Neural induction by the node and placode induction by head mesoderm share an initial state resembling neural plate border and ES cells Katherine E. Treversa,1,2, Ravindra S. Prajapatib,c,1, Mark Hintzeb,c,1, Matthew J. Stowera,1,3, Anna C. Strobla,4, Monica Tambalob,c,5, Ramya Ranganathanb,c, Natalia Moncauta,6, Mohsin A. F. Khana,7, Claudio D. Sterna,8, and Andrea Streitb,c,8 aDepartment of Cell and Developmental Biology, University College London, London WC1E 6BT, United Kingdom; bDivision of Craniofacial Development and Stem Cell Biology, King’s College London, London SE1 9RT, United Kingdom; and cCentre for Craniofacial and Regenerative Biology, King’s College London, London SE1 9RT, United Kingdom Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved November 29, 2017 (received for review November 16, 2017) Around the time of gastrulation in higher vertebrate embryos, suggesting that they may initially share common properties. inductive interactions direct cells to form central nervous system Both neural induction and PPR induction require FGF activity, (neural plate) or sensory placodes. Grafts of different tissues into as well as antagonism to BMP and Wnt signaling (2, 11–14), the periphery of a chicken embryo elicit different responses: although the relative contribution of each signal still needs to Hensen’s node induces a neural plate whereas the head mesoderm be unraveled. induces placodes. How different are these processes? Transcrip- tome analysis in time course reveals that both processes start by Significance induction of a common set of genes, which later diverge. These genes are remarkably similar to those induced by an extraembry- onic tissue, the hypoblast, and are normally expressed in the pre- It is generally believed that the outcome of many inductive gastrulation stage epiblast. Explants of this epiblast grown in the interactions occurring during development is largely de- absence of further signals develop as neural plate border deriva- pendent on the responding tissue, the source of the signals tives and eventually express lens markers. We designate this state playing a relatively minor part. Here, we compare induction of as “preborder”; its transcriptome resembles embryonic stem cells. the neural plate by the node, and of placodes by the head Finally, using sequential transplantation experiments, we show mesoderm, and show that both inducing tissues elicit a similar that the node, head mesoderm, and hypoblast are interchangeable initial response but that they later diverge. We characterize the to begin any of these inductions while the final outcome depends initial common state by a variety of methods and show its on the tissue emitting the later signals. similarity to ES cells, suggesting that these inductions may begin with a common “reprogramming” step. This initial state also shares many features in common with the border of the embryonic induction | organizer | gastrulation | embryonic stem cells | neural plate, suggesting that this region retains features of a pluripotency “ground state.” uring development, cell fate decisions are controlled by in- Author contributions: C.D.S. and A.S. designed research; K.E.T., R.S.P., M.H., M.J.S., A.C.S., Dductive interactions: instructive signals from one tissue M.T., and N.M. performed research; K.E.T., R.S.P., M.H., M.T., R.R., M.A.F.K., C.D.S., and cause a change of fate in adjacent responding cells. For example, A.S. analyzed data; and C.D.S. and A.S. wrote the paper. signals from Spemann’s organizer (Hensen’s node in amniotes) The authors declare no conflict of interest. can induce neural tissue from cells that otherwise develop into This article is a PNAS Direct Submission. epidermis (1, 2), and the zone of polarizing activity (ZPA) in- Published under the PNAS license. duces and patterns digits in the limb bud (3). Thus, each inducer Data deposition: The data reported in this paper have been deposited in the National elicits a distinct and specific response. However, classical trans- Center for Biotechnology Information Gene Expression Omnibus (GEO) database, https:// plantation experiments indicate that some inducers are in- www.ncbi.nlm.nih.gov/geo {accession no. GSE106346 for the complete dataset and acces- terchangeable: Hensen’s node and the ZPA alike result in digit sion nos. GSM2836311 [cPS_RNASeq (central epiblast from pre-streak)], GSM2836312 [aPS_RNASeq (anterior epiblast from prestreak)], GSM2836313 (induced-5h_RNASeq), duplication when grafted into the anterior limb bud (4). These GSM2836314 (uninduced-5h_RNASeq), GSM2836315 (neural plate RNASeq), and similar observations have led to the suggestion that, while GSM2836316 (anterior PPR_RNASeq), GSM2836317 (posterior PPR_RNASeq), and inducing signals may be universal, it is the responding tissue that GSM2836318 (nonneural epiblast_RNASeq)}. confers specificity to inductive events (5–7). Here, we explore 1K.E.T., R.S.P., M.H., and M.J.S. contributed equally to this work. two well-characterized inductive events that occur at approxi- 2Present address: Centre for Developmental Neurobiology, King’s College London, mately the same time, but under the influence of signals from London SE1 1UL, United Kingdom. different tissues: the induction of the neural plate (NP) by 3Present address: Department of Physiology, Anatomy and Genetics, University of Oxford, Hensen’s node and the induction of placode progenitors by the Oxford OX1 3QX, United Kingdom. lateral head mesoderm (lHM). 