Table 1.
List of abbreviations used in the text and figure legends.
Figure 1.
Overview of post-embryonic stages of Pseudopallene sp. and corresponding anatomy of the VNC (PS 1 – sub-adult).
A: SEM micrographs (up to scale), ventral view. Overall body size increases considerably during post-embryonic development. White cross marks damaged distal part of walking leg 1 in PS 6. B: Anti-acetylated tubulin labeling (orange) with Hoechst counterstain (cyan). Imaris Surpass mode (volume, MIP). Images up to scale. New segmental ganglion anlagen are added during the first stages of post-embryonic development. The overall size of the VNC increases notably, although not nearly as dramatically as for overall body size. Abbreviations: pg = posterior ganglion anlage, PS = post-embryonic stage, seg = sub-esophageal ganglion, wlg = walking leg ganglion (anlage).
Figure 2.
Development and fusion of the posterior ganglion anlagen of Pseudopallene sp.
Acetylated tubulin (black, red)- and PH3 (yellow)-labeled VNCs with Hoechst (blue) counterstain (A–D) and histological sections (E–G). A–C: Ventral views of the posterior ganglion anlagen, Imaris volume (MIP). Clipping planes have been applied to remove non-target structures in more dorsal and ventral position, only acetylated tubulin labeling shown. Arrowheads mark commissures of posterior ganglion anlagen. Arrow marks postero-ventral commissure of walking leg ganglion 4. Note size decrease of the posterior ganglion anlagen during the fusion process. The paired connectives and proctodeal nerves approach their contra-lateral counterpart medially. D: Optical sagittal section through walking leg ganglion 4 and posterior ganglion anlagen in PS 3. Dashed outlines highlight (nascent) apical clusters of walking leg ganglion 4 and posterior ganglion anlage 1, the latter being already detached from the apical ectoderm. Note spindle-shaped cells at the apical side of posterior ganglion anlage 2. Asterisks mark large NSCs that are not in division. E: Sagittal section through walking leg ganglion 4 and posterior ganglion anlagen in PS 6. All ganglion anlagen are completely detached from the apical ectoderm. The posterior ganglion anlage 1 has started to fuse with walking leg ganglion 4. F&G: Sagittal sections through walking leg ganglion 4 in a sub-adult. The posterior ganglion anlagen and walking leg ganglion 4 are completely fused. A dorsal neural sheath (arrowhead in F) indicates the fusion line between them. The two commissural tracts (arrowheads in G) of the posterior ganglion anlagen lie closely spaced posterior to the dorsal sheath. White arrow (F) points at the posteriorly extending proctodeal nerve. Abbreviations: ad = adult, apc = apical cell cluster, con = connective, mg = midgut, pg = posterior ganglion anlage, pn = proctodeal nerve, PS = post-embryonic stage, sub-ad = sub-adult, wlg = walking leg ganglion.
Figure 3.
Cell counts in the developing walking leg ganglion 1 of Pseudopallene sp. (PS 1 – PS 6).
Each bar stands for a single analyzed specimen. Ganglion cells are shown in dark red. Cells of the (nascent) apical cell cluster are labeled in light red. A: Overview of counted cell numbers per hemi-ganglion in different stages. Note considerable increase of ganglion cell number in the course of development with a simultaneous decrease of cell numbers in the apical cell cluster. B&C: Spots-model of selected hemi-ganglion counts of PS 1 (early) and PS 2. Ventral view on the left side, medial view on the right side. Note the compaction of the (nascent) apical cell cluster (light red) after its complete detachment from the ventral ectoderm in PS 2. Abbreviations: EC = ectodermal cell, INP = intermediate neural precursor, PS = post-embryonic stage.
Figure 4.
Apical pits and general VNC structure in PS 1 of Pseudopallene sp.
