Heyland A and Hodin J (2014), A detailed staging scheme for late larval devel...

ECB-ART-43438

BMC Dev Biol 2014 May 19;14:22. doi: 10.1186/1471-213X-14-22.

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A detailed staging scheme for late larval development in Strongylocentrotus purpuratus focused on readily-visible juvenile structures within the rudiment.

Heyland A , Hodin J .

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BACKGROUND: The purple sea urchin, Strongylocentrotus purpuratus, has long been the focus of developmental and ecological studies, and its recently-sequenced genome has spawned a diversity of functional genomics approaches. S. purpuratus has an indirect developmental mode with a pluteus larva that transforms after 1-3 months in the plankton into a juvenile urchin. Compared to insects and frogs, mechanisms underlying the correspondingly dramatic metamorphosis in sea urchins remain poorly understood. In order to take advantage of modern techniques to further our understanding of juvenile morphogenesis, organ formation, metamorphosis and the evolution of the pentameral sea urchin body plan, it is critical to assess developmental progression and rate during the late larval phase. This requires a staging scheme that describes developmental landmarks that can quickly and consistently be used to identify the stage of individual living larvae, and can be tracked during the final two weeks of larval development, as the juvenile is forming. RESULTS: Notable structures that are easily observable in developing urchin larvae are the developing spines, test and tube feet within the juvenile rudiment that constitute much of the oral portion of the adult body plan. Here we present a detailed staging scheme of rudiment development in the purple urchin using soft structures of the rudiment and the primordia of these juvenile skeletal elements. We provide evidence that this scheme is robust and applicable across a range of temperature and feeding regimes. CONCLUSIONS: Our proposed staging scheme provides both a useful method to study late larval development in the purple urchin, and a framework for developing similar staging schemes across echinoderms. Such efforts will have a high impact on evolutionary developmental studies and larval ecology, and facilitate research on this important deuterostome group.

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Species referenced: Echinodermata
Genes referenced: impact LOC100887844 LOC105438433 LOC593824

