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Keywords:

  • brain;
  • central nervous system (CNS);
  • netrin;
  • planarian;
  • regeneration

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The planarian central nervous system (CNS) can be used as a model for studying neural regeneration in higher organisms. Despite its simple structure, recent studies have shown that the planarian CNS can be divided into several molecular and functional domains defined by the expression of different neural genes. Remarkably, a whole animal, including the molecularly complex CNS, can regenerate from a small piece of the planarian body. In this study, a collection of neural markers has been used to characterize at the molecular level how the planarian CNS is rebuilt. Planarian CNS is composed of an anterior brain and a pair of ventral nerve cords that are distinct and overlapping structures in the head region. During regeneration, 12 neural markers have been classified as early, mid-regeneration and late expression genes depending on when they are upregulated in the regenerative blastema. Interestingly, the results from this study show that the comparison of the expression patterns of different neural genes supports the view that at day one of regeneration, the new brain appears within the blastema, whereas the pre-existing ventral nerve cords remain in the old tissues. Three stages in planarian CNS regeneration are suggested.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The development and evolution of the central nervous system (CNS) are major issues in developmental biology research. In spite of the differences between the CNS of vertebrates and invertebrates, some axon guidance mechanisms and the basic genetic program for brain development have been highly conserved (Finkelstein & Boncinelli 1994; Acampora et al. 1998; Chisholm & Tessier-Lavigne 1999). Freshwater planarians (Platyhelminthes, Tricladida) are a useful system to study CNS formation in a developmental context different from normal embryogenesis: neural regeneration. Planarians have a simple body structure made from 12 to 15 differentiated cell types (Romero & Baguñà 1991). The CNS is also apparently simple from a morphological perspective, with cephalic ganglia in the anterior region and two longitudinal ventral nerve cords (VNC) along the body (for reviews see Rieger et al. 1991; Reuter & Gustafsson 1995). However, the planarian CNS has some ambiguous features. Many neurons have a neurosecretory function, and their integrative capacities seem intermediate between those of cnidarians and higher metazoans (Koopowitz 1986). In addition, some similarities to vertebrates, such as the presence of multipolar neurons in the CNS and bipolar nerve cells in planarian commissures have also been described (Hanström 1926; Sarnat & Netsky 1985; Reuter et al. 1995a). At the molecular level a more complex view emerges. Umesono et al. (1997, 1999) showed that the planarian brain could be divided into four molecular domains based on analyses of the expression of planarian homologues of the otd/Otx family genes. Recently, a more ambitious approach based on DNA microarrays has been used to isolate a large number of planarian neural genes and to define several molecular and functional domains in the brain (M. Nakazawa, unpubl. obs., 2001).

Planarians are best known for their high regenerative capabilities (for a general review, see Brøndsted 1969 and Baguñà 1998). However, how the regeneration of the various cellular systems is accomplished as well as the molecular basis of the re-patterning of the regenerating fragment are still unclear (for a review see Agata & Watanabe 1999). Concerning the regeneration of the CNS, few studies have been done and an old question remains to be answered: is the new brain regenerated from the outgrowth of the truncated VNC, or does the brain appear independently of the pre-existing nerve cords? (for reviews see Hanström 1928 and Brøndsted 1969). To analyze how the planarian complex CNS is regenerated and which genes are involved, we have studied the regeneration of the CNS by a combination of immunostaining with an antibody against a synaptotagmin homologue (Tazaki et al. 1999) and in situ hybridization for 12 neural genes expressed in different molecular domains in the intact brain. Our results show that during regeneration, the new brain primordium appears within the blastema at day one. At this stage, the pre-existing VNC remain truncated within the stump region. Moreover, three main stages in planarian CNS regeneration are proposed based upon morphological observations and the expression of various neural genes.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Planarians and culture conditions

A clonal strain of the planarian Dugesia japonica, originally obtained from the Iruma river (Gifu, Japan) and previously established in our laboratory, was used in this study. In all experiments, planarians 4–6 mm in length that had been starved for 2 weeks were used. For regeneration experiments, animals were cut at a prepharyngeal level and the anterior regeneration was monitored. Planarians were kept constantly at 21°C. (In all figures a representative image corresponding to at least 10 different samples is shown.)

