Department of Molecular and Cell Biology, University of California, 385 LSA, Berkeley, CA 94720-3200, USA
* Present address: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA
Author for correspondence (e-mail: weisblat{at}uclink4.berkeley.edu)
Accepted 5 May 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Leech, even-skipped, Segmentation, Annelid, Antisense oligonucleotides
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analyzing the mechanisms of segmentation in glossiphoniid leeches such as Helobdella robusta may contribute to resolving this paradox by either of two routes. For example, finding that segmentation is homologous between leeches (a highly derived but experimentally tractable taxon of segmented lophotrochozoans) and insects (a highly derived but experimentally tractable taxon of segmented ecdysozoans) would suggest that the urprotostome was already segmented, and would be consistent with the hypothesis that the urbilaterian was already segmented as well. Conversely, concluding that segmentation is not homologous between representatives of the two clades of protostomes would support the hypothesis that segmentation arose multiple times.
The primary pair-rule gene even-skipped (eve) is among the first genes to exhibit regular, spatially iterated patterns in Drosophila (Frasch et al., 1987; Macdonald et al., 1986
). Homologs of eve have also been described in arthropods, such as the beetle Tribolium castaneum and the grasshopper Schistocerca americana, and the spider Cupiennius salei, that form segments sequentially. Tribolium-eve and Cupiennius-eve are expressed in striped patterns that correlate with segmentation (Brown et al., 1997
; Damen et al., 2000
; Patel, 1994
), while Schistocerca-eve is expressed in the posterior growth zone without forming stripes (Patel et al., 1992
). Neuronal expression of eve-class genes is conserved throughout the arthropods (Damen et al., 2000
; Duman-Scheel and Patel, 1999
; Frasch et al., 1987
; Patel et al., 1992
).
We report the identification of eve-class genes from two species of glossiphoniid leeches and the initial characterization of Hro-eve, the eve homolog in Helobdella robusta. Semi-quantitative RT-PCR revealed that the highest levels of Hro-eve transcription occur during organogenesis. In situ hybridization revealed earlier expression, during cleavage and the early phases of segmentation, but we found no evidence for a pair-rule pattern. In early development, Hro-eve transcripts seem to be associated with mitotic chromatin in the stem cells of the posterior growth zone (teloblasts) and in their progeny (primary blast cells) that are the founder cells for the five distinct lineages of segmental mesoderm and ectoderm. In more advanced embryos, Hro-eve is expressed in segmentally iterated subsets of the neurons that arise from the N teloblasts. To assess the function of Hro-eve, we examined embryos in which selected blastomeres had been injected with antisense Hro-eve morpholino oligonucleotides (AS-Hro-eve MO). Focussing on the N lineage, we found that teloblasts injected with AS-Hro-eve MO continued to divide at about the normal rate, but other aspects of their division were disturbed, and primary blast cell divisions were also disrupted. Presumably because of these early effects on teloblasts and blast cells, segmentation was also perturbed in the injected lineage, as evidenced by misalignment of left and right hemiganglia, fusion of serially adjacent hemiganglia and/or missing groups of cells. Ganglionic neurons expressing Hro-eve arose, though in an abnormal pattern; serotonergic neurons failed to form. In the O lineage, injection of AS-Hro-eve MO disrupted both neural and epidermal cell fates. Our results suggest that Hro-eve is important in regulating cell divisions in stem cells and segmental founder cells in early development and that it also regulates the differentiation of a subset of ganglionic neurons. However, we find no evidence that Hro-eve plays a pair rule function similar to that of eve-class genes in arthropod segmentation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To perturb Hro-eve expression, cells of interest were injected with an antisense morpholino oligonucleotide (MO; Genetools) complementary to the downstream end of the 5' UTR and first five bases of coding sequence of Hro-eve, designated as AS-Hro-eve MO (5'-ATCATTTTACTTTTCGATTCAGCGG-3'; anti-start codon underlined). For control injections, we used a generic MO (5'-CCTCTTACCTCAGTTACAATTTATA-3') or MM-Hro-eve MO (5'-ATCtTTTaACTTTTCGATaCAGgGG-3') that had the same overall nucleotide composition as AS-Hro-eve MO, but with four mismatched nucleotides (Nasevicius and Ekker, 2000).
MO concentrations ranged from 0.06-1.3 mM in the micropipette and the volume of material injected corresponds to roughly 1% of the teloblast (Bissen and Weisblat, 1987). Thus, we estimate that the MO concentrations ranged from 0.6-13 µM in the cytoplasm of the injected cells. MO was co-injected with lineage tracer (
50 mg/ml in the pipette) and Fast Green (
0.5% in the pipette).
