Planarian brain and ventral nerve cords are two distinct structures in the head region
As we reported previously, whole-mount immunostaining with a polyclonal antibody raised against a planarian synaptotagmin homologue, DjSYT (Tazaki et al. 1999), shows that the planarian CNS is basically constituted by an anterior ‘brain’ or cephalic ganglia and two longitudinal VNC that run throughout the length of the body (Fig. 1A). The planarian brain consists of two lobed ganglia connected exclusively in their most anterior part, and it can be divided into a central, structurally spongy region (neuropil; Morita & Best 1966; Baguñà & Ballester 1978) and several lateral branches that extend towards the head margins (Fig. 1A). This brain is not a simple thickening of the anterior end of the VNC, but a distinct structure located more dorsally, as can be clearly seen by combining in situ hybridization for various neural genes and immunostaining against DjSYT (Fig. 1B–E), and through histological cross-sections after in situ hybridization for neural genes (Fig. 1F–G). A collection of new planarian- specific neural genes has been isolated in studies using DNA microarrays (M. Nakazawa, unpubl. obs., 2001). One of these genes (clone 944-HH) shows homology to neural cell adhesion molecules (N-CAM) and is highly expressed in the planarian CNS (Fig. 1B). A second neural gene (clone 1791-HH, a planarian homologue of G-protein α-subunit genes) is specifically expressed in the distal part of brain lateral branches (Fig. 1D). After in situ hybridization with clone 944-HH (N-CAM homologue), the antibody against DjSYT could not immunoreact with hybridization-positive brain cells, probably because the precipitate obscures the fluorescent signal. DjSYT within longitudinal nerve tracts, however, could be detected, allowing us to clearly observe the VNC below the brain (Fig. 1C). This anterior portion of the VNC shows the same orthogonal pattern found all along the antero–posterior axis (Fig. 1A), with ganglionic knots connected by transverse commissures. In contrast, immunostaining with anti-DjSYT after in situ hybridization with clone 1791-HH (G protein homologue) completely stains the planarian spongy brain (Fig. 1E). Comparison of Fig. 1C,E reveals that the brain is located dorsal to VNC which run below it reaching the anterior end of the planarian. This relationship between the brain and the anterior VNC can be more clearly observed after cross-sectioning whole-mount in situ hybridizations. Figure 1F shows a histological section after in situ hybridizations for clone 944-HH (N-CAM homologue), which is expressed in both brain (arrow) and VNC (arrowhead) cells. Clone 721-HH (unknown), however, is highly expressed in brain cells (arrow) but no expression is detected in the VNC (arrowhead), which suggests that also at the gene expression level, brain and VNC cells can be distinguished (Fig. 1G). All of these results indicate that planarian brain and VNC, although closely associated, can be considered as distinct and overlapping structures.
Figure 1. . Planarian central nervous system (CNS) structure. (A) Whole- mount immunostaining with anti-DjSYT antibody. The two lobes of the brain are connected by a single anterior commissure. The ventral nerve cords (VNC) have regularly spaced ganglionic knots (arrow) from which transverse commissures (TC) and lateral branches (LB) extend. Double in situ hybridization for clone 944-HH (B,C) or 1791-HH (D,E) and immunostaining with anti-DjSYT antibody (green fluorescence). Clone 944-HH (N-CAM homologue) is highly expressed in the brain (B). Clone 1791-HH (G-protein homologue) is specifically expressed in the distal part of brain branches (D). Histological cross-sections after in situ hybridization for clones 944-HH (F) and 721-HH (G). Clone 944-HH is expressed in both brain (arrowhead) and VNC (arrow) cells (F). Clone 721-HH is expressed in brain (arrowhead) but not VNC (arrow) cells (G). A–E, anterior to the top; F,G, dorsal to the top. ph, pharynx. Bars (A) 500 μm, (B–E) 300 μm and (F,G) 500 μm.
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Planarian netrin homologue is expressed in the overlapping region of brain and ventral nerve cords
Planarian clone 5189-HH shows sequence similarity to netrin family (Fig. 2) and is expressed in few brain and VNC cells giving rise to a unique pattern, as positive VNC cells are exclusively localized in the region where VNC run below the brain (Fig. 3A). In cross-sectional view, these two cell populations can be distinguished (Fig. 3B,F). Dorsal positive cells (arrowhead) show strong signals when compared to the ventral positive cells (arrow) that appear to be clustered around longitudinal nerve tracts (Fig. 3C), in a similar pattern found for other clones also expressed in VNC cells such as 944-HH (N-CAM homologue) and 517-HH (receptor protein tyrosine phosphatase (rPTP) homologue; data not shown). In contrast, dorsal positive cells form small, irregularly spaced clusters placed in the dorsal part of the brain, as revealed by double in situ hybridization and immunostaining with anti-DjSYT (Fig. 3D,E). Outside the CNS, this gene is also expressed in the ventral muscle fibers.