SEM micrographs (A–D) and histological sections (E–H). Stars mark ganglionic neuropil. White arrows indicate lumen of apical pits. Big gray arrows mark pycnotic bodies at the basal side of the nascent apical cell clusters. A: Lateral view of PS 1, distal portions of proboscis, chelifores and walking leg anlagen removed, anterior to the top, dorsal to the left. Arrows point to the apical ventral pits in walking leg segment 1 under detached cuticle. B: Detail of cuticle of walking leg segment 1, ventral view. Cuticular depressions (arrowheads) correspond in position to the underlying ectodermal pits. C&D: Ventral detail of apical pits in the palpal and ovigeral segments (C) and in walking leg segments 1 and 2 (D). The epidermis covers already the complete ventral side except for the segmentally paired pits (arrows). E: Composite sagittal section of the VNC of early PS 1 (stippled line indicates border of two images). Sections lie lateral to the midline region. The palpal and ovigeral neuromeres are already fused to form the sub-esophageal ganglion. The apical pit in walking leg segment 2 is not in the plane of the section. The less developed ganglion anlage of walking leg segment 3 still lacks a distinct apical pit. White arrowhead highlights a NSC division. F–H: Transverse sections through the ventral ‘embryonic’ ganglion anlagen of a PS 1 specimen slightly older than the one shown in E. Note distinct separation (black arrowheads) of the nascent apical cell clusters and the underlying hemi-ganglion anlagen proper (stippled outline in G). Abbreviations: cec = circum-esophageal connective, ch = chelifore, mg = midgut, GC = ganglion cell, ov = ovigeral neuromere, seg = sub-esophageal ganglion (anlage), wl = walking leg (segment), wlg = walking leg ganglion (anlage).
Figure 5.
Apical cluster detachment and mitotic activity during PS 1 of Pseudopallene sp.
A&B: Ventral overview of the VNC in early and late PS 1. Imaris volumes (MIP) of acetylated tubulin (A) and tyrosine-tubulin (B) immunolabeling (both red) and with additional PH3-staining (yellow). White circles and ovals mark apical segmentally paired pits. Stippled ovals indicate shallow apical invagination of the ganglion anlage in walking leg segment 3. Asterisks indicate central cavity of completely detached apical cell clusters. Note decrease of overall mitotic activity from early to late PS 1. C–G Apical pits and detached apical clusters in the VNC. Optical transverse sections of tubulin (red)- and PH3 (yellow)-labeled embryos with Hoechst (blue) counterstain. Composite images in which the left side shows early PS 1 (acetylated tubulin) and the right one the corresponding region in late PS 1 (tyrosinated tubulin). Arrows point at the lumen of the apical pits or shallow invaginations. Open white arrowheads indicate small flattened epidermis cells. Asterisks label selected large NSCs (not in division). Filled white arrowheads mark sub-apical divisions of INPs. Stars label neuropil or axonal pathways. Note decrease of mitotic activity from early to late PS 1 and the fully detached apical clusters with central cavity in three segments (ov-2 wl) in late PS 1. Abbreviations: ov = ovigeral neuromere, pa = palpal neuromere, PS = post-embryonic stage, seg = sub-esophageal ganglion, vpn = ventral proboscis nerve, wlg = walking leg ganglion (anlage).
Figure 6.
Mitotic activity of apical NSCs and sub-apical INPs in walking leg ganglia 1 and 2 during PS 1 of Pseudopallene sp.
