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, 41 (5), 483-93

Early Development in the Velvet Worm Euperipatoides Kanangrensis Reid 1996 (Onychophora: Peripatopsidae)

Affiliations

Early Development in the Velvet Worm Euperipatoides Kanangrensis Reid 1996 (Onychophora: Peripatopsidae)

Bo Joakim Eriksson et al. Arthropod Struct Dev.

Abstract

We present here a description of early development in the onychophoran Euperipatoides kanangrensis with emphasis on processes that are ambiguously described in older literature. Special focus has been on the pattern of early cleavage, blastoderm and germinal disc development and gastrulation. The formation of the blastopore, stomodeum and proctodeum is described from sectioned material using light and transmission electron microscopy as well as whole-mount material stained for nuclei and gene expression. The early cleavages were found to be superficial, contrary to earlier descriptions of cleavage in yolky, ovoviviparous onychophorans. Also, contrary to earlier descriptions, the embryonic anterior-posterior axis is not predetermined in the egg. Our data support the view of a blastopore that becomes elongated and slit-like, resembling some of the earliest descriptions. From gene expression data, we concluded that the position of the proctodeum is the most posterior pit in the developing embryo. This description of early development adds to our knowledge of the staging of embryonic development in onychophorans necessary for studies on the role of developmental changes in evolution.