Gene isolation
Degenerate oligonucleotides (upstream, 5'-MGIYAYMGIACIGCITT-3'; downstream, 5'-YGIYAYYTTRTCYTTCAT-3') corresponding to nucleotides 7-24 and 108-125 of the eve-class homeobox were designed by comparing the sequences for eve-class genes from zebrafish (Joly et al., 1993), fly (Macdonald et al., 1986
), mouse (Bastian and Gruss, 1990
), human (Faiella et al., 1991
), coral (Miles and Miller, 1992
), grasshopper (Patel et al., 1992
), nematode (Ahringer, 1996
) and frog (Ruiz i Altaba and Melton, 1989
). Candidate gene fragments were amplified from a cDNA library (Stratagene) of stage 7-10 embryos by degenerate PCR.
To obtain additional sequence of Hro-eve, we performed PCR on the cDNA library and on first strand cDNAs. For the first strand cDNA, 500 embryos from stages 6-10 were homogenized in RNAwiz (Ambion) and total RNA was extracted. Polyadenylated mRNAs were isolated using Oligotex mRNA mini kit (Qiagen). For rapid amplification of the cDNA ends, the Marathon cDNA amplification kit (Clontech) was used, with exact gene specific oligos designed from the gene fragments described above. Sequence analyses were performed by the MacDNASIS pro v3.5 program and the BLAST program (http://www.ncbi.nlm.nih.gov).
Semi-quantitative developmental RT-PCR
At each stage, total RNAs were extracted from 50 embryos and treated with DNase (DNA-free, Ambion), and then reverse-transcribed using random decamers (Ambion). As an internal standard to adjust for differences in efficiency of RNA extraction between samples, a 488 bp fragment of 18S rRNA was amplified in parallel to each sample. To quantitate PCR products, each sample was electrophoresed in 2% agarose gel and stained with ethidium bromide. Band intensity was measured with an Alphaimager (Alpha Innotech) using Alphaease (v3.3b) program.
To confirm the relative levels of expression, we amplified two separate regions of Hro-eve cDNA: nucleotides 784-1243 (which spans an intron site, to control for the possibility of genomic DNA contamination of the template) and nucleotides 1724-1939. Both produced equivalent results, and the identity of the amplified fragments was confirmed by sequencing one sample of each fragment.
In situ hybridization
Digoxigenin (Dig-11-UTP, Roche)-labeled riboprobes were made in vitro using MEGAscript kit (Ambion) and hydrolyzed. T7 RNA polymerase (Ambion, cat No. 1334) was used for all probes. Sequence for Hro-eve riboprobes includes 3' coding region and most of the 3'-UTR (nucleotides 1211-3140).
To localize Hro-eve mRNA in situ, two different protocols were used, depending on the age of the embryo. Embryos at stages 9-11 were processed as described previously (Harland, 1991; Nardelli-Haefliger and Shankland, 1992
). For early stages (through stage 8), embryos were fixed in 4% formaldehyde, 1xphosphate-buffered saline (PBS, diluted from 10x PBS stock, pH 7.4) for 1 hour at room temperature. After fixation, all incubations were performed at room temperature with constant rocking. Fixed embryos were rinsed with 0.1% PBTw (PBS, 0.1% Tween 20), devitellinized and rinsed in serial changes of 0.1% PBTw, 0.1% PBTw:HYB (1:1) and hybridization solution [HYB; 5xSSC, tRNA 0.5 mg/ml, heparin 50 µg/ml, 0.1% Tween 20, 50% formamide, pH 6.0 (adjusted with 1 M citric acid)], then pre-hybridized in HYB for 2 hours at 68°C. HYB was replaced with fresh HYB containing riboprobes and hybridized at 68°C for 28-40 hours. Embryos were washed with pre-warmed HYB:2xSSC/0.3% chaps (1:1), and then 2xSSC/0.3% Chaps, followed by successive 20 minute washes with 2x, 0.2x and 0.1x SSC/0.3% Chaps at 68°C. Embryos were incubated in blocking solution [0.1% PBTw, 2% sheep serum (Sigma, St Louis, MO), 2 mg/ml bovine serum albumin] for 2 hours at room temperature and further incubated in blocking solution containing anti-digoxigenin alkaline phosphatase-conjugated polyclonal Fab fragments (1:5000, Roche) at 4°C overnight. Embryos were rinsed with frequent changes of 0.1% PBTw for 5 hours at room temperature, followed by a brief rinse with alkaline phosphatase buffer (AP buffer; 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2, 0.1% Tween 20). Embryos were then incubated in AP buffer containing 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indoyl-phosphate (Roche) for color reaction. When the embryos reached the desired color, they were rinsed with double distilled H2O and dehydrated in a graded ethanol series, then cleared in benzyl benzoate:benzyl alcohol (3:2) for observation.