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	Figure 1. Examples of soft tissue developmental stages of S. purpuratus larvae as defined in Table1(roman numerals). All images (except as indicated) are close-up views of the rudiment in the same orientation as indicated in A, i.e., an abanal view of the larvae (sensu [37]). Insets in C-K show schematic views of the stages in question; see also Table 1. (A) Overview diagram of larva with corresponding DIC image of actual larva (B – anal view). (C-F) Black arrow- invaginating ectoderm (e); white arrow- hydrocoel (h). (C) Stage i, approximately 60% ectodermal invagination. (D) Stage i, approximately 90% ectodermal invagination (anal view). (E) Stage ii, contact of invaginating ectoderm with hydrocoel (anal view). (F) Stage iii, ectoderm flattening alongside hydrocoel (anal view). (G) Stage iv, 5-fold mesoderm (first visible sign of 5-fold symmetry); white arrowheads- 3 of the 5 primary podia anlage are visible in this view; black arrow- invaginating ectoderm. (H) Stage v, 5-fold ectoderm; arrowheads as in E. (I) Stage vi, primary podia stage, arrowheads indicate two of the forming podia. (J) Soft tissue Stage vii with folded primary podia. (K) Soft tissue Stage viii with primary podia bending at the tip and touching each other (anal view). Note that schematic in A does not show the pair of preoral arms for simplicity. Abbreviations: Ala – anterolateral arms; M – Mouth; St – stomach; PD – postdorsal arms; PO – postoral arms, PrO – preoral arms; h – hydrocoel; e – invaginating ectoderm (=vestibule); L – Left larval side; R – Right larval side; A – anterior; P – posterior; An – anal; Ab – abanal. Scale bars: B- 200 μm C, D, E – 35 μm; F - 25 μm; G, H, I - 40 μm, J - 30 μm; K - 40 μm.
	Figure 2. Examples of skeletogenic developmental stages of S. purpuratus larvae as defined in Table2(arabic numerals). All images (except as indicated) are rudiment close-up views in same orientation (i.e., abanal view, sensu [37]) as indicated in A (earlier stages) and N (later stages). E, F, G, K, M are anal views. Insets in C-M: schematic views of defining skeletogenic features for stage in question, not drawn to scale; see also Table 2. Drawing (A) and corresponding live image (B) of representative early skeletogenic stage larva. (C) Stage 1: spicule dot (white arrow). (D) Stage 2: spicule (white arrow). (E) Stage 3: tube foot spicule dot (white arrow). (F) Stage 4: tri-radiate tube foot spicule (white arrowhead); multi-branched spicule (white arrow), fated to form ocular plate 1 (see [34,35]). White asterisk indicates ocular plate 5 –a non-rudiment skeletal character not included in our scheme– which forms off of the left PO rod (see [43]). (G) Stage 5: spine primordium (6-sided star- white arrow; note also spine lumen visible at this and later stages). (H) Also Stage 5: incomplete TF ring (black arrow); multi-branched spicule indicated (black arrowhead) is fated to form the interambulacral skeleton at the base of an adult spine in interambulacrum 1 (sensu [34,35]; but see [44]). Note this view is looking down on the left side of larva. (I) Stage 6: spine primordium (white arrowhead) with base of spine (black arrowhead) extending orthogonal to that. (J) Stage 7: pre-spine (fronds present with no cross hatches; white arrowhead), and TF ring with 2nd ring <1/2 complete (white arrow). Larva viewed from left side. (K) Stage 8: adult spine with cross hatches (white arrowhead); juvenile spine (white arrow) indicated for comparison; anal view. (L) Stage 9: TF ring with 2nd ring >1/2 complete (white arrow is pointing directly to a gap in 2nd ring). (M) Stage 10: TF ring with 2nd ring complete (white arrow); anal view. (N) schematic of larva as seen in panels I-M with larger rudiment and spines becoming recognizable. (O) Stage 10. Whole larva (oriented as in N) showing, in addition to the rudiment, several of the non-rudiment juvenile skeletal characters that we do not incorporate in our staging scheme (see also Additional file 1: Figure S1 and [43]): genital plate 5 (white arrow), posterior juvenile spines articulating with genital plate 4 (white arrowhead) and right side posterior juvenile spine (black arrow; see [45]). Ocular 5 is indicated in panel F. Note that schematic does not show PO arms for simplicity. Abbreviations: Ala – anterolateral arms; M – Mouth; St – stomach; PD – postdorsal arms; PO – postoral arms, PrO – preoral arms. Scale bars B – 300 μm; C-D: 70 μm; E- 80 μm; F-M – 70 μm; O - 200 μm
	Figure 3. Temporal progression of skeletal development in S. purpuratus larvae. A) Mean stage length as a function of skeletal stage. Error bars are one standard error of the mean. B) Cumulative time as a function of skeletal stage, starting at the onset of Stage 1 (first appearance of any skeleton in rudiment; see Table 2). Boxes indicate the approximate cumulative time interval during which a larva is in a given stage; error bars indicate 95% confidence interval.
	Figure 4. Comparison of cumulative time to reach specific stages at three different temperatures. The temporal progression through these stages was clearly slowest at 12°C, whereas no difference is apparent between 14°C and 16°C. Note that the data for 12°C and 16°C originate from the Seattle dataset while the data for 14°C originate from the Guelph dataset. Several other differences existed between these two experiments (see Methods). Error bars indicate 95% confidence interval.
	Figure 5. Spine elongation in S. purpuratus rudiment. A) single developing adult spine with cross hatches; this spine has 3 cross hatches on the indicated face, but notice that other faces have fewer. B) Spine elongation measured by the average (Avg) and maximum (Max) number of cross hatches in the adult spines of developing S. purpuratus larvae at Stage 8 or later (see Table 2). We detected no significant difference in spine elongation between the first and second 24 hours of development in well plates during the course of the experiment at 14°C. Error bars are one standard error of the mean.

References [+] :

Amador-Cano, Role of protein kinase C, G-protein coupled receptors, and calcium flux during metamorphosis of the sea urchin Strongylocentrotus purpuratus. 2006, Pubmed, Echinobase