Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the hemichordate Schizocardium californicum

Abstract

Background: There are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in the development of indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when and where the tissue that will give rise to the adult originates. How do the processes of cell division and cell death redesign the body plans of indirect developers? In this study, we present patterns of cell proliferation and cell death during larval body plan development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium (Cameron and Perez in Zootaxa 3569:79-88, 2012) to answer these questions.

Results: We identified distinct patterns of cell proliferation between larval and adult body plan formation of S. californicum. We found that some adult tissues proliferate during the late larval phase prior to the start of overt metamorphosis. In addition, using an irradiation and transcriptomic approach, we describe a genetic signature of proliferative cells that is shared across the life history states, as well as markers that are unique to larval or juvenile states. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis.

Conclusions: Cell proliferation during the development of S. californicum has distinct patterns in the formation of larval and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferated and gave rise to the larvae and juveniles have a genetic signature that suggested a heterogeneous pool of proliferative progenitors, rather than a set-aside population of pluripotent cells. Taken together, we propose that the gradual morphological transformation of S. californicum is mirrored at the cellular level and may be more representative of the development strategies that characterize metamorphosis in many metazoan animals.

Keywords: Cell death; Cell proliferation; Hemichordate; Metamorphosis; Tornaria.

Fig. 1

Larval development and metamorphosis of S. californicum. A Schematic of the complex life cycle of the indirect developing S. californicum (modified from Ref. [35]. B–J Light microscopy of the complex life cycle of the indirect developing S. californicum (from [35]). B early tornaria larva, C mid tornaria larva, D late tornaria larva, E–I process of metamorphosis, J juvenile

Fig. 2

Cell proliferation throughout early larval development of S. californicum. All: anterior up; blue = Hoechst, green = EdU; scale bar is 100um. Schematics of ventral (A), dorsal (D), and medial (E) views with key structures labeled. EdU staining with maximum intensity projections showing ventral (B), dorsal (E), and medial H sections. C–I insets. White arrowheads denote preoral ciliary band and postoral ciliary band

Fig. 3

Cell proliferation throughout mid larval development of S. californicum. All: anterior up; blue = Hoechst, green = EdU; scale bar is 100um. Schematics of ventral (A), dorsal (D), and medial (E) views with key structures labeled. EdU staining with maximum intensity projection showing ventral (B), dorsal (E), and medial (H) sections. C–I insets. White arrowhead in (B and C) denote postoral ciliary band, white asterisk denotes telotroch. White arrowhead in (F) denotes protocoel pore

Fig. 4

Cell proliferation in late larval development of S. californicum. All: anterior up; blue = Hoechst, green = EdU; scale bar is 100 um. Schematics of ventral (A), dorsal (D), and medial (G) views with key structures labeled. EdU staining with maximum intensity projection ventral (B), dorsal (E), and medial (H) sections. B late larva ventral surface, C inset of (B), highlights ventral posterior epidermis and ciliary band E late larva dorsal surface, F inset of (E) highlights dorsal cord, marked by white asterisk, H late larva medial section, arrowheads highlight regions that will give rise to protocoel, mesocoel, and metacoel. I inset of lateral view of late larva medial section, arrowhead highlights gill bars. J protocoel, mesoderm that will form the proboscis, K mesocoel, mesoderm that will form the collar, L metacoel, mesoderm that will form the trunk. M, N distribution of anti-histone H3 (phospho S10) and EdU positive cells in ciliary bands, magenta = pHH3.3, grey = acetylated tubulin

Fig. 5

Cell proliferation throughout the metamorphosis of S. californicum. All: anterior up; dorsal view; blue = Hoechst, green = Edu; scale bar is 100um. Schematics of ventral (A), dorsal (D), and medial (G) views with key structures labeled. EdU staining with maximum intensity projection: B Early in metamorphosis, white asterisk marks lateral food groove C Inset of (B), EdU positive cells are distributed around and in the dorsal cord. E Middle of metamorphosis, white arrowhead marks lateral food groove. F Inset of (E), EdU positive cells are found in the dorsal cord and mesocoel. H End of metamorphosis, asterisk marks dorsal cord. I Inset of (H), white arrowhead highlights EdU positive cells are distributed throughout the lateral grooves

Fig. 6

Cell proliferation in juveniles of S. californicum. All: anterior up; dorsal view; blue = Hoechst, green = Edu; scale bar is 100 um. Schematic of juvenile (A) with key structures labeled. EdU staining with maximum intensity projection: B End of metamorphosis. C Highlights regions of (B), EdU positive cells are distributed throughout the proboscis. D Highlights regions of (B), EdU positive cells are distributed throughout the gill bars. E Highlights regions of (B), EdU positive cells are distributed throughout the dorsal cord. F Continuing development of the juvenile body plan. Arrowheads mark the base of the collar and expanding trunk

Fig. 7

Genetic signature of irradiation sensitive EdU cells in both larvae and juveniles. All; blue = Hoechst, green = EdU; scale bar is 100um. A Control larva representing the normal EdU pattern at this stage, representative of 5/5 animals. B Experimental larva representing the EdU pattern at this stage after receiving 120 Gy of X-ray irradiation, representative of 5/5 animals. C Control juvenile representing the normal EdU pattern at this stage, representative of 4/4 animals. D Experimental juvenile representing the EdU pattern at this stage after receiving 200 Gy of X-ray irradiation, representative of 2/2 animals. E Volcano plot showing expression differences in control versus irradiated larva. n = 3 for each group. F Volcano plot showing expression differences in control versus irradiated juvenile. n = 3 for each group. G A larva with HCR probes for Fgfr-B. H Higher magnification of ciliary band with Fgfr-B transcripts. I A juvenile with HCR probes for Spindle-E. J, K Higher magnification of Spindle-E transcripts expressed between the ectoderm and the gills bars. L A larva with HCR probes for Lbr-1. M Inset of H with Lbr-1 transcripts distributed throughout the ciliary band. N A juvenile with HCR probes for Lbr-1. O Inset of I with Lbr-1 transcripts distributed throughout the lateral grooves. P Lbr-1 transcripts distributed in the gill bars

Fig. 8

Cell death throughout the metamorphosis of S. californicum. All: anterior up; blue = Hoechst, grey = TUNEL; scale bar is 100um. Schematics of early in metamorphosis (A), during metamorphosis (D), and end of metamorphosis (G) with key structures labeled. B Start of metamorphosis with an increase in TUNEL⁺ cells. C Highlights regions of (B), specifically around lateral grooves. E Middle of metamorphosis. F Highlights regions of E, specifically around the dorsal cord. H End of metamorphosis. I Highlights regions of (H), specifically around the dorsal chord and collar. J Overlap of serotonin⁺ cells and TUNEL⁺ cells. K Inset of (G), highlighting TUNEL⁺ and serotonin⁺ positive cells. L Overlap of Elav⁺ cells and TUNEL⁺ cells. M Inset of (L), highlighting TUNEL⁺ and Elav⁺ cells