4Present address: Department of Pathology, University College London Cancer Institute, The neural plate gives rise to the entire central nervous London WC1E 6DD, United Kingdom. 5 system while sensory placodes contribute to the sense organs Present address: The Francis Crick Institute, London NW1 1AT, United Kingdom. and cranial ganglia and arise from the ectoderm surrounding 6Present address: Cancer Research UK Manchester Institute, The University of Manchester, the anterior neural plate [the preplacodal region (PPR)] (8, 9). Manchester M20 4BX, United Kingdom. 7 The neural plate is first defined by the appearance of Sox2,and Present addresses: Department of Experimental Cardiology and Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, 1105 AZ Am- the PPR becomes molecularly distinct at head fold stages by the sterdam, The Netherlands. Six1 Six4 Eya1 -2 expression of , ,and or .Atthisstage,neural 8To whom correspondence may be addressed. Email: [email protected] or andrea.streit@ and placode precursors continue to be intermingled with each kcl.ac.uk. BIOLOGY other as well as with future neural crest and epidermal cells in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. DEVELOPMENTAL a territory denominated the “neural plate border” (9, 10), 1073/pnas.1719674115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1719674115 PNAS | January 9, 2018 | vol. 115 | no. 2 | 355–360 Downloaded by guest on September 29, 2021 We have previously identified response genes to mesoderm- Fifty control and experimental tissues were collected, processed derived signals (15); we now use transcriptional profiling of for RNAseq, and analyzed for differentially expressed genes node-induced tissue, together with neural and preplacodal tissue (1.5-fold change, P < 0.05), which revealed a total of 2,477 dif- from normal embryos, to explore the similarities and differences ferentially expressed genes: 1,166 up-regulated and 1,311 down- between the two inductive events. We find that, despite the regulated in the induced epiblast (Fig. 1B and Dataset S1). difference in inducing tissues and in the ultimate outcome, the We noticed that many transcripts up-regulated in response to initial response to the node is largely identical to the response to an organizer graft (252, of which 25 are transcription factors) the lHM; this defines a distinct, common transcriptional state, were also identified in a recent screen for genes involved in which is very similar to the early prestreak epiblast and to the sensory placode induction [grafts of lateral head mesoderm neural plate border. When cells from the early epiblast are (lHM)] (15). This suggests considerable similarity between the allowed to develop in vitro without further induction, they dif- initial steps in neural induction by the node and PPR induction ferentiate into neural plate border derivatives: lens, neural crest, by the head mesoderm. To test this directly, we designed a and neural plate. To demonstrate that this “preborder” state is NanoString probe set containing known probes for neural, pre- common to neural and preplacodal induction, we show that in- placodal, neural plate border, and nonneural ectoderm cells, as well as other genes identified in our RNAseq data (Dataset S2). duction of this state by short exposure to either the node or the − Hensen’s node from HH4 or lHM from HH5/6 donors was lHM, as well as by the hypoblast (extraembryonic endoderm), − can be subsequently directed to each set of fates by short expo- grafted into the area opaca of HH4 hosts; after 3 and 5 h, the sure to the appropriate tissue. We propose that inductions by adjacent epiblast and noninduced epiblast from the contralateral different inducing tissues start by a reprogramming step that side were collected and processed for NanoString analysis. The converts cells to a common, preborder-like state which is also node induced 9 genes after 3 h, and the lHM induced 14; 8 of the similar to very early (pregastrulation) epiblast and to embryonic transcripts were represented in both sets (Fig. S1 and Dataset stem cells. Specific lineages diverge under the influence of later, S3). Five hours after grafting, the responses to the two tissues specific signals. started to diverge; 33% of the 123 induced genes were shared, and the lHM now induced many genes (51; 41%) that were not Results induced by the node (Dataset S3). Analysis of Genes Regulated by an Organizer Graft Reveals In summary, many early response genes induced by organizer Similarities Between Neural and Placodal Induction.