Heyland A et al. (2014), Manipulation of developing juvenile structures ...

ECB-ART-43719

PLoS One 2014 Dec 01;912:e113866. doi: 10.1371/journal.pone.0113866.

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Manipulation of developing juvenile structures in purple sea urchins (Strongylocentrotus purpuratus) by morpholino injection into late stage larvae.

Heyland A , Hodin J , Bishop C .

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Sea urchins have been used as experimental organisms for developmental biology for over a century. Yet, as is the case for many other marine invertebrates, understanding the development of the juveniles and adults has lagged far behind that of their embryos and larvae. The reasons for this are, in large part, due to the difficulty of experimentally manipulating juvenile development. Here we develop and validate a technique for injecting compounds into juvenile rudiments of the purple sea urchin, Strongylocentrotus purpuratus. We first document the distribution of rhodaminated dextran injected into different compartments of the juvenile rudiment of sea urchin larvae. Then, to test the potential of this technique to manipulate development, we injected Vivo-Morpholinos (vMOs) designed to knock down p58b and p16, two proteins involved in the elongation of S. purpuratus larval skeleton. Rudiments injected with these vMOs showed a delay in the growth of some juvenile skeletal elements relative to controls. These data provide the first evidence that vMOs, which are designed to cross cell membranes, can be used to transiently manipulate gene function in later developmental stages in sea urchins. We therefore propose that injection of vMOs into juvenile rudiments, as shown here, is a viable approach to testing hypotheses about gene function during development, including metamorphosis.

