ECB-ART-47448PLoS Biol January 1, 2019; 17 (9): e3000460.
The evolution of a new cell type was associated with competition for a signaling ligand.
There is presently a very limited understanding of the mechanisms that underlie the evolution of new cell types. The skeleton-forming primary mesenchyme cells (PMCs) of euechinoid sea urchins, derived from the micromeres of the 16-cell embryo, are an example of a recently evolved cell type. All adult echinoderms have a calcite-based endoskeleton, a synapomorphy of the Ambulacraria. Only euechinoids have a micromere-PMC lineage, however, which evolved through the co-option of the adult skeletogenic program into the embryo. During normal development, PMCs alone secrete the embryonic skeleton. Other mesoderm cells, known as blastocoelar cells (BCs), have the potential to produce a skeleton, but a PMC-derived signal ordinarily prevents these cells from expressing a skeletogenic fate and directs them into an alternative developmental pathway. Recently, it was shown that vascular endothelial growth factor (VEGF) signaling plays an important role in PMC differentiation and is part of a conserved program of skeletogenesis among echinoderms. Here, we report that VEGF signaling, acting through ectoderm-derived VEGF3 and its cognate receptor, VEGF receptor (VEGFR)-10-Ig, is also essential for the deployment of the skeletogenic program in BCs. This VEGF-dependent program includes the activation of aristaless-like homeobox 1 (alx1), a conserved transcriptional regulator of skeletogenic specification across echinoderms and an example of a "terminal selector" gene that controls cell identity. We show that PMCs control BC fate by sequestering VEGF3, thereby preventing activation of alx1 and the downstream skeletogenic network in BCs. Our findings provide an example of the regulation of early embryonic cell fates by direct competition for a secreted signaling ligand, a developmental mechanism that has not been widely recognized. Moreover, they reveal that a novel cell type evolved by outcompeting other embryonic cell lineages for an essential signaling ligand that regulates the expression of a gene controlling cell identity.
PubMed ID: 31532765
PMC ID: PMC6768484
Article link: PLoS Biol
Species referenced: Echinodermata
Genes referenced: alx1 aqr hoxb1l hpd LOC115919139 LOC594353 LOC594566 p58a pmar1 skiv2l vegfc
Morpholinos: flt1 MO1
Article Images: [+] show captions
|Fig 1. PMC-derived signals suppress the skeletogenic potential of BCs.Upper panel: Each pBC is multipotent and can adopt either a skeletogenic (PMC-like) or BC (immunocyte) fate. Ordinarily, a signal from PMCs (red bar) suppresses the skeletogenic potential of pBCs and causes them to adopt an immunocyte fate. Lower panels: Ablation of PMCs (red cells) at the late (mesenchyme) blastula stage leads to transfating of BCs (green cells). In these PMC(−) embryos, BCs ingress late in gastrulation but migrate to PMC-specific target sites on the blastocoel wall (indicated by white arrows), fuse to form a syncytium, and synthesize a correctly patterned skeleton, beginning with the formation of two triradiate skeletal primordia (shown in yellow). BC, blastocoelar cell; pBC, presumptive BC; PMC, primary mesenchyme cell.|