Anti-serotonin staining
Embryos at stage 11 were fixed in 4% formaldehyde in 0.75xPBS overnight at 4°C and washed in 1xHepes-buffered saline (HBS, diluted from 2xstock), then incubated for 24 hours in HBS containing 0.5 units/ml chitinase (Sigma C-1525, St Louis, MO) at RT with gentle shaking. Embryos were rinsed in HBS and incubated in a solution of PBS, 1% Triton X-100 and 10% NGS (PTN) overnight at 4°C, then further incubated in PTN containing rabbit anti-serotonin (1:300, Sigma S-5545, St. Louis, MO) overnight at 4°C. Embryos were rinsed in PTN and incubated in PTN containing peroxidase-conjugated goat anti-rabbit (1:300, Jackson ImmunoResearch Labs 111-036-003, West Grove, PA) overnight at 4°C. Embryos were rinsed in PTN, then in PBS and incubated in 0.75xPBS containing 0.5 mg/ml 3, 3'-diaminobenzidine (Sigma) and 0.008% NiCl2 for 15 minutes at room temperature. The color reaction was initiated by adding 0.06% H2O2 and monitored visually. For observation, germinal plates were dissected and mounted in 75% glycerol.
Histology and microscopy
For more detailed analyses, selected embryos were sectioned prior to microscopic examination. For this purpose, embryos labeled with lineage tracer only were dehydrated and embedded in glycol methacrylate resin (JB-4, Polysciences) according to the manufacturers instructions. This resin dissolves the colored in situ hybridization reaction product. Thus, in situ-stained embryos were embedded in an epoxide resin (Polybed 812, Polysciences). Embedded embryos were sectioned at 10 µm thickness (MT2-B Ultramicrotome, Sorvall) and mounted on glass slides using a non-fluorescent media (Fluoromount, BDH Laboratory Supplies, England). Intact, dissected or sectioned embryos were examined and photographed with DIC and/or epifluorescence optics (Zeiss Axiophot and 35 mm film camera or Nikon E800 and Princeton Instruments cooled CCD camera controlled by UIC Metamorph software) or by confocal microscopy (BioRad MRC 1024). Images were processed, montaged and composed digitally (Metamorph, UIC; Photoshop 5.0, Adobe).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
For Hro-eve, 3.1 kb of cDNA was obtained, encoding an ORF of at least 761 amino acids, taking the 5'-most in frame AUG as the start codon. There was no in-frame stop codon in the 171 bp upstream of the candidate start codon; three possible polyadenylation sites occurred in the 676 bp of 3'-UTR. In addition to the homeodomain, the Hro-eve polypeptide contains a polyalanine stretch and another region rich in serine and proline residues, both of which are common features in other eve-class genes (Ahringer, 1996
; Bastian and Gruss, 1990
; Brown et al., 1997
; Faiella et al., 1991
; Macdonald et al., 1986
; Patel et al., 1992
; Ruiz i Altaba and Melton, 1989
). Comparison with genomic DNA revealed one 257 bp intron at the same site within the homeodomain as in other eve-class genes and another, 100 bp intron following amino acid residue 51 of the putative polypeptide.
To compare Ttr-eve and Hro-eve to other eve-class genes, cladograms were constructed [PAUP 4.0 b4a (PPC)] using only homeodomains. Both Ttr-eve and Hro-eve clearly fell within a strongly supported clade of eve-class genes (98% bootstrap value), but there was no well-supported resolution within this clade (data not shown but available upon request). Ttr-eve and Hro-eve showed less amino acid identity than expected for orthologous genes from closely related species (Fig. 2) and were highly diverged outside the homeodomain. These results could reflect either an ancient duplication of the protostome eve-class gene or the rapid divergence of the Ttr-eve; we cannot distinguish between these possibilities. In any event, for the following analyses of gene expression and function, we focussed exclusively on Hro-eve.
Hro-eve is not expressed in a pair rule pattern during segmentation
In Helobdella, each segment arises from the interdigitating clones of seven distinct classes of bilaterally paired segmental founder cells (m, nf, ns, o, p, qf and qs blast cells) (reviewed by Weisblat and Huang, 2001) (Fig. 1). Blast cells arise in columns (bandlets) by unequal divisions from five bilateral pairs of large identified stem cells: the M, N, O, P and Q teloblasts. Teloblasts divide with a cell cycle time of about 1 hour. Each lineage contributes a stereotyped set of neurons and other cell types to each segment. In the N (and Q) lineages, two classes of blast cells, nf and ns (qf and qs) arise in exact alternation; one of each is required to generate one segments worth of progeny (Fig. 1). In the M, O and P lineages, each blast cell makes one segments worth of progeny. In each teloblast lineage, the first-born blast cells contribute to anterior segments and blast cells born later contribute to progressively more posterior segments.
Thus, in leech, in contrast to vertebrates and insects, there is a strict correlation between the cell cycle clock, by which blast cells arise from the teloblasts, and the segmentation clock, by which segmental tissues arise in anteroposterior progression. From this description, what might one expect to see if Hro-eve was expressed in a pair-rule pattern? One possibility was that Hro-eve would be expressed in alternate blast cells, or blast cell clones, in the M, O and P lineages, and in alternate pairs of blasts cells in the N and Q lineages. Another possibility was that it would be expressed in alternate cells within the N and Q lineages, for example, in all nf and qf cells.
