of mesodermal tissue in both mice and zebrafish (href="#bib108" rid="bib108" class=" bibr popnode">Yoshikawa et al., 1997, href="#bib51" rid="bib51" class=" bibr popnode">Martin and Kimelman, 2008, href="#bib29" rid="bib29" class=" bibr popnode">Garriock et al., 2015).Primitive streak and node cells transiently express the retinoic acid (RA)-synthesizing enzyme Aldh1a2 (href="#bib71" rid="bib71" class=" bibr popnode">Ribes et al., 2009). Mouse embryos lacking Aldh1a2 are truncated, suggesting a role for RA in axis elongation (href="#bib62" rid="bib62" class=" bibr popnode">Niederreither et al., 1999, href="#bib17" rid="bib17" class=" bibr popnode">Cunningham et al., 2015, href="#bib60" rid="bib60" class=" bibr popnode">Niederreither and Dolle, 2008, href="#bib25" rid="bib25" class=" bibr popnode">Duester, 2008) nevertheless, the role of RA in the establishment of NMPs remains unclear. At later stages of development, RA emanating from Aldh1a2-expressing somitic cells promotes the expression of genes characteristic of neural progenitors in the spinal cord. In addition, RA inhibits expression of both Wnt3a and Fgf8, and exposure of the tail region to increased concentrations of RA can arrest axis elongation (href="#bib65" rid="bib65" class=" bibr popnode">Olivera-Martinez et al., 2012). Excess RA concentration in the tail bud is prevented by the RA-metabolizing enzyme Cyp26a1, which is induced by Cdx genes and T/Brachyury under the control of Wnt/Fgf signaling (href="#bib52" rid="bib52" class=" bibr popnode">Martin and Kimelman, 2010, href="#bib100" rid="bib100" class=" bibr popnode">Vidigal et al., 2010, href="#bib75" rid="bib75" class=" bibr popnode">Savory et al., 2009, href="#bib109" rid="bib109" class=" bibr popnode">Young et al., 2009, href="#bib99" rid="bib99" class=" bibr popnode">van Rooijen et al., 2012). However, the overlapping functions and proximity of events hindered assigning direct and indirect activities to individual signaling pathways (href="#bib41" rid="bib41" class=" bibr popnode">Kimelman, 2016, href="#bib33" rid="bib33" class=" bibr popnode">Henrique et al., 2015, href="#bib31" rid="bib31" class=" bibr popnode">Gouti et al., 2015, href="#bib58" rid="bib58" class=" bibr popnode">Neijts et al., 2014).The co-expression of the TFs T/Brachyury (T/Bra) and Sox2 is characteristic of NMPs (href="#bib65" rid="bib65" class=" bibr popnode">Olivera-Martinez et al., 2012, href="#bib30" rid="bib30" class=" bibr popnode">Gouti et al., 2014, href="#bib94" rid="bib94" class=" bibr popnode">Tsakiridis et al., 2015, href="#bib102" rid="bib102" class=" bibr popnode">Wymeersch et al., 2016). Both Wnt and FGF signaling have been implicated as inducers of T/Bra in NMPs (href="#bib104" rid="bib104" class=" bibr popnode">Yamaguchi et al., 1999, href="#bib51" rid="bib51" class=" bibr popnode">Martin and Kimelman, 2008). Moreover, Cdx-binding regions have been found upstream of the T/Bra gene (href="#bib75" rid="bib75" class=" bibr popnode">Savory et al., 2009). This and subsequent analysis (href="#bib99" rid="bib99" class=" bibr popnode">van Rooijen et al., 2012) has led to the suggestion that Cdx proteins, induced by Wnt signaling, maintain T/Bra expression in NMPs, but are dispensable for its initial induction. However, Cdx proteins also appear to regulate Wnt and FGF expression in NMPs (href="#bib109" rid="bib109" class=" bibr popnode">Young et al., 2009, href="#bib75" rid="bib75" class=" bibr popnode">Savory et al., 2009, href="#bib99" rid="bib99" class=" bibr popnode">van Rooijen et al., 2012), thus the loss of T/Bra expression in the absence of Cdx might be due to the loss of these signals and the depletion of NMPs.Neural cells differentiating from NMPs downregulate T/Bra but maintain Sox2 expression (href="#bib30" rid="bib30" class=" bibr popnode">Gouti et al., 2014, href="#bib31" rid="bib31" class=" bibr popnode">Gouti et al., 2015, href="#bib92" rid="bib92" class=" bibr popnode">Tsakiridis and Wilson, 