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Neurons and pancreatic endocrine cells have a common physiology and express a similar toolkit of transcription factors during development. To explain these common features, it has been hypothesized that pancreatic cells most likely co-opted a pre-existing gene regulatory program from ancestral neurons. To test this idea, we looked for neurons with a "pre-pancreatic" program in an early-branched deuterostome, the sea urchin. Only vertebrates have a proper pancreas, however, our lab previously found that cells with a pancreatic-like signature are localized within the sea urchin embryonic gut. We also found that the pancreatic transcription factors Xlox/Pdx1 and Brn1/2/4 co-localize in a sub-population of ectodermal cells. Here, we find that the ectodermal SpLox+ SpBrn1/2/4 cells are specified as SpSoxC and SpPtf1a neuronal precursors that become the lateral ganglion and the apical organ neurons. Two of the SpLox+ SpBrn1/2/4 cells also express another pancreatic transcription factor, the LIM-homeodomain gene islet-1. Moreover, we find that SpLox neurons produce the neuropeptide SpANP2, and that SpLox regulates SpANP2 expression. Taken together, our data reveal that there is a subset of sea urchin larval neurons with a gene program that predated pancreatic cells. These findings suggest that pancreatic endocrine cells co-opted a regulatory signature from an ancestral neuron that was already present in an early-branched deuterostome.
Figure 1. SpLox is expressed in lateral ganglion neurons. (A–Câ€) SpLox (magenta) and SynB (green) protein localization in late larvae. Nuclei are labeled blue with DAPI. In (Aâ€,Bâ€,Câ€) nuclei are omitted in order to see the long and interconnected network formed by the neurite projections. Insets on the right show SpLox expression in the nucleiof left lateral ganglia (A,B) and right lateral ganglia (A,C). (D–F) Are fluorescent in situ hybridization (FISH) showing the localization of SpLox mRNA in the apical plate in late gastrula (A), prism (B) and early larva (C) stages. White dashed line boxes are magnifications of the apical plate area. White arrowheads indicate SpLox localization in the foregut. All pictures are full projections of merged confocal stacks. Nuclei are stained with DAPI and depicted in blue. es, esophagus; in, intestine; mo, mouth; st, stomach; abv, aboral view; lv, lateral view; ov, oral view.
Figure 2. Co-expression analysis of markers of pancreatic transcription factors and SpBrn1/2/4 define unique neurons. (A–C) FISH of SpNgn, SpIsl, and SpNeuroD in gastrulae. (D–Fâ€) Double fluorescent in situ hybridization (FISH) of SpNgn, SpIsl, and SpNeuroD with SpBrn1/2/4 combined with nuclear staining (DAPI, blue) in early larvae. White dashed-line circles highlight the apical organ region. All images are obtained as stacks of merged confocal Z sections. Split and combined channels of single confocal sections are provided to show that genes are expressed in the same cells. abv, aboral view; av, aboral view; lv, lateral view; ov, oral view.
Figure 3. Cells with a pre-pancreatic regulatory state. (A,B) double FISH of SpLox and the proneural gene SpSoxC in middle to late gastrula. White arrowheads show SpLox co-localization with SpSoxC in the foregut, yellow arrowheads mark neurons in the apical plate and ciliary band that co-express SpSoxC and SpLox. (C) double FISH of SpLox and SpBrn1/2/4 in mid gastrula. Insets on the right are magnifications of (Continued)
Figure 4. Transcript expression and protein localization of the neuropeptide SpANP2. (A,B)†SpANP2 (magenta) and SynB (green) immunolocalization in late larvae. (A–A)†is a frontal view, (B–B)†is a dorsal view. (C,D) SpANP2 transcripts (FISH, in magenta) and protein (immunofluorescence, in green) localization in early larva (C) and in 1-week old larva (D). White dashed line boxes in (C) mark the apical organ region, the left and the right lateral ganglia that are magnified on the right. Note that the protein accumulates close to the neuritis extension. (E) Double FISH of SpAN (magenta) with SpMist (green) showing that cells producing SpAN mRNA are secretory endocrine cells. White arrows indicate co-expression. (F) Double FISH of SpAN (magenta) and SpLox (green) at prism stage (66 h). White dashed line box marks the apical organ region. Insets on the right show three distinct cells where SpLox and SpAN are co-expessed. (G) Triple FISH shows the expression pattern of SpBrn1/2/4 (cyan), SpLox (green), and Sppnp5 (magenta). Insets on the right show single channels for each gene. The 72 h pluteus is oriented abanal along the A/V axis. Pictures are all full projection of merged confocal stacks; nuclei are labeled blue with DAPI. AO, apical organ; cp, coelomic pouches; es, esophagus; LLG, left lateral ganglion; PON, post-oral neurons; st, stomach; RLG, right lateral ganglion; abv, aboral view); ov (oral view).
Figure 5. SpLox controls SpANP2 expression. (A)
SpAN mRNA detected by single-color in situ hybridization or SpANP2 protein detected by immunofluorescence localization in controls and in larvae injected with SpLox MOs directed against the translation of SpLox RNA. Note that injected embryos/larvae show the typical SpLox MO phenotype of a straight gut that does not have the pyloric sphincter (38). All images are obtained as stacks of merged confocal Z sections. Nuclei are labeled blue with DAPI. (B–D) Quantification of the number of SpANP2 cells in the SpLox morphants shows a ****p < 0.0001 by Chi squared test. Cartoons of early larvae on top of the graphs summarize the most frequent phenotypes. In the graph we put together data form SpANP2+ cells at prism stage (66 h), early larvae (70 h), and 1-week old larvae. For (C,D) we show percentages of lateral ganglia and post-oral neurons together. abv, aboral view; lv, lateral view; ov, oral view.
Figure 6. Summary of the regulatory state of the SpLox+ neurons. (A) Schematic representation of a sea urchin gastrula (left) and early larva (right) showing the neurons identified in this study. Neurons with the same pancreatic signature have the same color. (B) Cartoon showing decrease in SpANP2+ neurons in SpLox morphants. The three most frequent phenotypes are shown.
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