The pre-vertebrate origins of neurogenic placodes

Abstract

The sudden appearance of the neural crest and neurogenic placodes in early branching vertebrates has puzzled biologists for over a century. These embryonic tissues contribute to the development of the cranium and associated sensory organs, which were crucial for the evolution of the vertebrate "new head". A previous study suggests that rudimentary neural crest cells existed in ancestral chordates. However, the evolutionary origins of neurogenic placodes have remained obscure owing to a paucity of embryonic data from tunicates, the closest living relatives to those early vertebrates. Here we show that the tunicate Ciona intestinalis exhibits a proto-placodal ectoderm (PPE) that requires inhibition of bone morphogenetic protein (BMP) and expresses the key regulatory determinant Six1/2 and its co-factor Eya, a developmental process conserved across vertebrates. The Ciona PPE is shown to produce ciliated neurons that express genes for gonadotropin-releasing hormone (GnRH), a G-protein-coupled receptor for relaxin-3 (RXFP3) and a functional cyclic nucleotide-gated channel (CNGA), which suggests dual chemosensory and neurosecretory activities. These observations provide evidence that Ciona has a neurogenic proto-placode, which forms neurons that appear to be related to those derived from the olfactory placode and hypothalamic neurons of vertebrates. We discuss the possibility that the PPE-derived GnRH neurons of Ciona resemble an ancestral cell type, a progenitor to the complex neuronal circuit that integrates sensory information and neuroendocrine functions in vertebrates.

Extended Data Figure 1

Lineage information for Six1/2 expression in Ciona intestinalis from the gastrula to initial tailbud stage. a, Schematic of the anterior neural plate border at the mid-gastrula stage, including cell lineage nomenclature. Dmrt is initially activated in six blastomeres of 64-cell embryos (a7.9, a7.10 and a7.13). This lineage produces only the anterior neural plate (and the adjacent anterior neural plate border), which forms the PPE. The anterior-most ZicL⁺ cells of row IV (yellow) mark the boundary of the neural plate, which gives rise to the anterior sensory vesicle in tailbud embryos. The dotted line indicates the oral opening. b, Schematic of the anterior neural plate border during the gastrula–neurula transition, indicating the lineage-specific expression of Six1/2 (magenta). Six1/2 is initially expressed in eight cells comprising the posterior cells (row V) and the posterior lateral cells (row VI). c, Dorsal view of an initial tailbud embryo co-electroporated with Six1/2. mCherry, Dmrt. GFP and ZicL. CFP. At this stage, the cells initially expressing Six1/2 have divided once, giving rise to 16 cells in total. Brackets indicate the derivatives of the annotated lineages shown in b. Anterior is to the left.

Extended Data Figure 2

Six1/2⁺ cell morphogenesis in Ciona intestinalis from the initial tailbud stage to late tailbud I. a–c, Dorsal views of tailbud embryos electroporated with Six1/2. mCherry (magenta) and counterstained with phalloidin (blue). a, At the initial tailbud stage I (according to Hotta et al.), the Six1/2⁺ cells are arranged in a U shape, anterior to the neural plate. a9–c9, Underlined cell lineages are derived from the left side of the embryo. a9, Schematic indicates that the U shape is composed of 16 cells in total at the 11th generation (that is, a11.154, a11.138, etc.). There are no further divisions of these cells until after the late tailbud I stage. The green cells indicate a11.205, which are fated to become PPE-derived GnRH neurons. b, At the initial tailbud stage II, the lateral edges of the Six1/2⁺ cells begin to intercalate towards the midline. b9, The schematic shows a dotted circle where the future opening of the oral siphon forms. c, At the late tailbud I stage, theSix1/2⁺ cells have completed intercalation. The bright phalloidin signal in the centre of the pattern marks the apically localized actin of cells constricted towards the oral opening. The arrowheads indicate a PPE-derived cell fated to become a GnRH neuron. At this stage, the Six1/2⁺ cells are positioned on top of the anterior sensory vesicle over the ocellus. c9, The schematic shows a dotted circle where the oral opening forms. More anterior cells have undergone local cellular rearrangements. d–d0, Anterior lateral view of late tailbud I stage embryo electroporated with Six1/2. GFP and CNGA. mCherry. Asterisks indicate the ocellus. The arrowheads indicate a PPE-derived cell fated to become a GnRH neuron. d, Shows the Six1/2. GFP channel. d9, Shows the CNGA. mCherry channel (see Fig. 3c for larval expression). d0, Shows the merged image of Six1/2. GFP and CNGA. mCherry.