2015, href="#bib31" rid="bib31" class=" bibr popnode">Gouti et al., 2015). By contrast, as NMPs differentiate into mesoderm, expression of Sox2 is downregulated and Msgn1 and Tbx6 are upregulated to form nascent mesodermal progenitor cells (MPCs) (href="#bib13" rid="bib13" class=" bibr popnode">Chalamalasetty et al., 2011). Then, as cells commit to a PSM identity, expression of T/Bra is downregulated. In embryos lacking T/Bra, mesoderm induction fails and axis elongation halts (href="#bib34" rid="bib34" class=" bibr popnode">Herrmann et al., 1990). This is, at least in part, explained by a requirement for T/Bra for the induction of Msgn1 and Tbx6 (href="#bib104" rid="bib104" class=" bibr popnode">Yamaguchi et al., 1999, href="#bib103" rid="bib103" class=" bibr popnode">Yabe and Takada, 2012). Moreover, the loss of PSM tissue in the absence of Tbx6 or Msgn1 is accompanied by ectopic generation of neural tissue (href="#bib16" rid="bib16" class=" bibr popnode">Chapman and Papaioannou, 1998, href="#bib107" rid="bib107" class=" bibr popnode">Yoon et al., 2000, href="#bib14" rid="bib14" class=" bibr popnode">Chalamalasetty et al., 2014), raising the question of the role that the induction of these TFs plays in balancing neural and mesodermal production from NMPs.Taken together, the data suggest complex regulatory mechanisms with multiple interactions and feedbacks. It has proven challenging, however, to assemble a definitive network that explains the generation of NMPs and their balanced allocation toward mesodermal and neural tissue. These difficulties arise from the necessity of analyzing in vivo experimental perturbations in which axis elongation fails or the expression of signals is lost. To circumvent this, we have taken advantage of the in vitro directed differentiation that we and others have recently developed to generate NMPs from pluripotent stem cells (href="#bib30" rid="bib30" class=" bibr popnode">Gouti et al., 2014, href="#bib94" rid="bib94" class=" bibr popnode">Tsakiridis et al., 2015, href="#bib95" rid="bib95" class=" bibr popnode">Turner et al., 2014, href="#bib47" rid="bib47" class=" bibr popnode">Lippmann et al., 2015). This system decouples the development of NMPs and trunk cell types from the specific tissue architecture associated with axis elongation, thereby avoiding the difficulty of interpreting data from chimeric or morphologically abnormal embryos. Furthermore, it allows exogenous control of the supply and timing of signaling molecules. Thus, aspects of the gene regulatory network that are tightly linked in vivo can be separated and assayed in vitro.Using single-cell transcriptome analysis we first established the similarity between in vivo and in vitro derived NMPs. We then reverse engineered the transcriptional network responsible for NMP induction and differentiation. This revealed a network comprising the TFs Cdx1, 2, 4, T/Bra, Sox2, Msgn1, and Tbx6, which integrate Wnt and RA signaling to regulate entry to and exit from the NMP state. Mutation of individual or multiple components validated the network. Within the network, RA plays dual roles. Initially, RA is required for the expression of Sox2 and generation of NMPs; later, increased levels of RA drive neural differentiation. Cdx genes not only posteriorize cells but also, by restraining RA signaling, maintain T/Bra expression to allow mesoderm induction. Msgn1 and Tbx6 control the timing and outcome of NMP differentiation by cell-autonomously repressing T/Bra and Sox2 to propel mesoderm differentiation and by inducing the RA-producing enzyme Aldh1a2. The latter increases RA levels to non-autonomously promote neural differentiation and thereby provides regulative feedback that balances neural and mesoderm production. A dynamic model capturing these interactions demonstrates how the network coordinates the generation of the two cell types from the bipotential progenitor and ensures the well-proportioned generation of tissues necessary to build the vertebrate trunk.