Figure 3. . Whole-mount in situ hybridization for Djnet1. (A) Positive cells are detected in the region where the brain and ventral nerve cords (VNC) overlap (arrowheads). Weak signals observed throughout the body as small dots correspond to the expression of this clone in muscle fibers. (B) Histological cross- section showing dorsal brain (arrowhead) and ventral (arrow) positive cells. (C) Ventral positive cells appear to be clustered (arrow) around nerve tracts (clear region, asterisk). Double in situ hybridization for Djnet1 (D) and immunostaining with the anti-DjSYT antibody (E) to show positive cells in the dorsal brain. (F) Schematic drawing of a cross-section of planarian CNS showing the localization of Djnet1-positive cells. Red dots correspond to dorsal positive cells and blue dots correspond to ventral positive cells. b, brain; vnc, ventral nerve cord. (B,F) dorsal to the top. Bars (A) 1 mm, (B) 400 μm, (C) 50 μm and (D,E) 200 μm.
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Central nervous system formation during anterior regeneration
With the aim of studying how planarian CNS is rebuilt during regeneration and which genes could be involved, we analyzed the expression patterns of synaptotagmin and a set of other neural-specific genes during head regeneration.
Anti-DjSYT immunostaining detects the newly formed brain from day two of regenerationFigure 4 shows CNS formation as revealed by immunostaining with the anti-DjSYT antibody. Just after amputation, neoblasts (undifferentiated, totipotent stem cells) close to the wound proliferate, giving rise to the blastema where new structures will be formed. In 1-day regenerants, no staining is detected within the blastema (arrowheads) and the VNC appear to remain truncated in the stump region (Fig. 4; day one). After 2 days of regeneration, a small, thin and weakly stained brain appears within the blastema (Fig. 4; day two). The brain structure can be distinguished by the small lateral branches. At day three of regeneration, the new brain is bigger; however, it is difficult to determine whether the VNC have grown into the blastema as no clear commissures between VNC can be detected in the new cephalic region at this stage (Fig. 4; day three). After 4–5 days of regeneration in contrast, VNC connected by transverse commissures appear in the head region below the newly formed brain (Fig. 4; day five). At day seven of regeneration, the new brain clearly shows the spongy structure characteristic of the brains of intact organisms.
Figure 4. . Planarian central nervous system (CNS) regeneration as revealed by immunostaining with the anti-DjSYT antibody. At day one no signal is detected within the regenerative blastema (arrowheads). At day two a small brain can be recognized within the blastema, dorsal to the ventral nerve cords (VNC) of the stump region, which are out of focus. At day three the new brain is bigger but no transverse commissures between VNC can be clearly detected. At day five, however, transverse commissures (arrow) connect the left and right VNC below the newly formed brain. Finally, at day seven the new brain shows a spongy structure similar to that of the brains of intact organisms. Dashed lines separate the newly formed blastema from the old stump region. Anterior to the top. Bars, 300 μm.
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Neural genes can be classified as early, mid- regeneration and late expression genes Recently, many neural genes have been isolated through planarian DNA microarray (M. Nakazawa, unpubl. obs., 2001) and expressed sequence tags (EST; S. Gaudieri, unpubl. obs., 2001 and K. Mineta, unpubl. obs., 2001) projects. Some of the genes used in this study show sequence similarity to known genes in other organisms, such as N-CAM, rPTP, G-protein α-subunit, lethal giant larva, very low-density lipoprotein receptor (VLDLR), and human brain protein 239AB. Other clones, however, show no clear homology to any sequence in the current databases. According to their upregulation during head regeneration, these neural genes can be classified as early, mid-regeneration or late expression genes. Table 1 summarizes the clones used, their sequence homologies and expression patterns in intact organisms as well as when they are first detected within the regenerative blastema.