Whole-mount immunostaining with anti-DjSYT antibody

Immunostaining was carried out essentially as described in Sánchez Alvarado and Newmark (1999). Planarians were treated with 2% hydrochloric acid in distilled water for 30 s and then fixed in Carnoy’s solution (ethanol, chloroform and glacial acetic acid in proportions 6:3:1) for 2 h at room temperature (RT). After fixation, they were washed with 75% methanol in phosphate-buffered saline (PBS) for 5 min, and then bleached in 6% H2O2 in methanol for 4–6 h under light. After they were washed with methanol three times for 5 min each, the animals were rehydrated in a decreasing series of methanol in PBS with 0.3% Triton X-100 (PBST) for 5 min at each step and blocked in 0.25% bovine serum albumin (BSA) in PBST for 2 h at RT. The planarians were then incubated with the polyclonal antibody anti-DjSYT (Tazaki et al. 1999) diluted 1:2000 in 0.25% BSA in PBST for 16–24 h at RT, with shaking. The samples were washed with 0.25% BSA in PBST for 6–12 h (several changes of the medium) and anti-DjSYT was detected with Alexa Fluor 488 goat antimouse IgG (Molecular Probes, Eugene, OR, USA) diluted 1:400 in 0.25% BSA in PBST for 16–20 h in the dark. After the samples were washed for several hours with several changes of PBST, fluorescence was detected with an Olympus (Osaka, Japan) BX62 microscope. Images were obtained using UplanApo 4× and 10× objectives and a Hamamatsu digital camera (C4742-95; Hamamatsu, Japan).

Whole-mount in situ hybridization

Planarians were treated with 2% hydrochloric acid in Holtfreter’s solution for 5 min at 4°C and then fixed in Carnoy’s solution for 2 h at 4°C. Hybridization was carried out using about 20 ng/mL digoxygenin (DIG)-labeled riboprobes, as described in Umesono et al. (1997) and Agata et al. (1998). Images were obtained using an AxioCam digital camera (Zeiss, Tokyo, Japan). After in situ hybridization experiments, some animals were further processed for immunostaining with anti-DjSYT antibody. Once the in situ hybridization development was stopped, samples were washed with PBS two times for 15 min each, fixed in 4% paraformaldehyde for 1 h and washed with PBST two times for 30 min each. Next, the samples were blocked and immunostaining was done as described above. After in situ hybridization, some animals were fixed in 1% glutaraldehyde at 4°C for 16 h and sectioned for histological analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

image

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.

image

Figure 2. . Sequence alignment of the partial planarian netrin homologue, Djnet1 (clone 5189-HH), with leech netrin 1 (Gan et al. 1999), C. elegans UNC-6 (Ishii et al. 1992), Drosophila netrins A and B (Harris et al. 1996; Mitchell et al. 1996), chicken netrin 1 (Serafini et al. 1994) and human netrin 1 (Serafini et al. 1996) using ClustalW. The three epidermal growth factor (EGF) repeats (similar to laminin domain V) are indicated. Conserved amino acidic residues are shown in bold. The partial cDNA sequence of Djnet1 has been deposited in DDBJ/EMBL/GenBank databases with the accession number AB064947.

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image

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.

image

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
ClonePutative homologueExpression in adult organismsFirst expression within blastema
  1. CNS, central nervous system; LDL, low-density lipoprotein; N-CAM, neural cell adhesion molecule; PTP, protein tyrosine phosphatase; VLDL, very low-density lipoprotein; VNC, ventral nerve cord.

953-HHVLDL/LDL receptorGeneral expression in CNSDay 1
721-HHUnknownBrain and other head cellsDay 1
1791-HHG-protein α-subunitDistal part of brain branchesDay 1
517-HHPTP receptorGeneral expression in CNSDay 1
2467-HHHuman brain protein 239ABBrain and lower expression in VNC and other mesenchymal cellsDay 1
1242-HHHuman giant larvaBrain and other mesenchymal cellsDay 1
1008-HHUnknownBrain branchesDay 2
944-HHN-CAMGeneral expression in CNSDay 2
Eye793UnknownBrain (except branches) and VNCDay 2
5189-HHNetrinOverlapping region of brain and VNC; ventral muscle fibersDay 3
Eye53UnknownBrain (except branches); visual cells; cells surrounding VNCDay 4–5
1020-HHUnknownBrain (except branches); very few cells along VNCDay 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.

image

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.

image

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).

image

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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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Brain primordium appears within the blastema at day one of regeneration

