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BMC Evol Biol
2016 May 26;161:117. doi: 10.1186/s12862-016-0686-0.
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A pancreatic exocrine-like cell regulatory circuit operating in the upper stomach of the sea urchin Strongylocentrotus purpuratus larva.
Perillo M
,
Wang YJ
,
Leach SD
,
Arnone MI
.
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BACKGROUND: Digestive cells are present in all metazoans and provide the energy necessary for the whole organism. Pancreatic exocrine cells are a unique vertebrate cell type involved in extracellular digestion of a wide range of nutrients. Although the organization and regulation of this cell type is intensively studied in vertebrates, its evolutionary history is still unknown. In order to understand which are the elements that define the pancreatic exocrine phenotype, we have analyzed the expression of genes that contribute to specification and function of this cell-type in an early branching deuterostome, the sea urchin Strongylocentrotus purpuratus.
RESULTS: We defined the spatial and temporal expression of sea urchin orthologs of pancreatic exocrine genes and described a unique population of cells clustered in the upper stomach of the sea urchin embryo where exocrine markers are co-expressed. We used a combination of perturbation analysis, drug and feeding experiments and found that in these cells of the sea urchin embryo gene expression and gene regulatory interactions resemble that of bona fide pancreatic exocrine cells. We show that the sea urchin Ptf1a, a key transcriptional activator of digestive enzymes in pancreatic exocrine cells, can substitute for its vertebrate ortholog in activating downstream genes.
CONCLUSIONS: Collectively, our study is the first to show with molecular tools that defining features of a vertebrate cell-type, the pancreatic exocrine cell, are shared by a non-vertebrate deuterostome. Our results indicate that the functional cell-type unit of the vertebrate pancreas may evolutionarily predate the emergence of the pancreas as a discrete organ. From an evolutionary perspective, these results encourage to further explore the homologs of other vertebrate cell-types in traditional or newly emerging deuterostome systems.
Fig. 1. Expression analysis of regulatory and terminal differentiation orthologs of known pancreatic genes. mRNA localization of sea urchin regulatory (a-f, green) and terminal differentiation (h-m, magenta) genes. For all the figures in this paper, every picture is a full projection of merged confocal Z stacks and nuclei are stained with DAPI and depicted in blue. Red circles in f show Mist1 RNA localization in alternating cells of the apical organ. Inset in panel k is a representative single confocal section of the upper stomach of a late larva showing that SpCpa2L transcripts are abundantly expressed in the entire cell. g, n Temporal expression profiles of pancreatic regulatory and terminal differentiation genes during sea urchin development. The graphs show the relative transcript abundance normalized against ubiquitin mRNA. The results are expressed as percentage of the maximum value, corresponding to the stage with the highest level of expression. Standard deviations of three technical replicas are all <0.5. For the sake of simplicity, for each panel, the species in the gene name has been omitted. Abbreviations: d, days; dv, dorsal view; h, hours post fertilization; vv, ventral view
Fig. 2. Co-expression analysis of markers of pancreatic exocrine cell-types. Double FISH of selected pancreatic genes in the sea urchin embryos and larva (dv). On the right of each panel, split and combined channels of single confocal sections of the gut domain expressing the two genes (the region of the embryo shown in the right insets is underlined by a yellow square ) are provided to confirm that the two genes are indeed expressed in the same cells
Fig. 3. Spatial analysis of gene expression after Notch signaling perturbation. Hnf1 (a, b), Ptf1a (c, d), Mist1 (e, f) and Fng (g, h) transcript localization tested by FISH in control animals (a, c, e, g) and in animals treated with DAPT (b, d, f, h). In all the experiments, Ptf1a (in green) was used as second probe in double FISH (in a, b, e, f, g, h) since the increase of Ptf1a + cells in treated larvae confirms the phenotype. Red arrow in D indicates Ptf1 ectopic expression in a few cells of the anus. Yellow circles in E show that Mist1 is expressed in alternated cells of the apical organ in control larvae, while arrows in F indicate that in treated larvae Mist1 is expressed in adjacent cells of the apical organ (yellow arrow) and in a few cells of the esophagus (white arrow). For each analyzed gene, quantification of the phenotypes is shown on the right. Abbreviations: AO, apical organ; es, esophagus; lv, lateral view; MG, midgut; st, stomach, v, ventral