Figures

S. Fig. 1
S. Fig. 1
Euperipatoides kanangrensis. The fused ovaries (ov) lead to paired oviducts (od) each broadening into a hairpin-bent uterus (u) containing numerous embryos. This female had one batch of segmenting embryos in each uterus. The proximal end (white arrow heads) of each uterus contains relatively undeveloped embryos (asterisk) while the distal end (white arrows) contains embryos at a slightly later stage of development (star). The distal end of each uterus has been cut at the point where they fuse to form the unpaired vagina leading to the gonopore between the last pair of legs. Scale bar = 1 mm.
S. Fig. 2
S. Fig. 2
Euperipatoides kanangrensis. Stained with SYBR green. A dissected blastoderm stage embryo showing blastoderm (bd), egg membrane (m) and nuclei (arrows) within yolk clusters (y). Scale bar = 100 μm.
S. Fig. 3
S. Fig. 3
Euperipatoides kanangrensis. Stained with SYBR green. A surface view of a dissected early stage of gastrulation showing the paired germ band and blastopore. The initial phase of somite formation is often asynchronous with the left side slightly ahead with two somites formed (left arrows) while the right side only has one (right arrow). a = anterior part of blastopore, p = posterior part of blastopore, u = unsegmented germ band. Scale bar = 50 μm.
S. Fig. 4
S. Fig. 4
Euperipatoides kanangrensis. Stained with methylene blue. Cross section through the mid-part of the blastopore of an early segmenting embryo showing flask-shaped cells (stars), the surface ectoderm (ec) and the endoderm (en). Arrows mark the border between endoderm and ectoderm. y = yolk. Scale bar = 40 μm.
S. Fig. 5
S. Fig. 5
Euperipatoides kanangrensis. Stained with methylene blue. Cross section of the same embryo as above at the level of the pit of the posterior part of the blastopore to show the multi-layered appearance of the area. Arrows mark the border between the ectoderm (ec) and mesoderm (m). en = endoderm, y = yolk. Scale bar = 30 μm.
S. Fig. 6
S. Fig. 6
Diagrammatic representation of cleavage and gastrulatory movements in onychophoran embryos of the yolk-free South African species Peripatopsis balfouri, P. moseleyi, P. sedgwicki and P. capensis as well as the yolk-rich Australasian species Peripatoides novaezealandiae, Eoperipatus weldoni and Euperipatoides kanangrensis. (A) Early cleavage is similar in the South African species and E. kanangrensis with cleavage nuclei or blastomeres being generated at the periphery of the egg while cleavage nuclei are initially generated within the yolk mass in P. novaezealandiae and E. weldoni. (B) The first divisions result in a pattern of nuclei at the surface of the egg with a variable orientation with regard to the long axis of the egg in the South African species and E. kanangrensis, there is no report of this phenomenon in P. novaezealandiae and E. weldoni but nuclei gather at one location at the yolk surface. (C) The dividing blastomeres initially cover part (a saddle) of the egg surface in Australasian and South African species. Cells at the peripheral part of the expanding cell mass (blue) are internalized in P. capensis and P. balfouri (yellow circles) and this has been described as precocious gastrulation. (D) The resulting blastoderm (blue) covers yolk granules in the Australasian species and fluid in the South African species P. sedgwicki and P. moseleyi, while a gastrula has formed in P. capensis and P. balfouri. In P. capensis, this gastrula is open to the outside by a blastopore and a cavity, the archaenteron (ar) lined by endoderm (en), is present inside. In P. balfouri, the internalised cells are vacuolated (vc) and later disappear leaving a blastula-like embryo. (E) Gastrulation begins in all species with the formation of a blastopore in the germinal disc (blue). In P. capensis and P. balfouri, a secondary blastopore is opened, from where mesoderm and endoderm (P. balfouri) or only mesoderm (P. capensis) are formed (arrows). In P. capensis, there is no formation of a germinal disc and all endoderm derives from the previously formed endoderm. In the Australasian species, endoderm is formed from the anterior part of the blastopore (arrow heads). The shape and timing of the separation of the anterior part of the blastopore varies among species. In P. capensis, the anterior part of the blastopore is relatively large and has protruding edges (black, thick-lined oval). The anterior part of the blastopore in P. balfouri is separated very early into the stomodeum and the middle part of the blastopore (white ovals). In P. moseley, P. sedgwicki, E. kanangrensis, P. novaezealandiae and E. weldoni, the anterior blastopore remains slit-like (white, vertical bar) for some time after the first phases of segmentation. Panel A–C & E represent surface views except for grey-shaded embryos that show sagittal sections. Panel D shows sagittal (ovals) and cross sections (circles). The blue lining in panel D represent the cellular surface layer, ectoderm or blastoderm. The present investigation concerns E. kanangrensis only while details of early development in the other species have been taken from the literature as cited.
S. Fig. 7
S. Fig. 7
Euperipatoides kanangrensis. Schematic illustrations of early developmental stages. (A) A single layer of cells, the blastoderm, covers the yolk. (B) The germinal disc (gd) has formed as an area with more densely packed cells. (C) The blastopore (bp) forms as an elevation of cells with a central pit (cp) within the germinal disc. (D) The elongation of the blastopore pit forms a furrow in the anterior part of the blastopore (ab) that is continuous with the posterior part of the blastopore (bp). (E) The early gastrula has started to form unsegmented mesoderm (u) lateral to the central pit. (F) A magnification of the box in (E) showing the areas of mesoderm formation (red line) and the area of endoderm formation (yellow line).
Fig. 1
Fig. 1
Euperipatoides kanangrensis. Diagrammatic representation of gastrulatory movements. (A) Ventral view of embryo showing germinal disc (gd) as encircled gray area within the surrounding blastoderm (bdm). Upper arrowhead indicates position of anterior end of the embryo. A mid-line groove (marked in yellow) is referred to as the blastopore by Sedgwick (1887) or the mouth-anus by Manton (1949) but here is called the anterior part of the blastopore. Upper two pairs of small, solid arrows show direction of migration of germinal disc cells as they move towards the midline then, as dashed arrows, as they descend into the groove and spread laterally taking on the function of vitellophages before becoming part of the anterior midgut. The posterior pair of small, solid arrows shows cells from the raised pit (dark grey circle), called here the posterior part of the blastopore, but referred to as the primitive streak by Sedgwick (1887) or blastopore area by Manton (1949). They become internalized and migrate anteriorly and laterally (dashed arrows), before ending up as part of the posterior midgut. Boxed area B is enlarged in ventral view B′ and in transverse section in B″. Lower arrowhead indicates position of the posterior end of the embryo. The large arrows on each side of the pit indicate the migration of the mesoderm as the germ bands on each side descend into the pit (dashed arrows) and migrate, at first laterally, and then anteriorly and parallel to the groove. Segmentation is first seen as the separation of the first somite, the antennal segment (a), from the