Some studies on the CNS of freshwater planarians have described the organization of the nervous system in intact organisms (Reuter et al. 1995a,b, 1996; Agata et al. 1998; Tazaki et al. 1999). However, little attention has been paid to how this CNS is formed during regeneration. Here, we have analyzed for the first time the process of planarian brain regeneration by using a collection of neural-specific markers. At the beginning of the last century, Bresslau (1904, 1909) suggested that VNC would appear after brain formation. In contrast, Micoletzky (1907) and Reisinger (1925) postulated that brain was formed after the fusion of the most anterior commissures of VNC (summarized in Hanström 1928). These hypotheses were based on the observation of intact organisms. In Fig. 1, we have shown that in intact animals, brain and VNC, although closely associated, can be considered as distinct structures. Taking together the different expression patterns of the neural markers used in this study, it appears that during regeneration brain primordium appears within the forming blastema from the first day of regeneration. The new brain cells form two bilateral clusters not connected to the pre-existing VNC, which remain truncated in the stump region and no apparent outgrowth from them can be detected at this stage. These early stages of brain formation within the regenerative blastema resemble those during embryogenesis in several platyhelminthes, where brain primordium cells also appear as two bilateral hemispheres in the anterior region, and later, longitudinal tracts grow from this cephalic ganglion towards the posterior regions of the embryo (Le Moigne 1963, 1966; Younossi-Hartenstein et al. 2000). Although some studies have suggested that the planarian brain is regenerated via extension of the nerve cords (Lender & Gripon 1962), the results obtained here are in agreement with the speculation of Brøndsted who wrote in 1969: ‘My personal view is that the new nerve cells arise from neoblasts in the blastema and that they connect with outgrowing nerve fibers from the old nervous system’.

Concerning the nature of the brain primordium cells at early stages of regeneration, and because of the lack of markers for planarian neural stem cells, it is at present difficult to discern whether these cells correspond to neural precursors or differentiated neurons. However, the fact that at this early stage the brain primordium cells express neural markers found in mature neurons suggests that it would contain, at least, differentiating neurons. Remarkably, the brain primordium appears always as two bilateral cell clusters within the blastema for the different neural markers used. Two alternative mechanisms could explain how neoblasts would be committed to brain cells as two bilateral clusters: (i) it is possible that the regenerative blastema itself possesses bilateral cues that could be sent to the neoblasts; or (ii) although the brain primordium does not apparently appear from the outgrowth of the truncated VNC of the stump region it cannot be dismissed that the VNC could have a role in the induction of the bilateral brain primordium. For instance, during the asexual reproduction of the platyhelminth Microstomum lineare, the new brain cells always appear close to the parental nerve cords (Reuter & Palmberg 1989). Finally, at day two of regeneration, the lateral and central domains of the new brain can already be segregated as the expression of clones 1791-HH (G-protein homologue) and 1008-HH (unknown) is detected in more lateral regions when compared to the expression of other markers which are expressed in the central region of the planarian brain (Figs 5,6). Although at day one specific neural markers for lateral and central brain regions are expressed within the brain primordium, we cannot conclude at present that these two domains have already been segregated at this initial stage.

Three main stages in planarian central nervous system regeneration

Combining all the data, three stages in the regeneration of the planarian CNS may be suggested.

Stage 1 This stage is defined by the appearance of the new brain primordium within the regenerative blastema. These two bilateral cell clusters probably originate from the proliferation of neoblasts in the stump region and the subsequent migration of these neoblasts within the blastema (Saló & Baguñà 1984; Baguñàet al. 1989; Newmark & Sánchez-Alvarado 2000). A fundamental question that must be answered is which genes are responsible for the commitment of these neoblasts to becoming new brain cells. The expression of planarian homologues of otd/Otx genes in two clusters within the blastema at 24–36 h of regeneration (Umesono et al. 1997, 1999) is consistent with the role that these genes play in brain specification in other organisms (Finkelstein & Boncinelli 1994; Acampora et al. 2000); however, the actual function of these expression domains has yet to be demonstrated in planarians.