Histological sections (A–D) and optical sections of tubulin (red)- and PH3 (yellow)-labeled specimens with Hoechst (blue) counterstain (E–H). A–D: Sagittal sections at different levels through walking leg ganglion 1 in early PS 1. Stars mark ganglionic neuropil. Arrows indicate lumen of apical pits. White arrowheads highlight divisions of selected NSCs, the morphological asymmetry of the divisions being visible in late mitotic stages (A,C). Black arrowheads mark symmetrical divisions of sub-apical INPs (A,D). Big gray arrow marks pycnotic body (C). E–H: Horizontal sections through apical pits in early PS 1 and detached apical cell clusters and streams in late PS 1. Acetylated tubulin (E,F) and tyrosine tubulin (G,H) labeling. Stippled outlines mark extensions of nascent apical clusters and ganglion anlagen proper (E,F) or anterior and posterior cell streams (H). Asterisks label NSCs. E: Walking leg ganglion 1, section slightly basal to the epidermal cells covering the rim of the pit. F: Walking leg ganglion 1, section through the bottom of the pit being characterized by massive proliferation of the lining NSCs (bottom half of image). Anteriorly, open arrowheads indicate divisions of INPs. At this stage, each nascent apical cluster extends across the postero-ventral side of the corresponding hemi-ganglion proper. G: Walking leg ganglion 2, section through detached apical cell clusters at the level of the central cavities. Note size differences of NSCs and the smaller intermingled cells with more brightly stained nuclei. White spots label epidermis cells. H: Walking leg ganglion 2, section through the more defined anterior and posterior cell streams that extend into the ganglion proper. Note INP divisions within the streams. Abbreviations: PS = post-embryonic stage, wlg = walking leg ganglion (anlage).
Figure 7.
Nucleus measurements in the apical cell clusters of walking leg ganglion 1 of Pseudopallene sp. (early PS 1 – ad).
The largest nuclei encountered in the (nascent) apical cell clusters have been measured along their elongated axis. Arithmetic means are shown, bars represent standard deviation. Small numbers below the values give the number of specimens analyzed per developmental stage. In early PS 1 the apical pit is still open and lined by numerous large NSCs with nuclei measuring about 15 µm. After apical closure of the pit and detachment of the apical cell clusters (stippled vertical line), nucleus size (and cell size) in the clusters drops considerably to mean values lower than 10 µm. Already from PS 2 onwards NSCs are not reliably identifiable any longer based on morphological characteristics alone. Abbreviations: ad = adult, PS = post-embryonic stage, sub-ad = sub-adult.
Figure 8.
Sensory setae, epidermal glands and VNC structure in PS 2 of Pseudopallene sp.
SEM micrographs (A,B,D), acetylated tubulin (orange) labeling with Hoechst (blue) counterstain (C) and histological sections (E–H). A: Walking leg segment 1 to anal tubercle, ventral view. Small fields of cuticular folds (arrowheads) indicate location of underlying segmental ganglion anlagen. B: Detail of area highlighted in A. The cuticle bears bifurcate setae (arrowhead) and minute slit-openings of epidermal glands (arrows). C: Anterior in-situ view of walking leg ganglion 1. Imaris volume (blend), clipping planes used to reveal target structures. The apical cell clusters (filled arrowheads) are completely detached from the ventral epidermis with no connection to cells of the slit glands (arrow) or setae (open arrowheads). Longitudinal muscles (orange-yellowish bands) are in part located between apical clusters and ventral epidermis. D Ventral view of VNC (primordium of 4 wlg missing). Arrowheads indicate detached apical clusters. Cross marks the original position of the left apical cluster of 2 wlg, having been posteriorly displaced during dissection. The nascent apical clusters of 3 wlg still cover its entire ventral side. E–H: Stars mark ganglionic neuropil. Arrows indicate regions where cell streams enter underlying ganglion proper. Black arrowheads indicate separation lines between (nascent) apical clusters and underlying ganglia proper. E: Composite sagittal section of the VNC (stippled line indicates border of two images), lateral to the midline region. Stippled ovals highlight detached apical clusters. The cluster of the palpal neuromere lies not in the plane of the section. Note apical attachment of the less-developed anlagen of ‘post-embryonic’ 3 wlg and 4 wlg. F–H: Transverse sections through different ventral ganglia (anlagen). Note decreased cell size in more anterior compared to more posterior apical clusters (F,G and H, respectively). White arrowhead in H marks NSC division. Abbreviations: cec = circum-esophageal connective, con = connective, eso = esophagus, mg = midgut, mgd = midgut diverticulum, NSC = neural stem cell, ov = ovigeral neuromere, seg = sub-esophageal ganglion, VNC = ventral nerve cord, wl = walking leg (bud), wlg = walking leg ganglion (anlage).
Figure 9.
Mitotic activity in detached and nascent apical CSSs in the VNC of Pseudopallene sp. (PS 2).