As a first step in characterizing the expression of Hro-eve, we used semi-quantitative RT-PCR to estimate the relative levels of expression of Hro-eve during development (Fig. 3). By this technique, Hro-eve transcripts were present in embryos at stage 7, which roughly corresponds to the onset of blast cell production. But in contrast to predictions based on pair-rule expression patterns, in situ hybridization of embryos at stages 7-8 revealed Hro-eve mRNA in all primary blast cells and in the micromere cap (Fig. 4A). The level of staining was uniform among different lineages, and among individual blast cells within each bandlet (Fig. 4A,B). Thus, we found no evidence for either lineage-specific or pair rule expression of Hro-eve.
|
|
|
Late expression of Hro-eve was in subsets of developing neurons
RT-PCR experiments indicated that Hro-eve expression increased sharply beginning in stage 9, by which time the germinal plate is complete and the differentiation of ventral ganglia and other segmental tissues is under way. In situ hybridization using embryos at stages 9-11 revealed Hro-eve expression in segmentally iterated sets of neurons in the ventral nerve cord (Fig. 6). The Hro-eve transcripts in these cells were cytoplasmic rather than nuclear (Fig. 6).
|
Neuronal expression of Hro-eve was first observed when n blast cell clones were 70 hours old. This corresponds to early stage 9 in the anterior germinal plate. Expression was seen in two spots, one anterior and another more posterior, in each hemiganglion (Fig. 6B). The anterior spot consisted of two or three adjacent cells in the ventrolateral portion of the ganglion; the more posterior spot lies about halfway back in the ganglion and consists of two or three cells in the dorsal portion of the ganglion, roughly midway between the ventral midline and the lateral edge of the ganglion. By clonal age
90-100 hours, the anterior spot of Hro-eve expression started to disappear, so that some ganglia had only one anterior spot (Fig. 6C). By clonal age
130 hours, the anterior spots had disappeared, but the posterior spot of Hro-eve expression remained in each ganglion through clonal age
130-150 hours. Therefore, the posterior spot was evident throughout the germinal plate in individual embryos at late stage 10 (Fig. 6D).
In glossiphoniid leeches, ganglionic neurons arise from each of the five teloblast lineages, with half or more arising from the N lineage (Kramer and Weisblat, 1985; Weisblat et al., 1984
). To identify the lineage(s) of origin of the neurons expressing Hro-eve, we carried out in situ hybridization on embryos in which one or more cells (N, OP or OPQ) had been injected with lineage tracer. The confocal images of sectioned embryos revealed that the in situ label colocalized with lineage tracer when the N lineage was labeled and not when any other lineage was labeled (Fig. 7). Thus, we conclude that the cells expressing Hro-eve arose from the N teloblasts. Moreover, by comparison with more detailed lineage analyses (Shain et al., 1998
), we concluded that anterior ventrolateral cells exhibiting transient expression of Hro-eve arose from the ns.a clone, and that the posterior neurons arose from the nf.a clone in each segment.
|
We estimate that the MO concentrations ranged from 0.6-13 µM in the cytoplasm of the injected teloblasts. When AS-Hro-eve MO was injected into N teloblasts at lower concentrations, the overall pattern of development was normal until stage 10 (140 hours after the injection). But fluorescence microscopy revealed marked abnormalities in the distribution of progeny from the experimental N teloblasts. The usual segmental pattern of RDA-labeled ganglionic neurons was disrupted and was often out of register with the control side (Fig. 8A-C). At higher concentrations, development was more severely perturbed; these embryos suffered higher than normal mortality during gastrulation (37/120 embryos died in six experiments), which was due to a failure of germinal plate formation and consequent rupture and leakage of the yolk cell. Many of the surviving embryos exhibited dorsolaterally directed hairpin loops in the posterior portion of the germinal plate (Fig. 9) that was not seen with control injections. Further analyses of this phenomenon are described later.
|
|
Further examination of lineage tracer in cells derived from the experimental N teloblasts revealed that they had failed to generate appreciable numbers of neurites. In normal development, three prominent segmental nerves exiting each side of the ganglion (Ort et al., 1974; Stent et al., 1992
), contain neurites derived from contralateral N-derived neurons (Shain et al., 1998
); these nerves were not detected contralateral to AS-Hro-eve MO-injected N teloblasts, in contrast to the case with control injections (data not shown). Observations of ganglionic Hro-eve expression were made at stage 10, whereas scoring for serotonergic neurons and segmental nerve formation was carried out at stage 11. Together, these results suggest that AS-Hro-eve MO injections resulted in a widespread failure of neural development at some point prior to terminal differentiation. Consistent with the fact that Hro-eve is expressed in all five teloblast lineages in early development, the defects induced by AS-Hro-eve MO injections were not restricted to neuronal tissues. For example, the O lineage normally makes a mixture of neurons and epidermal cells (Fig. 8G). Here, AS-Hro-eve MO injections resulted in an almost total loss of epidermal cells; neural precursors could be recognized by their ganglionic locations, but as in the N lineage, failed to complete differentiation (Fig. 8H).
