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Fig. 1. Early stages of arm regeneration in the brittle star Amphiura filiformis. A) Schematic diagram of early arm regeneration stages and average timing to those stages, based on morphological landmarks. Stage 1-wound healing and re-epithelialization. Stage 2-regenerative bud formation. Stage 3-appearance of the radial water canal, coelomic cavities (aboral coelomic canal, ectoneural and hyponeural sinuses) and radial nerve in the regenerative bud. Stage 4-appearance of first metameric units (arm segments). Stage 5-advanced extension of arm, formation of several metameric units at proximal end. B–E) Oral view of fixed arms at stages 2 (B), 3 (C), 4 (D) and 5 (E) shown using differential interference contrast (DIC) microscopy. C’) Detail of C focusing on dermal layer. D’) Detail of D. E’) Detail of E. Arrows - the dermal layer, asterisk - cuticle, D - distal, L - left, OV - oral view, P - proximal, R - right, RWC - radial water canal, S - spicule
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Fig. 2. Histological sections of the earliest stages of arm regeneration in the brittle star Amphiura filiformis. Sagittal (A and A’) and parasagittal (A”) paraffin sections at stage 1 and sagittal paraffin sections at stage 2 (B) stained with Milligan’s trichrome technique. Collagen stained cyan, all cells labeled pink/magenta. A) The wound is completely healed and re-epithelialization occurs. A’) Detail on the new thin epithelium with a recognizable cuticle (asterisk). A”) Histolysis (arrowheads) of intervertebral muscle bundles proximal to the amputation plane. B) The radial nerve, the radial water canal and the coelomic cavities start regenerating beneath the new epidermis. B’) Detail of the mesenchymal cells (arrow). B”) Detail of the mesenchymal cells at the level of the aboral dermal layer (arrow). A - aboral, ACC - aboral coelomic cavity, CT - connective tissue, D - distal, E - epidermis, ES - ectoneural sinus, intervertebral muscles - IM, N - radial nerve, O - oral, P - proximal, Po - podia, PS - parasagittal section, RWC - radial water canal, SS - sagittal section, V - vertebra
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Fig. 3. Histological sections of regenerating arms at stages 3, 4 and 5. Sagittal and transverse paraffin sections at stage 3 (A and A”), and sagittal sections of stages 4 (B), and 5 (C) stained with Milligan’s trichrome technique. Collagen stained cyan, all cells labeled pink/magenta. Red dashed line indicates amputation plane. A) The three regenerating axial structures are enveloped by the dermal layer (arrow) and the new epidermis. A') Detail of A showing mesenchymal cells at the level of the aboral dermal layer (arrow) covered by the new epidermis with its cuticle (asterisk). A”) Transverse section of a stage 3 regenerate showing the dermal layer (arrow), developing radial nerve, radial water canal and aboral coelomic cavity. B) The stage 4 regenerate is longer and the radial water canal shows the first signs of podia regeneration. B') Detail of B showing the developing podia (dotted line). B'') Detail of B showing the scattered mesenchymal cells in the aboral dermal layer (arrow). C) Further development and differentiation of the three axial structures at stage 5. C') Detail of developing podia (dotted line). A - aboral, ACC - aboral coelomic cavity, CT - connective tissue, D - distal, E - epidermis, ES - ectoneural sinus, HS - hyponeural sinus, L - left, N - radial nerve, O - oral, P - proximal, Po - developing podia, RWC - radial water canal, R - right, SS - sagittal section, TS - transverse section
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Fig. 4. Skeletogenesis during early and late regeneration stages. a) DIC (top panels) and fluorescent images (bottom panels) of calcein-labeled spicules at stages 3, 4 and 5. White arrows show individual spicules, which can be compared in both images. Insets show detail of single spicules. b) High magnification DIC images showing single spicules localized in the dermal layer. c) 50 % differentiated arm showing the formation of the skeletal elements (reflective structures). The highly calcified distal cap (terminal ossicle) forms at the distal-most end of the regenerating arm. The spicules, which will form the lateral shields and spines, appear in the first metameric unit (see inset for detail). Proximally, eight metameric units later, first vertebral spicules can be observed (see inset for detail). At the proximal end of the regenerate skeletogenesis is already very advanced and forms the individual stereomic skeletal elements including the oral, aboral and lateral shields, spines and vertebrae (see insets for detail). d) Differentiated skeletal elements in an adult non-regenerating arm. Arrows - calcein-labeled spicules, asterisk - terminal ossicle, E - epidermis, De - dermis. L - left, R - right, P - proximal, D - distal. Red dashed line - amputation plane
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Fig. 5.