Acetylated tubulin (red) and PH3 (yellow) labeling of VNCs with Hoechst (blue) counterstain. A: Composite image of VNC, ventral view. Imaris volume (MIP), Hoechst counterstain not shown, stippled lines mark borders of separate images. Note that mitoses in the anterior ‘embryonic’ ganglia (seg-2 wlg) are restricted to the detached apical clusters. B–F: Optical sections through detached and nascent apical clusters and underlying segmental ganglia (anlagen). Sagittal (B,D,F) or transverse (C,E) sections above dashed lines, horizontal sections below dashed lines. Stars indicate ganglionic neuropil. White arrows mark anterior and posterior cell streams. Open arrowheads indicate sub-apical mitoses close to or within the cell streams. Asterisks (E,F) highlight selected apical NSCs that are not in mitosis. Note tiny central cavities and still detectable differences in cell sizes and nuclear staining intensities in the detached anterior clusters (pa-2 wlg). The paired nascent apical clusters of the anlage of walking leg ganglion 3 are characterized by a nuclei-free apical invagination. H: Optical sections through primordium of walking leg ganglion 4. Transverse section above dashed line, horizontal section below dashed line. Arrows indicate basal anlagen of the longitudinal connectives. Note tangential apical mitosis and lack of large NSCs in this early neurogenic phase. Abbreviations: cec = circum-esophageal connective, ov = ovigeral neuromere, pa = palpal neuromere, seg = sub-esophageal ganglion, wlg = walking leg ganglion (anlage).
Figure 10.
Persistence of apical CSSs into the adult of Pseudopallene sp.
Sagittal histological sections. Black arrowheads highlight the neural sheath. Arrows indicate fibrous cell streams that penetrate through the neural sheath into the ganglionic somata cortex. White arrowheads mark mitoses in apical cell clusters. A&B: Composite overview images of the VNC of PS 6 (A) and the adult (B). Stippled lines indicate borders between separate images. Adjacent original images show sections at slightly different levels in order to depict more apical cell clusters. Stippled circles highlight apical clusters that are not shown in a separate detail. The palpal cluster is not shown in any overview, owing to its further lateral position (see also Fig. 7D). Note the similar or occasionally darker nuclear staining of the cluster cells compared to differentiated neuronal nuclei. C&D: PS 6. C: Ovigeral CSS. Note cell division in the apical cluster. D: CSS of walking leg ganglion 2. Note the tiny central cavity (stained in pink) in the apical cluster. E: Ovigeral CSS in an adult. F&G: Sub-adult. F: CSS of walking leg ganglion 1. Note elongated shape of the apical cluster. G: CSS of walking leg ganglion 4. Note mitotic profiles in the elongated and flattened apical cluster. Abbreviations: ad = adult, ov = ovigeral neuromere, PS = post-embryonic stage, seg = sub-esophageal ganglion, sub-ad = sub-adult, wlg = walking leg ganglion.
Figure 11.
Mitotic activity in detached CSSs during advanced post-embryonic development of Pseudopallene sp.
Optical sections of acetylated tubulin (red)- and PH3 (yellow)-labeled VNCs with Hoechst (blue) counterstain. White arrows label fibrous anterior and posterior cell streams. Black arrowheads mark sub-apical mitoses close to or within cell streams. White arrowheads indicate oblong nuclei of cells that migrate along the fibrous cell streams. Stars label ganglionic neuropil. Note the absence of distinctly larger cells in the apical clusters. A–C: PS 3. A: Slightly oblique transverse section through sub-esophageal ganglion at the level of the ovigeral cluster-stream-system (CSS). Note flattened nuclei (gray arrowheads) of glial cells in the neural sheath surrounding the ganglion. B&C: Sagittal (B) and horizontal section (C) through walking leg ganglion 4, respectively. Note the still diffuse, not clearly defined cell streams in this posterior walking leg ganglion. D–F: PS 4. D: Sagittal section through ovigeral CSS. E: Sagittal section through CSS of walking leg ganglion 2. F: Horizontal section through CSS of walking leg ganglion 3. G–I: PS 6. G: Transverse section showing detail of ovigeral CSS. Note the narrow neck of the nuclei (arrowheads) of cluster cells that start to extend into the cell stream. H: Horizontal section through CSS of walking leg ganglion 4. I: Horizontal section through CSS of walking leg ganglion 3. The white line extends between two newly forming nuclei during telophase. The cell division is morphologically symmetrical. Abbreviations: ov = ovigeral neuromere, PS = post-embryonic stage, wlg = walking leg ganglion (anlage).