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Species referenced: Echinodermata
Genes referenced: adcy10 LOC100887844 LOC100893907 LOC115919910 LOC115925415 P16
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	Figure 1. Results from incubation of post-gastrula embryos in p16 and p58b vMOs. A) Representative images of embryos exposed to p58b or p16 vMOs for 48 h, compared to control vMO (cMO) and MFASW. B) Quantification of embryonic skeletal length for all treatments. Table underneath panel B shows p-values from ANVOVA with post-hoc comparisons using Bonferroni correction. Significant differences (p<0.05) are indicated with darker shading. Note that no significant difference was found between MFASW and cMO at any concentration. Note also that µM MFASW indicates that the same amount of milliQ water was added to MFASW as to the concentrated vMOs. Error bars indicate one standard error of the mean. C) RT-PCR of embryos incubated in 15 µM p58b vMO. The visible bands corresponds to the correctly spliced variant of p58b. This variant is absent in the p58b morpholino treatment, indicating knock-down of the correctly spliced variant. Scale bar in A applies to all images and corresponds to 55 µm.
	Figure 2. Representative images of rudiments, depicting the most common injection locations.(A) A schematic diagram of juvenile rudiment tissue layers. Cavities within the rudiment are shown in white, the blastocoel in light grey, and the stomach in dark grey at left. (B–D) Schematic diagrams of RD fluorescence (orange) in (B) the vestibule, (C) the hydrocoel (including primary podia) and (D) the intercoelomic space. (B′–D′) Representative corresponding epi-fluorescent images of RD distribution in these respective regions, one minute after injection. The stomach is in dark grey at left. The area in medium grey in the center of the diagram, to the right of the stomach, connecting the two shown portions of the hydrocoel (H) indicates the out of focus radial canal. All larvae shown and drawn are in anal view sensu [30], posterior down; therefore the "left" side (where the rudiment is found) is seen to the right in these larvae. H = hydrocoel, V = vestibule, IS = intercoelomic space, PP = primary podium, DS = dental sac (derived from left somatocoel). IS is contiguous with the blastocoel, but we give it a distinct term for the reasons described in the text. Scale bars in B′–D′ 35 µm.
	Figure 3. Projections of confocal image stacks, showing the distribution of rhodaminated dextran (RD; green) injected within juvenile rudiments at various time points post-injection (PI), with DAPI (blue in A–C) or with RD alone (D–H).(A) Projection of three sections; larva fixed 10 min PI. Arrow points to vestibule labeling (see Fig. 2B): a thin layer of RD between the vestibular ectoderm and the floor of the vestibule surrounding a primary podium. (B) Projection of sixteen sections; larva fixed 24 hrs PI. (C) Projection of twenty-three sections; larva fixed 24 hrs PI. Arrows in B, C point to labeling in the lumen of primary podia, which is contiguous with the hydrocoel (see Fig. 2C). (D) Projection of thirty sections; larva fixed 24 hrs PI. Arrow points to punctate labeling of RD, which has accumulated in the right somatocoel. (E) Projection of twenty-four sections; larva fixed 5 min PI. Arrow points to the lumen of a developing spine. Note that in this common "vestibule" pattern, RD labeling surrounds every tube foot and spine element on the oral side of the rudiment. (F) Projection of thirteen sections; larva fixed 5 min PI. Arrow points to a thin layer of RD between vestibular ectoderm and the floor of the vestibule surrounding a primary podium; i.e., vestibule pattern. (G) Projection of 47 sections; larva fixed 10 min PI. Arrow points to RD accumulation in the lumen of a primary podium. (H) Higher magnification projection of nine sections; larva fixed 30 min PI. Arrow points to RD, which appears in a punctate pattern in a spine lumen. Larvae are all oriented approximately as in Figure 2, with the stomach (S) towards the left of each panel, and the rudiment towards the right. Scale bars: A = 100 µm; B = 166 µm; C, D = 133 µm; E−G = 110 µm; H = 63 µm.
	Figure 4. Change in the distribution of rhodaminated dextran (RD) injected into the hydrocoel of a juvenile rudiment during a period of 10 minutes.Note that in addition to strong initial RD accumulation into the lumen of the developing primary podia (white arrow), the RD also labeled the vestibular space in this larva (i.e. the enclosed cavity between the floor and the roof of the vestibule; white arrowhead). Images were not individually adjusted for brightness and contrast and so represent the original relative brightness of RD. The larva imaged was viewed from the anal side sensu [30], posterior down; therefore the "left" side (where the rudiment is found) is seen here to the right. The larva imaged was oriented as in Figure 2. Scale bar = 70 µm.
	Figure 5. Developing spine structures in S. purpuratus.(A) Close-up of the rudiment, focused on two of the adult spine cavities and the developing skeletal elements within. (B) Cartoon showing the relative arrangement of three adult spine cavities and the surrounding pair of primary podia. (C–F) Close-up views of the developing adult spine anlage at progressive stages, sensu [28]. (C) Stage 6 "spine primordium + base". In stage 6 larvae vertical spine fronds are not yet present in any of the 15 adult spine anlage. (D) Early Stage 7 "pre-spine". Note that six fronds (four or five of which are visible here; arrow) have now started to elongate vertically from the spine base (arrowhead). (E) Late Stage 7 "pre-spine". Note that the spine fronds (three of which [numbered] are in focus in this view, the other three are visible but out of focus in the background) have continued to elongate, but no cross bars ("cross hatches") are yet visible. (F) Early Stage 8 spine, defined by the presence of at least one complete cross hatch (arrow). In our vMO experiment, we only selected larvae that were at Stage 7; rejecting all Stage 6 and Stage 8 larvae. All images here are from abanal views sensu [30], with posterior to the left and the left (rudiment) side up.
	Figure 6. An example of one of our experimental larvae, compressed under cover glass to score skeleton at the end of the experiment (96 hrs after injection – see also Fig. 5 ).Arrow: an adult spine with two cross hatches. Arrowhead: an adult "pre-spine" - so called since it has zero cross hatches. Asterisk: tube foot end plate with two concentric rings. Double asterisk: a juvenile "pre-spine." This larva was injected with p16 vMO, and has under-developed adult spines compared to the control treatments (see Fig. 7). Scale bar = 89 µm
	Figure 7. Quantification of vMOs knock-down effect on three aspects of skeleton growth in juvenile rudiments.See Figures 5 and 6 for details on which skeletal elements were scored. A) qRT-PCR results for p58b and p16 in three development stages show expression of these genes in juvenile stages. B) spine elongation, measured by the number of cross hatches in adult type spines over a time period 96 h post-injection. C) number of adult type pre-spines present in the oral region of the juvenile rudiment 96 h post-injection D) number of adult type spines present in the oral region of the juvenile rudiment 96 h post-injection. Lines above bars indicate significant differences (p<0.05) between pairs of treatments.

References [+] :

Adomako-Ankomah, P58-A and P58-B: novel proteins that mediate skeletogenesis in the sea urchin embryo. 2011, Pubmed, Echinobase