Stage 2 At this stage the brain primordium differentiates into a small brain with the two halves already connected in the most anterior part and with few lateral branches projecting to the new head periphery. The VNC that at stage 1 remain in the stump region would grow now into the blastema and their association with the forming brain would be re-established. An interesting point here is to understand how the new connections between the regenerating brain and VNC are determined. In this sense it might be useful to further analyze the function of planarian netrin homologue. Netrins are diffusible proteins that may act as both attractive and repulsive cues depending on the receptors expressed in the axonal growth cones (reviewed in Chisholm & Tessier-Lavigne 1999 and Kennedy 2000). Because of the expression pattern of netrin homologue in the overlapping region of brain and VNC, it is tempting to speculate that one of the possible roles for planarian netrin could be related to the establishment of the proper association between brain and VNC cells in the head region. More data, however, are necessary to address which mechanisms are responsible for the brain and VNC connections during planarian CNS development.

Stage 3 The original CNS pattern is restored at least at the structural level, with a new spongy brain and VNC, connected through several transverse commissures running below it. Although the basic structure is rebuilt at this stage, this new brain is probably not completely functional, as the original pattern for some late expression genes has not yet been completely recovered (Fig. 7). Clones eye53 and 1020-HH are first detected within the blastema after 4–5 days of regeneration. As they show no homology to known genes, it is at present difficult to speculate about their function in the planarian CNS.

Planarian brain regeneration and brain development

The first studies carried out at the gene level revealed that the planarian CNS is highly complex despite its apparently simple structure (Umesono et al. 1997, 1999; M. Nakazawa, unpubl. obs., 2001). Such studies have identified several molecular and functional domains in the planarian brain distinguished by the distinct expression of many neural-specific genes. Some of these genes show sequence similarity to conserved factors in vertebrates and invertebrates which play important roles in neural development and/or function, such as netrin, PTP receptors (Stoker & Dutta 1998), G-protein (Nicholls et al. 2001) or VLDL receptor (Howell & Herz 2001). The analysis of the expression of several of these genes during regeneration has allowed us to monitor how the planarian CNS is regenerated. Thus, it has been possible to propose three main stages on the basis of both structural features and the upregulation of different neural genes. These stages can be used as a working model for future analyses of planarian CNS regeneration. Recently, RNAi-based gene silencing has been proved to be useful to analyze gene function in planarians (Sánchez Alvarado & Newmark 1999; Pineda et al. 2000). In the future, we plan to use RNAi to analyze the function of the neural genes used here as well as to study the relationships between them and the otd/Otx family genes, in order to better understand the genetic program responsible for CNS maintenance and regeneration in planarians.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Shigeru Kuratani and Yasunori Murakami for helpful discussions, Satoshi Koinuma for his help in sequencing and Chiyoko Kobayashi for her valuable help with the histological sections.

F. C. would like to sincerely thank all members of the laboratory of Dr Agata and Dr Kuratani at Okayama University for their kindness and help with adapting to daily life in Japan.