Fig. 4. Analysis of gene expression after MO perturbation. Ptf1a mRNA localization in control embryos (a) and in embryos injected with MO directed against the translation of SpHnf1 RNA (b). Cpa2L, Pnlp2/5, Amy3, Ptf1a and Fng transcripts were detected by single or double FISH in control larvae (c, e, g, i) and in larvae injected with two different MOs directed against the translation of SpPtf1a RNA (d, f, h, j). Note that f, h and j show larvae injected with SpPtf1a MO1. To confirm the effects of MO1, figure d shows that SpCpa2L transcripts are absent also in larvae injected with SpPtf1a MO2. Inset in j is a representative single confocal section of the dorsal upper stomach of a late larva and the white arrow shows that Ptf1a + and Fng + cells are adjacent and not overlapping. k qPCR analysis showing the effects of SpPtf1a MO1 perturbation on transcript levels of selected pancreatic genes at 70 h. Fold changes ≥ -2 and ≤ 2 are shaded in light grey and indicate non-significant changes in gene expression
Fig. 5. Sea urchin Ptf1a is active and can function together with mammalian Ptf1 partners. a 293 t cells were transiently transfected with luciferase reporter containing 4x Ptf1a-responsive element (vector indicated as 4x in the figure) and/or spPtf1a, E47, Rbpl construct; renilla vector was cotransfected for normalization. Results are displayed in the box plot as fold activation over the activity of 4x reporter alone. Transfections were done in triplicate and were repeated 5 times. Luciferase assays from all transfections show similar trend, one representative result is shown. t-test was carried out for group comparison. The bottom and top of the box are the first and third quartiles, and the band inside the box is the median. The whisker represents 1.5 IQR (interquantile range, the differences between the third and first quantile). b SpPtf1a has lower activity compared with mammalian Ptf1a in the in vitro luciferase assay. Experiment setting is the same as (a) except that rat rnPtf1a was transfected in a second plate side-by-side for comparison. We tested SpPtf1a activity in multiple cell lines, one representative result from HeLa cells is shown. c Alignment of the closest to the transcription start site (TSS) Ptf1a binding sites found by bioinformatic analysis on S. purpuratus (Sp) promoters with the canonical Ptf1a binding site of the RnCtbr1 gene (ref). nt, nucleotides
Fig. 6. Effect of different feeding regimes on pancreatic exocrine cell gene expression. a and (b) are bright field images of 15-day larvae that are representative for the fed or starved conditions. Larvae are viewed from lateral right side, mouth up. Stomach diameter was measured from ventral to dorsal, along the midline of the larval body. c Quantitative PCR analysis of SpPtf1a and digestive enzymes transcripts in feeding and fasting conditions. Data were normalized using ubiquitin as reference gene. Three biological replicas and three technical replicas were measured. The differences between groups that resulted significant by Student’s two tailed t-test are indicated as ** if p < 0.01 (highly significant)
Fig. 7. Proposed model of sea urchin pancreatic exocrine-like specification and differentiation. a The schemes show the development of exocrine-like cells from late gastrula (left) to larva (right). The right drawing also shows the localization of putative endocrine cells that are positive for antibody staining of a member of the insulin family (ILP-1+ cells). Note that we do not know if the ILP-1+ cells in the upper stomach are simply adjacent to the pancreatic exocrine-like cells or there are stomach cells which share both features. b Summary of regulatory interactions occurring in pancreatic exocrine-like cells unraveled by this study. Different genes are shown with different colors. The wiring among the genes is shown with solid lines. Arrows represent positive regulation, bars represent repression, and dashed lines indicate signaling events. Question marks in the food input means that the mechanism and the genes involved in the pathway are not known. Abbreviations: fg, foregut; hg, hindgut; in, intestine; mo, mouth
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