Injection of antisense Hro-eve oligonucleotide disrupted the normal pattern of teloblast and blast cell divisions
To investigate the origins of the hairpin loops that occurred in the experimental side of germinal plates, we further examined embryos in which N teloblasts were injected with AS-Hro-eve MO or MM-Hro-eve MO as above, but fixed earlier, at mid stage 8 (42-48 hours after the injection). At this stage, control germinal bands reached smoothly around the equator of the embryo, as in uninjected embryos (Fig. 10). By contrast, germinal bands on the experimental side often exhibited a prominent dorsally directed kink in the posterior part of the germinal band (Fig. 10).
|
This normal pattern was observed in bandlets derived from N teloblasts injected with MM-Hro-eve MO or with lineage tracer alone. Each control bandlet contained between 26 and 28 clones and, with two exceptions, the blast cells were labeled uniformly throughout each bandlet (Fig. 11). Of the two exceptional bandlets, one contained a single blast cell with noticeably fainter lineage tracer than the others, while in the second bandlet 18 out of 28 cells were noticeably fainter. In all but one of the control bandlets, the oldest clone had undergone its unequal first mitosis (Fig. 12). In two of these bandlets, another blast cell had divided, giving an average of 1.1 mitoses per bandlet.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Early expression of Hro-eve
In situ hybridization reveals that Hro-eve is expressed during cleavage and in all teloblast lineages up through the first division of the primary blast cells. No pair-rule or striped pattern is observed for Hro-eve during segmentation.
In teloblasts and primary blast cells, the in situ signal for Hro-eve is strongest over the chromatin of cells in mitosis. One interpretation of these results is that pre-existing Hro-eve transcripts localize to chromatin upon nuclear envelope breakdown during mitosis. An alternative explanation is that Hro-eve is transcribed during mitosis; in this case, the transcripts might remain associated with chromatin because the biochemical machinery required for splicing and polyadenylation is not operative during mitosis (Alberts et al., 1994). The generalization is sometimes made that transcription is shut down during mitosis, but this is not an absolute condition; in yeast,
300 genes have been identified as being transcribed during mitosis (Krebs et al., 2000
; Spellman et al., 1998
).
Neuronal expression of Hro-eve
Semi-quantitative RT-PCR reveals that Hro-eve transcripts are most abundant during the period in which ganglia are differentiating, and in situ hybridization reveals two sets of bilaterally paired, segmentally iterated neurons that express Hro-eve during neurogenesis. An anterior set of neurons, which are derived from the ns (probably from the ns.a) clone in each hemiganglion, expresses Hro-eve transiently, while a more posterior group of neurons, derived from the nf (probably from the nf.a) clone, continue to express Hro-eve in the oldest embryos studied (early stage 11).
Many genes that were first identified as regulating segmentation in Drosophila are also expressed later during neural development, including even-skipped (Doe et al., 1988; Frasch et al., 1987
; Patel et al., 1989
). The expression patterns of eve-class genes suggest similar dual functions in other arthropods (Damen et al., 2000
; Patel et al., 1992
). In nematodes (Ahringer, 1996
), a taxon of unsegmented ecdysozoans, and in segmented deuterostomes, including amphibians (Ruiz i Altaba, 1990
), fish (Thaeron et al., 2000
) and mammals (Dush et al., 1992
; Moran-Rivard et al., 2001
), eve homologs are involved in neurogenesis. These results, together with our present findings, are consistent with the idea that eve-class genes functioned in neurogenesis in the last common ancestor of the deuterostomes, ecdysozoans and lophotrochozoans. However, we cannot yet rule out an alternative explanation for these observations, based on convergent evolution. In this scenario, the evolution of the many neuronal phenotypes present in higher metazoans entailed the independent recruitment to neuronal functions of transcription factors that were used for other purposes in ancestral animals. Further information on this issue may be gained by studying the function of eve-class genes from cnidarians (Finnerty and Martindale, 1997
; Miles and Miller, 1992
).
Developmental role of Hro-eve expression
The expression of Hro-eve in the early embryos, including the teloblasts and primary blast cells (segmental founder cells) of all five lineages (M, N, O, P and Q) suggests the possibility that this gene is somehow linked to the general regulation of cell proliferation, but not the specification of teloblast lineages or segment identity.
Evidence for the function of Hro-eve in the early Helobdella embryo was obtained by knockdown experiments, in which antisense oligonucleotides were injected into the N teloblasts, along with fluorescent lineage tracers to monitor the development of the injected cells. Lacking an antibody for Hro-eve, we are unable to measure the extent of the resulting knockdown, but do observe disruptions of normal development, beginning with teloblast and primary blast cell divisions. AS-Hro-eve MO injections do not affect the average rate or general asymmetry of the teloblast divisions, but the blast cells produced by the experimental teloblasts show variations in lineage tracer content that are not seen in control MO injections. Moreover, primary blast cells produced by the N teloblasts injected with AS-Hro-eve MO sometimes divided several hours earlier than normal, and germinal bands containing bandlets derived from the experimental N teloblasts frequently exhibited a dorsally directed kink. We speculate that the kinks may result from mechanical deformations resulting from excess or ectopic N-derived cells that might be caused by the accelerated blast cell divisions, or from changes in biochemical properties that render these cells unable to undergo normal gastrulation movements.