This work was supported by a Grant-in-Aid for Creative Basic Research to K. A.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Acampora, D., Avantaggiato, V., Tuorto, F. et al. 1998. Murine Otx1 and Drosophila otd genes share conserved genetic functions required in invertebrates and vertebrates brain development. Development 125, 16911702.
  • Acampora, D., Pia Postiglione, P., Avantaggiato, V., Di Bonito, M. & Simeone, A. 2000. The role of Otx and Otp genes in brain development. Int. J. Dev. Biol. 44, 669677.
  • Agata, K., Soejima, Y., Kato, K., Kobayashi, C., Umesono, Y. & Watanabe, K. 1998. Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers. Zool. Sci. 15, 433440.
  • Agata, K. & Watanabe, K. 1999. Molecular and cellular aspects of planarian regeneration. Semin. Cell Dev. Biol. 10, 377383.
  • Baguñà, J. 1998. Planarians. In Cellular and Molecular Basis of Regeneration: from Invertebrates to Humans (Eds P. Ferretti & J. Géradieu), pp. 135–165. John Wiley & Sons, Chichester.
  • Baguñà, J. & Ballester, R. 1978. The nervous system in planarians: peripheral and gastrodermal plexuses, pharynx innervation, and the relationship between central nervous system structure and the acoelomate organization. J. Morph. 155, 237252.
  • Baguñà, J., Saló, E. & Auladell, C. 1989. Regeneration and pattern formation in planarians. III. Evidence that neoblasts are totipotent stem cells and the source of blastema cells. Development 107, 7786.
  • Bresslau, E. 1904. Beiträge zur Entwicklungsgeschichte usw. I. Die Entwicklung der Rhabdocölen und Alloeocölen. Zeitschr. f. wiss. Zool. Bd. 76.
  • Bresslau, E. 1909. Die Entwicklung der Acölen. Verhandl. d. dtsch. Zool. Ges. Bd. 19.
  • Brøndsted, H. V. 1969. Planarian Regeneration. Pergamon Press, Oxford.
  • Chisholm, A. & Tessier-Lavigne, M. 1999. Conservation and divergence of axon guidance mechanisms. Curr. Opin. Neurobiol. 9, 603615.
  • Finkelstein, R. & Boncinelli, E. 1994. From fly head to mammalian forebrain: the story of otd and Otx. Trends Genet. 10, 310315.
  • Gan, W. B., Wong, V. Y., Phillips, A., Ma, C., Gershon, T. R. & Macagno, E. R. 1999. Cellular expression of a leech netrin suggests roles in the formation of longitudinal nerve tracts and in regional innervation of peripheral targets. J. Neurobiol. 40, 103115.
  • Hanström, B. 1926. Über den feineren Bau des Nervensystems der Tricladen Turbellarien auf Grund von Untersuchungen an Bdelloura candida. Acta Zool. 7, 101115.
  • Hanström, B. 1928. Vergleichende Anatomie Des Nervensystems der Wirbellosen Tiere. Verlag Von Julius Springer, Berlin.
  • Harris, R., Sabatelli, L. M. & Seeger, M. A. 1996. Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila Netrin/UNC-6 homologs. Neuron 17, 217228.
  • Howell, B. W. & Herz, J. 2001. The LDL receptor gene family: signalling functions during development. Curr. Op. Neurobiol. 11, 7481.
  • Ishii, N., Wadsworth, W. G., Stern, B. D., Culotti, J. G. & Hedgecock, E. M. 1992. UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. Elegans. Neuron 9, 873881.
  • Kennedy, T. E. 2000. Cellular mechanisms of netrin functions: long-range and short-range actions. Biochem. Cell Biol. 78, 569575.
  • Koopowitz, H. 1986. On the evolution of central nervous systems: implications from polyclad turbellarian neurobiology. Hydrobiologia 132, 7987.
  • Le Moigne, A. 1963. Etude du développement embryonnaire de Polycelis nigra (Turbellarié, Triclade). Bull. Soc. Zool. Fr. 88, 403422.
  • Le Moigne, A. 1966. Etude du développement embryonnaire et recherches sur les cellules de régénération chez l’embryon de la Planaire Polycelis nigra (Turbellarié, Triclade). J. Embryol. Exp. Morph. 15, 3960.
  • Lender, T. H. & Gripon, P. 1962. La régénération des yeux et du cerveaux de Dugesia lugubris en présence de deux troncs nerveaux inégaux. Bull. Soc. Zool. Fr. 87, 387395.
  • Micoletzky, H. 1907. Zur Kenntnis des Nerven- und Exkretionssystems einiger Süßwassertricladen nebst anderen Beiträgen zur Anatomie von Planaria alpina. Zeitschr. f. wiss. Zool. Bd.87.
  • Mitchell, K. J., Doyle, J. L., Serafini, T. et al. 1996. Genetic analysis of netrin genes in Drosophila: netrins guide CNS commissural axons and peripheral motor axons. Neuron 17, 203215.
  • Morita, M. & Best, J. B. 1966. Electron microscopic studies of planaria. III. Some observations on the fine structure of planarian nervous tissue. J. Exp. Zool. 161, 391413.