|Fig 2. Axitinib blocks BC transfating.PMCs were removed from mesenchyme blastula–stage embryos, and the resultant PMC(−) embryos were separated into two cohorts. One cohort was left in plain seawater, whereas the other was transferred to 5 nM axitinib immediately after PMC removal. (A-G) Control PMC(−) embryos. (A’-G’) Axitinib-treated PMC(−) embryos. Axitinib treatment blocked the formation of birefringent skeletal elements (A-B’) and the expression of early skeletogenic regulatory genes by BCs as shown by WMISH analysis of Lv-alx1 (C,C’) and Lv-tbr (D,D’) expression. The expression of skeletogenic effector genes downstream of alx1 was also blocked, as indicated by WMISH analysis of Lv-p58a (E,E’) and Lv-msp130rel2 (F,F’), and by immunostaining with mAb 6a9 (G,G’). Arrowheads indicate expression of skeletogenic genes by transfating BCs. Panel H shows quantification of 6a9-positive cells in control and axitinib-treated PMC(−) embryos at 12 hpd (two independent trials from separate matings). Statistical significance of the data was assessed by two-sided t tests, and p-values <0.05 are indicated by asterisks. Raw data can be found in S1 Data. BC, blastocoelar cell; DIC, differential interference contrast microscopy; hpd, hours post–PMC depletion; Lv-alx1, L. variegatus aristaless-like 1; Lv-tbr, L. variegatus t-brain; mAb, monoclonal antibody; msp130rel2, L. variegatus mesenchyme specific protein 130-related 2; POL, polarized light microscopy; PMC, primary mesenchyme cell; WMISH, whole-mount in situ hybridization.|
|Fig 3. Time course of axitinib sensitivity during BC transfating.(A) Experimental design. Solid horizontal bars indicate the duration of axitinib (5 nM) treatment. Start times are shown as a 1-hour interval because microsurgical removal of PMCs required approximately 1 hour for each cohort of embryos. (B) Counts of 6a9-positive cells. Data shown were collected from several independent trials, and untreated controls from all trials were pooled. For all time periods tested, we observed a significant reduction in 6a9(+) cells in axitinib-treated PMC(−) embryos relative to untreated PMC(−) controls. Raw data can be found in S1 Data. (C-E) Signaling through VEGFR is required for the maintenance of Lv-alx1 expression in BCs during reprogramming. PMC(−) embryos were treated with axitinib for 5 hours beginning at 4–5 hpd. At the start of axitinib treatment, Lv-alx1 was expressed robustly by BCs at the tip of the archenteron (arrow in C, 10/11 embryos). Five hours later (9–10 hpd), alx1 continued to be expressed in untreated PMC(−) embryos (arrow in D, 5/5 embryos), but expression was undetectable in most axitinib-treated PMC(−) embryos (E, 10/13 embryos). BC, blastocoelar cell; hpd, hours post–PMC depletion; Lv-alx1, L. variegatus aristaless-like homeobox 1; PMC, primary mesenchyme cell; VEGFR, vascular endothelial growth factor receptor.|
|Fig 4. Lv-vegfr-10-Ig expression during BC transfating.(A) Control gastrula. Lv-vegfr-10-Ig is expressed at high levels by PMCs (arrow), as previously reported . (B) PMC(−) embryo, 2 hpd. Expression of Lv-vegfr-10-Ig is evident in the invaginating vegetal plate (arrow), a region that includes presumptive BCs (32/37 embryos). (C) PMC(−) embryo, 7 hpd. Expression of Lv-vegfr-10-Ig is apparent in the wall of the archenteron (arrow). (D) Emetine-treated PMC(−) embryo, 2 hpd. Inhibition of protein synthesis did not prevent the robust expression of Lv-vegfr-10-Ig in the vegetal plate following PMC removal (16/20 embryos). BC, blastocoelar cell; hpd, hours post–PMC depletion; Lv-vegfr-10-Ig, L. variegatus vascular endothelial growth factor receptor-10-Ig; PMC, primary mesenchyme cell.|
|Fig 5. Lv-VEGFR-10-Ig is required for BC transfating.(A) MO knockdown strategy. Following the approach of Duloquin and coworkers , an MO was directed at the exon 2/intron 2 splice junction of the Lv-vegfr-10-Ig primary transcript. (B) RT-PCR analysis of morphant embryos. The Lv-vegfr-10-Ig MO produced a significant reduction in the level of wild-type mRNA and the appearance of a prominent splicing isoform that included intron 2, as verified by cloning and sequencing of the PCR product. Inclusion of intron 2 resulted in multiple stop codons in all reading frames and the production of a truncated, nonfunctional