The definitive fates of cells arising within the blast cell clones are also perturbed by AS-Hro-eve MO injections: there appear to be fewer than the normal number of cells; the usual groupings of cells within the hemiganglia are no longer evident; the three N-derived peripheral neurons in each hemisegment cannot be found. Neurons expressing Hro-eve are produced, but in ectopic positions compared with their contralateral homologs; by contrast, the serotonergic neurons that arise from the N teloblasts fail to differentiate.
Thus, we conclude that interfering with the expression of Hro-eve perturbs cell divisions of teloblasts and the subsequent divisions within the blast cell clones, consistent with the expression of Hro-eve in these cells during early development. The extent to which the effects on teloblasts and blast cells represent separate roles for Hro-eve remains to be determined.
Segmentation in annelids, arthropods and vertebrates
Until recently, the notion that segmentation is homologous between annelids and arthropods was generally accepted, as were phylogenetic trees in which variously defined clades of annelids and arthropods were placed as sister taxa. In addition to the segmentation per se, similarities between the posterior teloblastic growth zones in annelids and crustacean arthropods (Dohle and Scholtz, 1988) were taken as evidence supporting these assumptions. Logically, however, using phylogenetic trees based on developmentally derived traits as the reference framework for interpreting developmental comparisons introduces an inherent circularity to the process.
Molecular phylogenies, which offer a partial escape from this circularity, suggest that protostomes comprise distinct ecdysozoan and lophotrochozoan clades (Aguinaldo et al., 1997; Ruiz-Trillo et al., 1999
) and that these have been separated since long before the Cambrian radiation (Adoutte et al., 2000
). If so, given the paucity of segmented taxa among Ecdysozoa and Lophotrochozoa, it is more parsimonious to assume that segmentation has arisen independently in annelids and arthropods.
Consistent with this interpretation, work to date has revealed little similarity between the embryological functions of Drosophila segmentation genes and those of their homologs in Helobdella. For example, although it was initially concluded that Helobdella engrailed-class genes play an essential role in early segmentation (Ramirez et al., 1995; Wedeen and Weisblat, 1991
), subsequent experimental analyses indicate that this is not the case (Seaver and Shankland, 1999
; Seaver and Shankland, 2000
; Shain et al., 1998
; Shain et al., 2000
). Also in contrast to Drosophila, the translation of maternally derived mRNA for the Helobdella nanos-class gene occurs after the AP axis is already established and correlates with the mesoderm-ectoderm fate decision (Pilon and Weisblat, 1997
). In the work reported here, we find that Hro-eve is not expressed in a pair-rule pattern as in Drosophila. Moreover, we find no evidence of a link between the early expression of eve- and engrailed-class genes in leech. The early expression of Hro-eve shows no spatial or temporal correlation with that of Hro-engrailed.
eve-class genes are expressed in the posterior segmentation zone of annelids (Lophotrochozoa), arthropods (Ecdysozoa) and vertebrates (Deuterostomia) (Brown et al., 1997; Damen et al., 2000
; Joly et al., 1993
; Patel, 1994
; Patel et al., 1992
). One explanation for this similarity is that it reflects evolutionary convergence, i.e. independent recruitment of this transcription factor into the segmentation mechanisms in the three taxa. Alternatively, similarities in gene expression patterns between arthropods and vertebrates have led some to propose that the last common ancestor of all bilaterally symmetric animals was already segmented (De Robertis, 1997
; Holland et al., 1997
; Palmeirim et al., 1997
). This latter explanation seems unlikely, on the basis of parsimony and, as reported here, because of the differences in the expression of eve-class genes between arthropods and leech.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adoutte, A., Balavoine, G., Lartillot, N., Lespinet, O., Prudhomme, B. and de Rosa, R. (2000). The new animal phylogeny: reliability and implications. Proc. Natl. Acad. Sci. USA 97, 4453-4456.
Aguinaldo, A. M., Turbeville, J. M., Linford, L. S., Rivera, M. C., Garey, J. R., Raff, R. A. and Lake, J. A. (1997). Evidence for a clade of nematodes, arthropods and other moulting animals. Nature 387, 489-493.[Medline]
Ahringer, J. (1996). Posterior patterning by the Caenorhabditis elegans even-skipped homolog vab-7. Genes Dev. 10, 1120-1130.[Abstract]
Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. and Watson, J. D. (1994). In Molecular Biology of the Cell. New York; Garland.