Afi-c-lectin expression during early arm regeneration stages. a) Schematic representation of a 3/4 stage regenerate. b–d) Chromogenic whole mount in situ hybridization (WMISH) showing expression of gene at stages 2 and 4. b) Afi-c-lectin is expressed at stage 2 in a broad, sub-epidermal domain. c) At stage 4 the expression of Afi-c-lectin becomes much more restricted to the sub-epidermal domain. d) Higher magnification of arm in c. e, f Fluorescent in situ hybridization of sections through stage 3/4 arm counterstained with the nuclear marker DAPI. e) Frontal paraffin section showing fluorescent ISH of Afi-c-lectin counterstained with DAPI. Afi-c-lectin is clearly localized to single cells just beneath the epidermis. Asterisk marks cells in sub-epidermal layer seen from oral view, due to the slanted plane of sectioning along the oral-aboral axis. f) Higher magnification of arm in e. Scale bars - 50μm
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Fig. 6.
Afi-c-lectin expression at 50 and 95 % differentiation stages. A) Chromogenic WMISH of the whole 50 % regenerating arm from proximal differentiated segments to distal undifferentiated segments and terminal cap. At distal-most end staining is localized to sub-epidermal cells. In differentiating metameric units the expression expands to scattered mesenchymal cells covering the areas of future formation of oral, aboral and lateral shields as well as spines. A’) Detail of gene expression in oral shields, aboral shields, lateral shields and vertebrae. B-D) Confocal images of fluorescent WMISH of Afi-c-lectin (red) in proximal segments of 95 % differentiated arms counterstained with nuclear stain DAPI (blue). B) Maximum projection showing Afi-c-lectin expression in three proximal segments of the arm is localized to cells in the aboral shields and spines. C) Single z-plane projection through one segment in B showing detail of Afi-c-lectin expression corresponding to the shape of the growing vertebra. D) Maximum projection through one segment in B showing detail of Afi-c-lectin expression along the spine. R - right, L - left, P - proximal, D - distal
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Fig. 7. Confocal images showing EdU labeling (red) of early stage regenerating arms counterstained with nuclear stain DAPI (blue). A) Stage 2 maximum projection of confocal z-stack showing widespread cell proliferation in regenerative bud. B) Maximum projection of confocal z-stack of stage 3 arm showing continuing cell proliferation in early regenerate. B’) Single z-plane of B. B”) Detail of B’ showing lack of EdU-labeled cells in dermal layer. Scale bars - 100μm
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Fig. 8.
Afi-c-lectin expression (green) combined with EdU labeling (red) and counterstained with nuclear stain DAPI (blue) showing that skeletogenic cells do not proliferate. A) Maximum projection of proximal segments of 50 % regenerating arm. A’ and A”) Single z-planes of A showing Afi-c-lectin expressing cells do not overlap with EdU-labeled cells. Yellow dashed box shows one case of yellow signal suggesting potential overlap of red and green signals. B) Magnified image in yellow dashed box from A showing cells in z-plane number 3 and z-plane number 15. The EdU labeled red nucleus is seen clearly in z-plane 3 where Afi-c-lectin expression is very faint, whereas on z-plane 15 where the whole cytoplasm of the green-labeled Afi-c-lectin cell is clearly visible, the nucleus of this cell is labeled with DAPI but not EdU (asterisk). C) Maximum projection of distal-most end of 50 % regenerating arm. White dashed half-circle indicates position of terminal ossicle. C’ and C”) Single z-planes of C showing Afi-c-lectin expressing cells do not overlap with EdU-labeled cells. Yellow dashed box shows one case of yellow signal suggesting potential overlap of red and green signals. D) Magnified image in yellow dashed box from C showing cells in single z-plane. Again the nucleus of the Afi-c-lectin expressing cell is labeled with DAPI (asterisk) and is in close proximity to the EdU-labeled nucleus causing the yellow overlap signal
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