receptor. Low levels of other mis-spliced forms of Lv-vegfr-10-Ig mRNA (asterisks) were also detected. (C-D) BC transfating and skeletogenesis were suppressed in PMC(−), morphant embryos, as revealed by DIC and polarization microscopy at 40 hpd (C-D’) and by immunostaining with mAb 6a9 at 12 hpd (E-F). BC reprogramming was not affected by an equivalent concentration of an MO directed against Lv-IgTM, a PMC-specific protein that regulates skeletal branching . Statistical significance of the data was assessed by two-sided t tests and p-values <0.05 are indicated by asterisks. Raw data can be found in S1 Data. BC, blastocoelar cell; DIC, differential interference contrast microscopy; hpd, hours post–PMC depletion; Lv-vegfr-10-Ig, L. variegatus vascular endothelial growth factor receptor-10-Ig; Lv-IgTM, L. variegatus Ig and transmembrane domain protein; mAb, monoclonal antibody; MO, morpholino; PMC, primary mesenchyme cell; POL, polarized light microscopy; RT-PCR, reverse transcription PCR.|
|Fig 6. Lv-VEGF3 is required for BC transfating.A previously characterized translation-blocking MO was used to interfere with Lv-vegf3 expression . PMC(−) morphant embryos failed to form skeletal elements even at 48 hpd (A-B’), and very few 6a9(+) cells were detectable at 12 hpd (C-D), indicating that BC transfating was largely blocked. Statistical significance of cell count data was assessed by two-sided t tests, and p-values <0.05 are indicated by asterisks. Raw data can be found in S1 Data. BC, blastocoelar cell; DIC, differential interference contrast microscopy; hpd, hours post–PMC depletion; Lv-vegf3, L. variegatus vascular endothelial growth factor 3; mAb, monoclonal antibody; MO, morpholino; PMC, primary mesenchyme cell; POL, polarized light microscopy.|
|Fig 7. Knockdown of Lv-VEGFR-10-Ig in PMCs reduces their ability to suppress BC transfating.(A) Experimental design. The entire complement of PMCs was removed from a recipient embryo and replaced with 15–40 RITC-labeled PMCs from control or Lv-VEGFR-10-Ig morphant donor embryos. Then, 10.5 hours after transplantation, recipient embryos were fixed and immunostained with mAb 6a9 and a DyLight 488 goat anti-mouse secondary antibody. Donor PMCs were identified by (red + green) fluorescence, whereas transfated BCs exhibited only green fluorescence. (B-E) A representative embryo after immunostaining, viewed with DIC (B) or epifluorescence optics. (C) mAb 6a9 immunostaining showing all skeletogenic cells (donor PMCs and transfated BCs). (D) Rhodamine fluorescence showing donor PMCs. (E) Overlay of the two fluorescent channels. Cells that are green but not red are transfated BCs. (F) Quantification of the numbers of transfated BCs following PMC transplantation. Control PMCs (orange) were more potent at suppressing BC transfating than PMCs that had reduced expression of Lv-VEGFR-10-Ig (teal). In both cases, the number of transfated cells was inversely related to the number of PMCs in the blastocoel. Each point represents a single recipient embryo. Lines of best fit are indicated, and 95% confidence intervals are shown in gray. Raw data can be found in S2 Data. BC, blastocoelar cell; DIC, differential interference contrast microscopy; Lv-VEGFR-10-Ig, L. variegatus vascular endothelial growth factor receptor-10-Ig; mAb, monoclonal antibody; pBC, presumptive BC; PMC, primary mesenchyme cell; RITC, rhodamine isothiocyanate.|