Bastian, H. and Gruss, P. (1990). A murine even-skipped homologue, Evx 1, is expressed during early embryogenesis and neurogenesis in a biphasic manner. EMBO J. 9, 1839-1852.[Abstract]
Bissen, S. T. and Weisblat, D. (1987). Early differences between alternate n blast cells in leech embryo. J. Neurobiol. 18, 251-270.[Medline]
Bissen, S. T. and Weisblat, D. A. (1989). The durations and compositions of cell cycles in embryos of the leech, Helobdella triserialis. Development 106, 105-118.[Abstract]
Blair, S. S. and Weisblat, D. A. (1984). Cell interactions in the developing epidermis of the leech Helobdella triserialis. Dev. Biol. 101, 318-325.[Medline]
Brown, S. J., Parrish, J. K., Beeman, R. W., Denell, R. E., Ruiz i Altaba, A. and Melton, D. A. (1997). Molecular characterization and embryonic expression of the even-skipped ortholog of Tribolium castaneum. Mech. Dev. 61, 165-173.[Medline]
Brusca, R. C. and Brusca, G. J. (1990). Invertebrates. Sunderland, MA; Sinauer.
Chen, Y. and Bissen, S. T. (1997). Regulation of cyclin A mRNA in leech embryonic stem cells. Dev. Genes Evol. 206, 407-415.
Damen, W., Weller, M. and Tautz, D. (2000). Expression patterns of hairy, even-skipped, and runt in the spider Cupiennius salei imply that these genes were segmentation genes in a basal arthropod. Proc. Natl. Acad. Sci. USA 97, 4515-4519.
Davis, G. K. and Patel, N. H. (1999). The origin and evolution of segmentation. Trends Cell Biol. 9, M68-M72.[Medline]
De Robertis, E. M. (1997). Evolutionary biology. The ancestry of segmentation. Nature 387, 25-26.[Medline]
Doe, C. Q., Hiromi, Y., Gehring, W. J. and Goodman, C. S. (1988). Expression and function of the segmentation gene fushi tarazu during Drosophila neurogenesis. Science 239, 170-175.[Medline]
Dohle, W. and Scholtz, G. (1988). Clonal analysis of the crustacean segment: the discordance between genealogical and segmental borders. Development 104 Suppl., 147-160.[Abstract]
Duman-Scheel, M. and Patel, N. H. (1999). Analysis of molecular marker expression reveals neuronal homology in distantly related arthropods. Development 126, 2327-2334.
Dush, M. K., Martin, G. R., Gutjahr, T., Patel, N. H., Li, X., Goodman, C. S. and Noll, M. (1992). Analysis of mouse Evx genes: Evx-1 displays graded expression in the primitive streak. Dev. Biol. 151, 273-287.[Medline]
Faiella, A., DEsposito, M., Rambaldi, M., Acampora, D., Balsofiore, S., Stornaiuolo, A., Mallamaci, A., Migliaccio, E., Gulisano, M., Simeone, A. et al. (1991). Isolation and mapping of evx1, a human homeobox gene homologous to even-skipped, localized at the 5' end of HOX1 locus on chromosome 7. Nucleic Acids Res. 19, 6541-6545.[Abstract]
Finnerty, J. R. and Martindale, M. Q. (1997). Homeoboxes in sea anemones (Cnidaria: Anthozoa): A PCR-based survey of Nematostella vectensis and Metridium senile. Biol. Bull. 193, 62-76.
Frasch, M., Hoey, T., Rushlow, C., Doyle, H., Levine, M., Lall, S. and Patel, N. H. (1987). Characterization and localization of the even-skipped protein of Drosophila. EMBO J. 6, 749-759.[Abstract]
Harland, R. M. (1991). In situ hybridization: An improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36, 685-695.[Medline]
Holland, L. Z., Kene, M., Williams, N. A. and Holland, N. D. (1997). Sequence and embryonic expression of the amphioxus engrailed gene (AmphiEn): the metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila. Development 124, 1723-1732.
Joly, J. S., Joly, C., Schulte-Merker, S., Boulekbache, H. and Condamine, H. (1993). The ventral and posterior expression of the zebrafish homeobox gene eve1 is perturbed in dorsalized and mutant embryos. Development 119, 1261-1275.
Kang, D., Pilon, M. and Weisblat, D. A. (2002). Maternal and zygotic expression of a nanos-class gene in the leech Helobdella robusta: primordial germ cells arise from segmental mesoderm. Dev. Biol. 245, 28-41.[Medline]
Kimmel, C. B. (1996). Was Urbilateria segmented? Trends Genet. 12, 329-331.[Medline]
Kramer, A. P. and Weisblat, D. A. (1985). Developmental neural kinship groups in the leech. J. Neurosci. 5, 388-407.[Abstract]
Krebs, J. E., Fry, C. J., Samuels, M. L. and Peterson, C. L. (2000). Global role for chromatin remodeling enzymes in mitotic gene expression. Cell 102, 587-598.[Medline]
Macdonald, P. M., Ingham, P. and Struhl, G. (1986). Isolation, structure, and expression of even-skipped: a second pair-rule gene of Drosophila containing a homeo box. Cell 47, 721-734.[Medline]
Miles, A. and Miller, D. J. (1992). Genomes of diploblastic organisms contain homeoboxes: sequence of eveC, an even-skipped homologue from the cnidarian Acropora formosa. Proc. R. Soc. Lond. B. Biol. Sci. 248, 159-161.[Medline]
Moran-Rivard, L., Kagawa, T., Saueressig, H., Gross, M. K., Burrill, J. and Goulding, M. (2001). Evx1 is a postmitotic determinant of v0 interneuron identity in the spinal cord. Neuron 29, 385-399.[Medline]
Nardelli-Haefliger, D. and Shankland, M. (1992). Lox2 a putative leech segment identity gene is expressed in the same segmental domain in different stem cell lineages. Development 116, 697-710.