unsegmented mesoderm (u). Boxed area C is enlarged in ventral view in C′ and transverse section in C″. (B′) Ventral view of the anterior part of the blastopore (ab), box B in A. The small, grey open circles represent germinal disc cells, on each side of the groove, before they become internalized and the grey area represents underlying yolk. (B″) Transverse section of the anterior part of the blastopore (ab), box B in A. Small, grey open circles indicate germinal disc cells at the surface and grey, open tear-like shapes indicate cells being internalized. Small, yellow open circles indicate internalized endodermal cells (en) migrating laterally. Large, grey-filled circles indicate yolk spheres. (C′) Ventral view of the posterior part of the blastopore (pb), box C in A. Grey circle indicates the posterior blastopore area (pb) and the lighter grey circle the central pit. The yellow arrows show the migration path of endoderm cells (en), while the red arrows show the migration of the mesoderm (m). (C″) Transverse section of the posterior part of the blastopore, box C in A. Open, grey circles indicate cells in the blastopore area (pb) and grey, open tear-like shapes indicate cells being internalized. Yellow, small open circles indicate internalized endodermal cells (en) migrating anteriorly, corresponding to yellow arrows in C′ and lower, small dashed arrows in A. Large, grey-filled circles indicate yolk spheres. Anterior is to the top in A, B′ and C′. Ventral is to the top in B″ and C″. This study recommends that the groove be henceforth referred to as the anterior part of the blastopore and the pit the posterior part of the blastopore, which removes the inappropriate use of vertebrate terminology.
Fig. 2
Fig. 2
Euperipatoides kanangrensis. (A) An egg (e) situated in the ovary (o) and connected to its flat side with a stalk (arrow). (B) An embryo with approximately 60 nuclei, the nuclei rise to the flattened surface of the egg (arrow). Scale bars: A = 200 μm; B = 300.
Fig. 3
Fig. 3
Euperipatoides kanangrensis. (A–I) Cleavage stages of unstained live material. (J–K) specimens fixed and then stained with DAPI. (A) Two nuclei at the surface (arrows). (B) Four nuclei stage, each nucleus is composed of a clear, circular area (arrowhead) surrounded by a white zone (arrows). (C) Eight nuclei (1–8) stage with cleavage planes indicated by arrowheads. (D) Same embryo as in C a few hours later when the newly divided nuclei have been rearranged into two rows; each numbered nucleus corresponds to the nucleus with the same number in C. The left arrow indicates the pattern of nuclei and the right arrow indicates the long axis of the egg. (E) Another eight nuclei embryo with the rows of nuclei arranged in a different orientation (left arrow) compared with long axis of the egg (right arrow). (F) 16 nuclei stage with the nuclei organized in an alignment (arrows). (G) 32 nuclei stage, the nuclei are starting to loose their organization and synchronization. The boundaries around the nuclei are starting to be sharp (arrowhead), indicating forming cell membranes (see also Fig. 3L). (H) An embryo with approximately fifty cells and the yolk starting to segment (arrows) forming yolk granules without nuclei. The cells containing a nucleus appear white (arrowhead). (I) Embryo with approximately 150 cells and yolk granules formed (arrow). (J) An embryo with the number of cells estimated to be around 1000 forming a saddle of blastoderm cells on one side of the egg (b). (K) An embryo with a blastoderm (b) covering the yolk granules (y). (L) A dissected cell from a 32 cell stage embryo stained with DAPI showing the nucleus (n) and granules (g). Scale bars: A–K 300 μm, L 50 μm.
Fig. 4
Fig. 4
Euperipatoides kanangrensis. A and C–G embryos stained with SYBR-green, (B) embryo stained with DAPI. (A) An embryo with the blastopore (between arrows) formed in the germinal disc (gd). The border between the germinal disc and the rest of the blastoderm (bd) is not sharp but nuclei start to become smaller and more densely packed within the germinal disc, compare both sides of white line. (B) The blastopore has elongated and the posterior (p) end can be distinguished from the anterior (a) by the former having a circular aggregate of cells (arrowhead) surrounding the central pit (asterisk) and the latter is an elongated concentration of cells on both sides of a cell-free furrow (arrow). (C–E) The anterior–posterior axis is, at the early germ band stages, not fixed in relation to the long axis of the egg. (F) An embryo with eight somites formed as indicated by the segmental grooves (arrowheads). The blastopore has been split up into three parts, the mouth (m), a middle opening (mo), and the proctodeum (pd). The first somite, the antennal segment, (s1) has already started to grow considerably larger than the following jaw (J) and slime papilla (sp) somites. (G) An embryo with approximately 15 segments out of a total of 18 formed. The brain lobes have formed as a result of extensive cell proliferation of the neuroectoderm that covers most of the segment. The split germ band is separated by dorsal and ventral extra-embryonic ectoderm (dee and vee). Scale bars = 250 μm. Anterior is to the top in panels F–G otherwise as indicated in the images.
Fig. 5
Fig. 5
Euperipatoides kanangrensis. (A) Cross section of an embryo at the same stage as Fig. 4, A–B. The section is cut level with the blastopore (bp) just posterior to the central pit. (y = yolk). (B) Cross section at the level of the first somite (s1). The ectoderm over the somite is approximately two cell layers deep (upper arrow) as compared to the ectoderm on the flanking sides that is one cell layer thick (lower arrow). The anterior part of the blastopore (asterisk) appears to produce cells that migrate inwards (en) and then laterally beneath the ectoderm. (y = yolk) (C) Cross section at the level of the presomitic mesoderm that has not yet formed a somitic cavity (arrows). The anterior part of the blastopore (asterisk) provides free passage between the outside and the yolk. The continuous sheet of cells (en) stretching from the outside to the inside splits up into scattered cells (arrowheads) located below the mesoderm. (y = yolk). (D) Cross section at the level of the posterior part of the blastopore (pbp). There appear to be three cell groups originating from this area, the ectoderm (ec), the mesoderm (m) and the endoderm (en). Scale bars: A, D = 50 μm; B–C = 100 μm. Dorsal is to the top in all panels.
Fig. 6
Fig. 6
Euperipatoides kanangrensis. (A) TEM micrograph of the zone between the endoderm (en) and mesoderm (m). The endoderm contains electron dense vesicles (dv) presumed to be yolk inclusions and cellular processes (cp) that might facilitate transfer of nutrients from the yolk. The mesoderm is epithelial-like with a surrounding basal lamina (arrowheads). (B) TEM micrograph of the zone between the mesoderm (m) and ectoderm (e) showing the basal lamina around the mesoderm (lower arrowheads) and ectoderm (upper arrowheads). mt = mitochondrion; n = nucleus. Scale bars = 1 μm.
Fig. 7
Fig. 7
Euperipatoides kanangrensis. The posterior part of an embryo hybridized with a probe directed against wingless. Wingless expression can be seen around the presumed proctodeum (pd). Note that there is no staining around the opening (mo) previously believed to form the proctodeum. us = presomitic mesoderm; gb = germ band. Scale bar = 200 μm.

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