  • Newmark, P. A. & Sánchez-Alvarado, A. 2000. Bromodeoxyuridine specifically labels the regenerative stem cells of planarians. Dev. Biol. 220, 142153.DOI: 10.1006/dbio.2000.9645
  • Nicholls, J. G., Martin, A. R., Wallace, B. G. & Fuchs, P. A. 2001. From Neuron to Brain, 4th edn. Sinauer Associates, Sunderland.
  • Pineda, D., González, J., Callaerts, P., Ikeo, K., Gehring, W. J. & Saló, E. 2000. Searching for the prototypic eye genetic network: Sine oculis is essential for eye regeneration in planarians. Proc. Natl Acad. Sci. USA 97, 45254529.
  • Reisinger, E. 1925. Untersuchungen am Nervensystem der Bothrioplana semperi. Zeitschr. F. Morph. U. Ökol. Bd. 5, 119149.
  • Reuter, M. & Gustafsson, M. K. S. 1995. The flatworm nervous system: Pattern and phylogeny. In The Nervous System of Invertebrates: an Evolutionary and Comparative Approach (Eds O. Breidbach & W. Kutsch), pp. 25–59. Birkhäuser-Verlag, Basel.
  • Reuter, M., Gustafsson, M. K. S., Mäntylä, K. & Grimmelikhuijzen, C. J. P. 1996. The nervous system of Tricladida. III. Neuroanatomy of Dendrocoelum lacteum and Polycelis tenuis (Plathelminthes, Paludicola): an immunocytochemical study. Zoomorphology 116, 111122.
  • Reuter, M., Gustafsson, M. K. S., Sahlgren, C., Halton, D. W., Maule, A. G. & Shaw, C. 1995b. The nervous system of Tricladida. I. Neuroanatomy of Procerodes littoralis (Maricola, Procerodidae): an immunocytochemical study. Invertebrate Neurosci. 1, 113122.
  • Reuter, M., Gustafsson, M. K. S., Sheiman, I. M. et al. 1995a. The nervous system of Tricladida. II. Neuroanatomy of Dugesia tigrina (Paludicola, Dugesiidae): an immunocytochemical study. Invertebrate Neurosci. 1, 133143.
  • Reuter, M. & Palmberg, I. 1989. Development and differentiation of neuronal subsets in asexually reproducing Microstomum lineare. Immunocytochemistry of 5-HT, RF-amide and SCPB. Histochemistry 91, 123131.
  • Rieger, R. M., Tyler, S., Smith, J. P. S. III & Rieger, G. 1991. Platyhelminthes: Turbellaria. In Microscopic Anatomy of Invertebrates: Platyhelminthes and Nemertinea (Eds F. W. Harrison & B. J. Bogitsh), pp. 7–140. Wiley-Liss, New York.
  • Romero, R. & Baguñà, J. 1991. Quantitative cellular analysis of growth and reproduction in freshwater planarians (Turbellaria; Tricladida). I. A cellular description of the intact organism. Invert. Report Dev.19, 157–165.
  • Saló, E. & Baguñà, J. 1984. Regeneration and pattern formation in planarians. I. The pattern of mitosis in anterior and posterior regeneration in Dugesia (G) tigrina, and a new proposal for blastema formation. J. Embryol. Exp. Morph. 83, 6380.
  • Sánchez Alvarado, A. & Newmark, P. A. 1999. Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc. Natl Acad. Sci. USA 96, 50495054.
  • Sarnat, H. B. & Netsky, M. G. 1985. The brain of the planarian as the ancestor of the human brain. Can. J. Neurol. Sci. 12, 296302.
  • Serafini, T., Colamarino, S. A., Leonardo, E. D. et al. 1996. Netrin 1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell 87, 10011014.
  • Serafini, T., Kennedy, T. E., Galko, M. J., Mirzayan, C., Jessell, T. M. & Tessier-Lavigne, M. 1994. The netrins define a family of axon outgrowth promoting proteins homologous to C.elegans UNC-6. Cell 78, 409424.
  • Stoker, A. & Dutta, R. 1998. Protein tyrosine phosphatases and neural development. Bioessays 20, 463472.
  • Tazaki, A., Gaudieri, S., Ikeo, K., Gojobori, T., Watanabe, K. & Agata, K. 1999. Neural network in planarian revealed by an antibody against planarian synaptotagmin homologue. Biochem. Biophys. Res. Commun. 260, 426432.DOI: 10.1006/bbrc.1999.0933
  • Umesono, Y., Watanabe, K . & Agata, K. 1997. A planarian orthopedia homolog is specifically expressed in the branch region of both the mature and regenerating brain. Dev. Growth Differ. 39, 723727.
  • Umesono, Y., Watanabe, K. & Agata, K. 1999. Distinct structural domains in the planarian brain defined by the expression of evolutionary conserved homeobox genes. Dev. Genes Evol. 209, 3139.
  • Younossi-Hartenstein, A., Ehlers, U. & Hartenstein, V. 2000. Embryonic development of the nervous system of the Rhabdocoel flatworm Mesostoma lingua (Abildgaard, 1789). J. Comp. Neurol. 416, 461474.DOI: 10.1002/(sici)1096-9861(20000124)416:4<461::aid-cne4>3.0.co;2-a