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene knockdown in zebrafish. Nat. Genet. 26, 216-220.[Medline]
Ort, C. A., Kristan, W. B. and Stent, G. S. (1974). Neuronal control of swimming in the medicinal leech. J. Comp. Physiol. 94, 121-154.
Palmeirim, I., Henrique, D., Ish-Horowicz, D. and Pourquie, O. (1997). Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91, 639-648.[Medline]
Patel, N. H. (1994). Developmental evolution: insights from studies of insect segmentation. Science 266, 581-590.[Medline]
Patel, N. H., Ball, E. E. and Goodman, C. S. (1992). Changing role of even-skipped during the evolution of insect pattern formation. Nature 357, 339-342.[Medline]
Patel, N. H., Schafer, B., Goodman, C. S. and Holmgren, R. (1989). The role of segment polarity genes during Drosophila neurogenesis. Genes Dev. 3, 890-904.[Abstract]
Pilon, M. and Weisblat, D. A. (1997). A nanos homolog in leech. Development 124, 1771-1780.
Ramirez, F.-A., Wedeen, C. J., Stuart, D. K., Lans, D. and Weisblat, D. A. (1995). Identification of a neurogenic sublineage required for CNS segmentation in an Annelid. Development 121, 2091-2097.
Ruiz i Altaba, A. (1990). Neural expression of the Xenopus homeobox gene Xhox3: evidence for a patterning neural signal that spreads through the ectoderm. Development 108, 595-604.[Abstract]
Ruiz i Altaba, A. and Melton, D. A. (1989). Bimodal and graded expression of the Xenopus homeobox gene Xhox3 during embryonic development. Development 106, 173-183.[Abstract]
Ruiz-Trillo, I., Riutort, M., Littlewood, D. T., Herniou, E. A. and Baguna, J. (1999). Acoel flatworms: earliest extant bilaterian Metazoans, not members of Platyhelminthes. Science 283, 1919-1923.
Seaver, E. C. and Shankland, M. (1999). Autonomous development during segment formation in leech: Evidence contradicting a role for engrailed as initiator of a signalling pathway in the segment primordium. Dev. Biol. 210, 207.
Seaver, E. C. and Shankland, M. (2000). Leech segmental repeats develop normally in the absence of signals from either anterior or posterior segments. Dev. Biol. 224, 339-353.[Medline]
Shain, D. H., Ramirez-Weber, F.-A., Hsu, J. and Weisblat, D. A. (1998). Gangliogenesis in leech: Morphogenetic processes leading to segmentation in the central nervous system. Dev. Genes Evol. 208, 28-36.[Medline]
Shain, D. H., Stuart, D. K., Huang, F. Z. and Weisblat, D. A. (2000). Segmentation of the central nervous system in leech. Development. 127, 735-744.
Smith, C. M. and Weisblat, D. A. (1994). Micromere fate maps in leech embryos: Lineage-specific differences in rates of cell proliferation. Development 120, 3427-3438.
Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., Brown, P. O., Botstein, D. and Futcher, B. (1998). Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol. Biol. Cell 9, 3273-3297.
Stent, G. S., Kristan, W. B., Jr, Torrence, S. A., French, K. A. and Weisblat, D. A. (1992). Development of the Leech Nervous System. Int. Rev. Neurobiol. 33, 109-193.[Medline]
Stuart, D. K., Blair, S. S. and Weisblat, D. A. (1987). Cell lineage, cell death, and the developmental origin of identified serotonin- and dopamine-containing neurons in the leech. J. Neurosci. 7, 1107-1122.[Abstract]
Thaeron, C., Avaron, F., Casane, D., Borday, V., Thisse, B., Thisse, C., Boulekbache, H. and Laurenti, P. (2000). Zebrafish evx1 is dynamically expressed during embryogenesis in subsets of interneurones, posterior gut and urogenital system. Mech. Dev. 99, 167-172.[Medline]
Wedeen, C. J. and Weisblat, D. A. (1991). Segmental expression of an engrailed-class gene during early development and neurogenesis in an annelid. Development 113, 805-814.[Abstract]
Weisblat, D. and Huang, F. (2001). An overview of glossiphoniid leech development. Can. J. Zool. 79, 218-232.
Weisblat, D. A., Kim, S. Y. and Stent, G. S. (1984). Embryonic origins of cells in the leech Helobdella triserialis. Dev. Biol. 104, 65-85.[Medline]
Zackson, S. L. (1984). Cell lineage, cell-cell interaction, and segment formation in the ectoderm of a glossiphoniid leech embryo. Dev. Biol. 104, 143-160.[Medline]