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Sci Rep
2018 Mar 05;81:4002. doi: 10.1038/s41598-018-22397-4.
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Sea urchin histamine receptor 1 regulates programmed cell death in larval Strongylocentrotus purpuratus.
Lutek K
,
Dhaliwal RS
,
Van Raay TJ
,
Heyland A
.
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Settlement is a rapid process in many marine invertebrate species, transitioning a planktonic larva into a benthic juvenile. In indirectly developing sea urchins, this ecological transition correlates with a morphological, developmental and physiological transition (metamorphosis) during which apoptosis is essential for the resorption and remodelling of larval and juvenile structures. While settlement is initiated by environmental cues (i.e. habitat-specific or benthic substrate cues), metamorphosis is regulated by developmental endocrine signals, such as histamine (HA), thyroid hormones (THs) and nitric oxide (NO). In the purple sea urchin, Strongylocentrotus purpuratus, we found that suH1R mRNA levels increase during larval development and peak during metamorphic competence. SuH1R positive cell clusters are prominently visible in the mouth region of sea urchin larvae, but the protein appears to be expressed at low levels throughout the larval arms and epidermis. SuH1R knock-down experiments in larval stages show that the function of suH1R is in inhibiting apoptosis. Our results therefore suggest that suH1R is regulating the metamorphic transition by inhibiting apoptosis. These results provide new insights into metamorphic mechanisms and have implications for our understanding of settlement and metamorphosis in the marine environment.
Figure 1. SuH1R antibody. (A) A fragment of the suH1R gene containing the suH1R-1 epitope was tagged with 6 × myc tags and 800 pg (lane 2) or 1600 pg (lane3) of mRNA was expressed in zebrafish eggs for protein production. Uninjected (U) eggs served as a negative control and anti-myc served as a positive control. Actin served as a loading control. Both the anti-myc and anti-suH1R-1 antibodies detect a band of ~75 kD, which is the predicted size of the construct. (B) The equivalent of 10 competent stage S. purpuratus larvae were loaded per lane and probed with the suH1R-1 antibody, which detected 3 bands. All but the high molecular weight 250 kD band disappear by the pre-incubation of the antibody with the immuno-peptide (+pep). The predicted size of suH1R is 76.4 kD. For 1 A original gels of which cropped versions are represented are provided in the Supplement. For 1B both extracts were exposed for the same amount of time.
Figure 2. suH1R expression in early (A–G) and late (H–N) stage S. purpuratus pluteus larvae (suH1R: green; nuclear stain: blue) using compound (A,H) and confocal (B–G; I–N) microscopy. The average number of suH1R labelled cells on one side of the mouth at different points of larval development were quantified (O). Specific staining was found on either side of the mouth in a symmetrical pattern. Note that the cells stained for suH1R are surrounded by a 6–7 cells (e.g. red asterisks in G). Red squares indicate the mouth region or specific cells highlighted in subsequent images. (A and H) whole mount larva under DIC light. (B and I) DAPI labelled cells in the mouth region of the larvae. (C and J) suH1R antibody staining in the mouth region. (D and K) combined image of B and C or I and J, respectively. (E and L) DAPI labelling around suH1R labelled cells. (F and M) suH1R labelling in specific cells around the mouth. (G and N) the combination of E and F or L and M, respectively. (O) There is an exponential increase in the cell count until larvae reach competence (at 25–30 days post fertilization). Mo: mouth region. St: stomach. (A and H) single z-plane DIC image, (B–G and I–N) are z-projections through the larvae.
Figure 3. Sea urchin histamine receptor 1 (suH1R; A) and histidine decarboxylase (HDC; B) expression levels both increase as sea urchin larvae develop towards metamorphic competence. Gene expression levels of both genes were calculated based on three independent biological replicates with two day old larvae as a reference.
Figure 4. Gene expression levels of sea urchin histamine receptor 1 (suH1R) and histidine decarboxylase for metamorphically competent larvae, juveniles and not competent larvae (which did not settle in response to KCl) relative to two week old larvae. Note an increase in suH1R expression in competent larvae compared to the pre-competent and juvenile samples. Letters denote means that are statistically the same.
Figure 5. Knock-down of sea urchin histamine receptor 1 (suH1R) decreases suH1R positive cell number in the mouth region of larva. suH1RMO refers to individuals injected with a solution containing a translation inhibiting MO that targets suH1R (22 µM). Ctrl refers to individuals injected with a solution containing nuclease-free water and NI refers to individuals not injected. Due to low sample size no statistical analysis of this data was performed. Quantitative reduction of protein levels is demonstrated by Western Blot (see the Supplement). Error bars represent +/− 1 standard error of the mean.
Figure 6. Sea urchin histamine receptor 1 morpholino (suH1RMO) results in cell expulsion at the arm tips of S. purpuratus. (A and B) DIC images of a pre-competent larva treated with suH1RMO. (C) DIC image of the arm tips of an untreated larva. Cells at the arm tips appear to be coming away from the rest of the arm tissue in suH1RMO treated larvae, while this phenotype is not observed in untreated larvae. Arrows indicate cells considered expelled or undergoing cell death.
Figure 9. Competent larvae treated with 80 mM KCl exhibit significantly higher YO-PRO signal than untreated (control) larvae (p < 0.05; log-transformed YO-PRO signal). YO-PRO signal was quantified as the number of bright spots detected for each optical slice through the larvae divided by the surface area of the larvae at it’s widest point (Supplement) This value was averaged across all optical sections for each larva. N = 5 for all treatments.
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