|Fig 8. Overexpression of Lv-VEGF3 induces BC transfating.Capped mRNA encoding Lv-VEGF3 was injected into fertilized eggs. Many supernumerary 6a9(+) cells were observed in such embryos 24 hpf, when sibling control embryos had reached the late prism stage (A, A’). In control embryos, expression of Lv-alx1 and Lv-tbr was restricted to PMCs, as expected (arrows in B and C), whereas injection of Lv-vegf3 mRNA resulted in the ectopic activation of Lv-alx1 and Lv-tbr in the wall of the archenteron (arrows in B’ and C’). The effect of Lv-vegf3 mRNA was dose-dependent (D). Raw data can be found in S1 Data. BC, blastocoelar cell; hpf, hours postfertilization; Lv-alx1, L. variegatus aristaless-like 1; Lv-tbr, L. variegatus t-brain; Lv-VEGF3, L. variegatus vascular endothelial growth factor 3; mAb, monoclonal antibody; PMC, primary mesenchyme cell.|
|Fig 9. A model of the PMC–BC interaction.During normal development (top panel), PMCs migrate into the blastocoel at the onset of gastrulation, and VEGFR-Ig-10 on their surfaces sequesters VEGF3, which is expressed by ventrolateral ectoderm cells [9,11]. BCs are hypothesized to express low but functional levels of VEGFR-10-Ig. In PMC(−) embryos (bottom panel), VEGF3 is free to diffuse through the blastocoel and interacts with BCs. As a consequence of this signal, the key selector gene, alx1, is activated along with its many targets. At the same time, alx1 suppresses competing regulatory states [24,29]. alx1, aristaless-like homeobox 1; BC, blastocoelar cell; PMC, primary mesenchyme cell; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.|
|Fig 10. A provisional model of skeletogenic cell type evolution in euechinoids.(A) Ancestral skeletogenic cell, present in the adult of the last common ancestor of all echinoderms. Ancient roles for alx1, vegf3, and vegfr-10-Ig in echinoderm skeletogenesis are inferred from the conserved expression of these genes in adult and embryonic skeletogenic centers in multiple echinoderm clades and from experimental perturbations of gene function in echinoid and holothuroid embryos (see references in ). Red lines are inferred from experimentally determined inputs of VEGF3 signaling into vegfr-10-Ig and biomineralization genes in euechinoid PMCs [9,11,13]. The brokenness of the lines indicates that intervening transcription factors have not been identified. Regulation of alx1 by VEGF signaling is hypothesized based on data presented in this study and from the restricted, ventral expression of alx1 at the late gastrula stage in euechinoids . Inputs from alx1 into vegfr-10-Ig and other biomineralization genes (blue arrows) have been revealed by knockdown/overexpression of alx1 in euechinoid and asteroid embryos [24,29,59,67]. (B) During echinoid evolution, the ancestral skeletogenic gene regulatory system was transferred into mesoderm-derived cells of the larva or late embryo. This may have only required a shift in vegf3 expression in the ectoderm if a widely expressed receptor was linked to a feedback mechanism that up-regulated vegfr-10-Ig. In modern euechinoids, at postgastrula stages of development (“late embryo”), alx1, vegfr-10-Ig, and many biomineralization genes are regulated by VEGF signaling, which we suggest reflects the ancestral mode. Essentially the same regulatory machinery operates in BCs when VEGF3 is available, i.e., in PMC(−) embryos. (C) The evolution of PMCs involved the transfer of alx1 expression into the large micromere lineage (“early embryo”) by linking the activation of this gene to maternal β-catenin, its direct target, pmar1, and unequal cleavage [28,68,69]. The early, cell-autonomous activation of alx1 in the large micromere-PMC lineage resulted in the precocious expression of VEGFR-10-Ig, which in modern euechinoids sequesters VEGF3 and isolates BCs from the exclusionary influence of Alx1, allowing these cells to express an alternative regulatory state. Alx1, Aristaless-like homeobox 1; BC, blastocoelar cell; pmar1, paired-class micromere anti-repressor; PMC, primary mesenchyme cell; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.|
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