Extended Data Figure 3

Dorsal–ventral BMP patterning during PPE specification in Ciona intestinalis. a, Schematic of the anterior neural plate border during the gastrula–neurula transition. Cell lineage nomenclature is used. Chordin and Six1/2 are co-expressed in the lateral posterior derivatives of rows V and VI (dark blue; also see Fig. 1b). b, Ventral view of mid-gastrula stage embryo hybridized with a BMP2/4 mRNA probe and merged with the nuclear counterstain 49,6-diamidino-2-phenylindole (DAPI). c, Lateral view of an embryo during the gastrula–neurula transition hybridized with a BMP2/4 mRNA probe. Anterior is to the left. d, Dorsal view of an embryo during the gastrula–neurula transition hybridized with a Chordin mRNA probe and merged with a DAPI nuclear counterstain.

Extended Data Figure 4

Endogenous expression of newly described reporter genes in hatched Ciona larvae. a–d, Bright field anterior lateral views. a, Animal hybridized with a GnRH2 mRNA probe. White arrowhead indicates the position of the oral opening. b, Animal hybridized with an RXFP3 mRNA probe. c, Animal hybridized with a SOG/Chemokine-like mRNA probe. The heavily stained tunic was manually removed to reveal the expression signal. d, Animal hybridized with a CNGA mRNA probe. Red arrows mark areas of comparable expression throughout the panels in presumed PPE-derived neurons. An adjacent signal in the ocellus-associated photoreceptors makes it difficult to discriminate expression in PPE-derived neurons in panel d.

Extended Data Figure 5

GnRH expression requires BMP attenuation. a–d, Lateral view of a larva electroporated with GnRH. YFP and counterstained with phalloidin (violet). a, Larva co-electroporated with Dmrt. BMP2/4. Of the 200 larvae, 196 had no GnRH. YFP expression in aATENs and displayed mild to severe morphogenetic defects. Bracket shows mild anterior morphological defect. b, Larva co-electroporated with Dmrt. BMP5/7. Of the 200 larvae, 124 had GnRH. YFP expression in aATENs. c, Larva co-electroporated with Dmrt. BMPR1^CA. Of the 200 larvae, 197 larvae had no GnRH. YFP expression in aATENs. d, Larva co-electroporated with Dmrt. TGF-βR^CA. Of the 200 larvae, 130 had GnRH. YFP expression in aATENs and most displayed severe anterior neural tube defects. Arrowheads in b and d indicate the position of GnRH expression in aATENs. Anterior is to the left in all images.

Extended Data Figure 6

Initial phylogenetic analysis of two GPCRs expressed in the PPE-derived neurons of Ciona intestinalis. This broad survey tree, constructed according to maximum likelihood, shows the approximate placement of the two GPCRs of interest (boxed in blue) within the rhodopsin-class G-protein-coupled receptors. The numbered Ciona sequences and their receptor-type identifications in parentheses are from Kamesh et al.. All non-Ciona sequences were downloaded in bulk from http://pfam.xfam.org; they comprise the Pfam ‘seed’ alignment of seven-transmembrane receptors for the GPCRs. The rhodopsin subclass names are given to the right of each coloured group. The asterisk and double asterisk indicate the genes labelled RXFP3 and SOG/Chemokine-like, respectively, in Fig. 2e.We judge robust nodal support as bootstrap percentages. 70 for minimum evolution (ME) and maximum likelihood (ML) and posterior probability percentages. 95 for Bayesian inference (BI).

Extended Data Figure 7

Refined maximum likelihood phylogeny of two GPCRs expressed in the PPE-derived neurons of Ciona intestinalis. Relative to the initial survey tree (see Extended Data Fig. 6), analysis of this focused set of ‘chemokine cluster’ sequences further clarifies the phylogenetic affinities of two Ciona GPCRs expressed in the aATEN–GnRH neurons. The latter sequences are highlighted with violet boxes; all other Ciona sequences are from Kamesh et al.. The three nodal support values are (in order): ME bootstrap percentage, ML bootstrap percentage and BI posterior probability (only values. 50 are shown). Branch lengths are proportional to molecular change (amino acid substitutions per site) between nodes; see scale bar for measurement.