Figure 12.
Glomerulus-like neuropils in the VNC of Pseudopallene sp.
Histological sections through ventral ganglia. Stippled ovals highlight ventro-lateral neuropil regions housing glomerulus-like neuropils (GNs). Arrows point at selected GNs. Arrowheads indicate apical cell clusters. Ganglion identity as indicated in bottom left corner of each image. A&B: PS 6. A: Sagittal section. B: Transverse section. C–E: Adult. C: Sagittal section. D: Transverse section. E: Magnification of GN-containing neuropil region, sagittal section. F: Sub-adult. Transverse section showing two ventro-lateral GNs and medial to them the apical cell cluster with one of its cell streams, which penetrates the neural sheath. One cell in the slender cell stream is in mitosis (black outline). Abbreviations: ad = adult, mg = midgut, PS = post-embryonic stage, pvc = postero-ventral commissure, sub-ad = sub-adult, wlg = walking leg ganglion.
Figure 13.
Cell numbers in apical clusters during post-embryonic development of Pseudopallene sp.
Developmental stages are color-coded as shown in the legend in the upper left corner. Segmental affiliation of the clusters is shown on the x-axis. Each column represents the median of counted cell numbers per cluster and developmental stage. The sample size of counted clusters is shown at the bottom of each column. Sample size differences between different clusters of the same developmental stage relate to cluster damage or loss during dissection. ‘Error’ bars indicate maximum and minimum cell counts. Complete lack of values (PS 2–3 wlg, 4 wlg) is related to the early differentiation state of the respective ganglion anlagen with no clearly separated clusters on the ventral side. Abbreviations: ad = adult, ov = ovigeral neuromere, pa = palpal neuromere, PS = post-embryonic stage, sub-ad = sub-adult, wl = walking leg neuromere.
Figure 14.
Formation of the apical CSSs during post-embryonic development of Pseudopallene sp.
Schematic drawings in sagittal view. A: A deep apical invagination has formed in each hemi-segment, cells of the prospective epidermis (EPCs) covering its apical rim. Large spindle-shaped NSCs line the deep invagination and divide in asymmetrical fashion. Interspersed between the NSCs smaller flask-shaped cells are found, being in the process of basal immigration. NSCs and the interspersed cells form together the nascent apical cell cluster. Sub-apical to it, at least some of the immigrating cells divide, representing intermediate neural precursors (INPs). Characteristically, INPs are found in the still wide, nascent anterior and posterior cell streams that lead into the underlying hemi-ganglion anlage proper. B: All cells lining the deep central invagination detach from the ventral ectoderm, forming a fully separated apical cluster with a small central cavity at the ventral side of the hemi-ganglion. After the detachment process, the epidermis is apically closed. The cluster remains in contact with the underlying hemi-ganglion via well-defined anterior and a posterior cell streams. Along these streams, additional cell material migrates into the hemi-ganglion, being produced in the clusters as well as in the streams themselves. C: With ongoing development, the NSCs in the apical clusters decrease in size and become (at least morphologically) unidentifiable. The cell streams diminish to fibrous strands. Mitotic activity of (as yet uncharacterized) neural precursors persists in cluster and streams, but at a lower rate. The ganglionic neuropil increases considerably and ventrally glomerulus-like neuropils (GNs) are formed therein. Abbreviations: con = connective, EPC = epidermis cell, GN = glomerulus-like neuropil, INP = intermediate neural precursor, NP = neural precursor, NSC = neural stem cell.