Table 1. . Summary of planarian neural clones used in the study
|Clone||Putative homologue||Expression in adult organisms||First expression within blastema|
|953-HH||VLDL/LDL receptor||General expression in CNS||Day 1|
|721-HH||Unknown||Brain and other head cells||Day 1|
|1791-HH||G-protein α-subunit||Distal part of brain branches||Day 1|
|517-HH||PTP receptor||General expression in CNS||Day 1|
|2467-HH||Human brain protein 239AB||Brain and lower expression in VNC and other mesenchymal cells||Day 1|
|1242-HH||Human giant larva||Brain and other mesenchymal cells||Day 1|
|1008-HH||Unknown||Brain branches||Day 2|
|944-HH||N-CAM||General expression in CNS||Day 2|
|Eye793||Unknown||Brain (except branches) and VNC||Day 2|
|5189-HH||Netrin||Overlapping region of brain and VNC; ventral muscle fibers||Day 3|
|Eye53||Unknown||Brain (except branches); visual cells; cells surrounding VNC||Day 4–5|
|1020-HH||Unknown||Brain (except branches); very few cells along VNC||Day 4–5|
Early expression genes Six of the neural genes examined here are expressed in the emerging blastema from the first day of regeneration (Fig. 5). These genes include putative planarian homologues of rPTP (517-HH), G-protein α-subunit (1791-HH), VLDLR (953-HH), human brain protein 239AB (2467-HH) and lethal giant larva (1242-HH). Independently of their different expression patterns in intact organisms, all six of these genes are expressed in a few cells grouped in two small clusters within the newly formed blastema (Fig. 5; day one). In the case of clone 953-HH (VLDLR homologue) these two small clusters of cells are not apparently connected to the truncated VNC of the stump region (Fig. 5; arrowhead and arrow). Looking at the expression dynamics of all these genes in the following days, it seems clear that the two cell clusters detected at day one can be defined as the new brain primordium. For clone 721-HH (unknown), the positive cells within the blastema define not only the forming brain but also the other head cells detected in intact organisms outside the brain, as suggested by the higher number of positive cells compared with those positive for the other genes, especially during the first days of regeneration (Fig. 5). After 5–7 days of regeneration the basic planarian CNS structural pattern is restored.
Figure 5. . Whole-mount in situ hybridization for early expression genes. These genes include clones 953-HH (very low-density lipoprotein receptor (VLDLR) homologue), 721-HH (unknown), 1791-HH (G-protein homologue), 517-HH (receptor protein tyrosine phosphatase (rPTP) homologue), 2467-HH (human brain protein 239AB homologue) and 1242-HH (lethal giant larva homologue). For all of these genes, brain primordium can be observed within the blastema from the first day of regeneration. For clone 953-HH, brain primordium within the blastema (arrowhead) is not connected to the ventral nerve cords (VNC) of the stump region (arrow). Dashed lines separate the newly formed blastema from the old stump region. Anterior to the top. Bars, 400 μm.
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Mid-regeneration expression genes Three neural clones, including a putative planarian homologue of N-CAM, are expressed within the blastema from the second day of regeneration (Fig. 6). At day one, no signal is detected in the new tissues and for clones 944-HH (N-CAM homologue) and eye793 (unknown) the VNC appear truncated in the stump region: these are similar results to those obtained with the anti-DjSYT antibody (Fig. 6; day one; arrow). At day two, the first signal within the blastema appears as two clusters of weakly stained cells, probably corresponding to the new brain cells. Other mid-regeneration genes are clones 1008-HH (unknown), which are expressed within the blastema at day two of regeneration as two bilateral clusters of cells, and clone 5189-HH (netrin homologue), which is first expressed within the blastema from day three of regeneration after the formation of the brain primordium.
Figure 6. . Whole-mount in situ hybridization for mid-regeneration expression genes. These genes include clones 944-HH (N-CAM homologue), eye793 (unknown), 1008-HH (unknown) and 5189-HH (netrin homologue). The expression of these clones within the blastema is first detected at day two or three of regeneration. For clones 944-HH and eye793, the ventral nerve cords (VNC) appear truncated in the stump region at day one (arrows). Dashed lines separate the newly formed blastema from the old stump region. Anterior to the top. Bars, 400 μm.
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Late expression genes Two neural clones with no clear homology to known proteins can be classified as late expression genes as they are not expressed within the new head region until days four to five of regeneration (Fig. 7). In intact animals, these two genes show similar expression patterns in the brain region. They are expressed in the central portion of the brain, but no positive cells are detected in the lateral branches. Some differences are observed, however, along the VNC. Clone eye53 (unknown) is expressed in many cells localized around the VNC throughout the length of the body. Moreover, this gene is also expressed in visual cells and in a few cells in the tip of the head (Fig. 7). In contrast, clone 1020-HH (unknown) is expressed in a few irregularly scattered cells closely associated with VNC (Fig. 7).
Figure 7. . Whole-mount in situ hybridization for late expression genes. These genes include clones 1020-HH (unknown) and eye53 (unknown). Weak signals are first detected in the new brain after 4–5 days of regeneration. Dashed lines separate the newly formed blastema from the old stump region. Anterior to the top. Bars, 400 μm.
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