Extended Data Figure 8

Elevation of intracellular Ca²⁺ concentration in cells expressing Ciona CNG channels in response to cyclic nucleotides. a, Intracellular Ca²⁺ was visualized by Fura-2 ratiometric calcium imaging. Ca²⁺ influx into HEK 293 cells transfected with CNGA and CNGC was induced by 10 mM 8-Br-cGMP and 1 mM 8-Br-cAMP. In contrast, no Ca²⁺ influx was observed when HEK 293 cells transfected with CNGB were treated with either 10 mM 8-Br-cGMP or 1 mM 8-Br-cAMP. Coloured numbers in the ‘Before’ panels indicate cells that were subjected to a quantitative measurement of fluorescence. b, Dose-dependent response of HEK 293 cells transfected with CNGA to 8-Br-cAMP. Data were obtained from at least seven different cells in each of 16 different transfections. c, Dose-dependent response of HEK 293 cells transfected with CNGC to 8-Br-cAMP. Data were obtained from at least seven different cells in each of seven different transfections.

Figure 1

Six1/2 expression requires BMP attenuation. a, Dorsal view of an embryo at early neurula stage electroporated with ZicL. GFP and hybridized with Six1/2 and Eya mRNA probes. b, Dorsal view of an embryo at mid-neurula stage co-electroporated with Six1/2. mCherry and Chordin. GFP after an additional division (16 Six1/2⁺ cells in total). c–f, Tailbud electroporated with Six1/2. mCherry and Dmrt. GFP; counterstained with phalloidin (grey). c, Tailbud co-electroporated with Dmrt. BMP2/4 (131 of 135 embryos showed no Six1/2. mCherry expression). d, Tailbud co-electroporated with Dmrt. BMP5/7 (120 of 135 embryos displayed a full expression pattern for Six1/2. mCherry). e, Tailbud co-electroporated with Dmrt. BMPR1^CA (95 of 135 embryos had partial anterior Six1/2. mCherry expression). f, Tailbud co-electroporated with Dmrt. TGF-βR^CA (93 of 135 embryos displayed a full expression pattern for Six1/2. mCherry and anterior neural tube defects). Arrowheads in d and e flank the derivatives of the anterior ectoderm that normally co-express Six1/2. mCherry and Chordin. GFP in control embryos (see panel b). Arrows show extrusion of sensory vesicle cells in f.

Figure 2

PPE-derived GnRH neurons express RXFP3. a, Tailbud electroporated with Six1/2. mCherry. Arrowhead indicates the posterior-most Six1/2⁺ cell that undergoes neurogenesis. b, Larva co-electroporated with GnRH. GFP and Six1/2. mCherry. Insets show Six1/2 expression (left) and GnRH expression (right) surrounding the oral opening (dotted line). The GnRH⁺ neuron is marked with an asterisk. c, Dorsal view of larva electroporated with SOG/Chemokine-like. mCherry. The dotted line indicates the oral opening. d, Larva electroporated with RXFP3. mCherry. e, Phylogenetic placement among human rhodopsin-class GPCRs of Ciona RXFP3 and SOG/Chemokine-like, indicated by the starred clades. Green clades comprise Ciona and human orthologues, whereas no human orthologue was found for the red clade and no known Ciona GPCR grouped within the blue clades. Support values are Bayesian posterior probability percentages. f, Juvenile co-electroporated with GnRH. GFP and Six1/2. mCherry. The dotted line indicates the oral opening. Ciona were counterstained with phalloidin (grey) in panels a–d and f. Brackets in panels b–d and f indicate PPE-derived GnRH neurons. Panels a, b, d and f are lateral anterior views.

Figure 3

Ciliated GnRH neurons express functional CNGA. a–c, Larvae stained with phalloidin (grey). a, Dorsal view of a larva stained with an acetylated α-tubulin antibody; dotted line indicates the oral opening. b, Lateral view of a larva electroporated with GnRH. mCherry and stained with an acetylated α-tubulin antibody. c, Lateral view of a larva electroporated with CNGA. mCherry. Arrowheads indicate PPE-derived GnRH neurons in all panels. d, Ratiometric calcium imaging of HEK 293 cells transfected with Ciona CNGA, assayed by the intracellular Ca²⁺ indicator Fura-2. The graph shows the efficiency of response of CNGA to the cyclic nucleotide analogues 8-Br-cGMP and 8-Br-cAMP in seven individual cells (coloured lines) over a 120-min period. Indicated are the time points (red arrows) and durations (pink bars) of each analogue perfusion. e, Schematic that compares the PPE-derived GnRH neurons in tunicates such as Ciona (left) and olfactory-derived neurons in vertebrates (right). CNS, central nervous system; HEK, human embryonic kidney.