1 Graduate Group in Biophysics, LSA 385, University of California, Berkeley, CA
94720-3200, USA
2 Department of Molecular and Cell Biology, LSA 385, University of California,
Berkeley, CA 94720-3200, USA
* Author for correspondence (e-mail: weisblat{at}berkeley.edu)
Accepted 23 February 2005
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SUMMARY |
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Key words: Leech, mRNA injection, Cell lineage, Asymmetric division, GFP
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Introduction |
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In contrast to known vertebrate and arthropod systems, segmentation in
Helobdella proceeds via stereotyped lineages, beginning with the
first mitosis and continuing, so far as is known, through the formation of
terminally differentiated, segmentally iterated cells
(Whitman, 1878;
Zackson, 1984
;
Kramer and Weisblat, 1985
;
Weisblat and Shankland, 1985
;
Bissen and Weisblat, 1989
;
Braun and Stent, 1989
;
Shankland 1987
;
Muller et al., 1981
). This
assertion is qualified by the fact that no complete lineages for any
definitive segmental progeny have been published.
In Helobdella, segmentation proceeds sequentially from a posterior
growth zone comprising five bilateral pairs of large, identifiable
segmentation stem cells (M, N, O, P and Q teloblasts;
Fig. 1). Each teloblast lineage
generates a stereotyped set of mesodermal (M) or ectodermal (N, O, P, Q)
progeny to each segment, via coherent columns of segmental founder cells
(blast cells). Previous studies have revealed two major differences at the
cellular level between the segmentation processes in leeches and arthropods.
First, compartments as defined for Drosophila
(Garcia-Bellido et al., 1973)
are not observed in Helobdella. Instead, the stereotyped clones
arising from individual blast cells interdigitate both mediolaterally (between
lineages) and anteroposteriorly (within given lineages, across segment
boundaries) (Weisblat and Shankland,
1985
). Second, clitellate annelid embryos exhibit two distinct
modes of stem cell divisions: the M, O and P teloblasts follow a parental mode
in which each blast cell generates an entire segmental complement of progeny
for its lineage; but the N and Q teloblasts follow a grandparental mode, in
which two blast cells are required to generate each segmental complement, and
the blast cells within each column follow distinct fates in exact alternation
(Weisblat et al., 1980
;
Weisblat and Shankland, 1985
;
Storey, 1989
;
Arai et al., 2001
).
|
Microinjection of fluorescent dextrans
(Gimlich and Braun, 1985) has
been the technique of choice for analyzing cell lineages in
Helobdella, but it becomes difficult to distinguish individual cells
in the marked clones as the cells increase in number and decrease in size,
especially when they lie adjacent to one another. More recently, diverse
applications employing fluorescent proteins (GFP, YFP, CFP and DsRed;
designated collectively here as XFPs) have been used to label specific cells
in vivo (Tsien, 1998
;
Lippincott-Schwartz and Patterson,
2003
). These fluorescent proteins can be fused to target genes,
allowing inferences to be drawn about their expression and localization in
live embryos of various sorts (Chalfie et
al., 1994
; Lee and Luo,
1999
; Amsterdam et al.,
1995
; Feng et al.,
2000
).
Here, we have injected mRNAs encoding various XFP constructs in a combination of lineage tracing and reporter construct technologies, focusing on the primary neurogenic (N teloblast) lineage in H. robusta. We found that these mRNAs meet all the criteria for being microinjectable lineage tracers. In particular, the spatially restricted fluorescence of nuclearly localized XFP (nXFP) enabled us to extend our knowledge of the nf and ns blast cell lineages and revealed dramatic differences between them. We have also characterized the degree of asymmetry of the primary nf and ns blast cell divisions by using nXFP fluorescence to measure the nuclear volumes of the daughter cells. To follow the spindle dynamics of dividing nf and ns blast cells, we injected mRNA encoding tau::GFP. The spindle dynamics in the dividing ns and nf cells showed both similarities and differences to those in the asymmetric divisions of the C. elegans zygote and Drosophila neuroblasts.
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Materials and methods |
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Plasmid constructs, mRNA synthesis and mRNA injection
eGFP mRNA was transcribed in vitro from linearized pCS2P-eGFP-X/P plasmid.
Nuclear localized GFP and ß-galactosidase (nGFP and nLacZ) and tau::GFP
mRNAs were transcribed from pCS2P-nls-eGFP, pCS2-nls-ßgal and
pCS2-tau-GFP plasmids, respectively. To make nuclear localized versions of
other fluorescent proteins, plasmids pECFP-N1, pEYFP-N1 and pDsRed2-N1
(Clontech) were used as templates (details available on request). RNA was
injected from standard glass pipets treated to avoid RNase contamination
(details available upon request). The concentration of mRNAs in the needle are
0.4 µg/µl (GFP, nGFP, nCFP, nYFP, nRFP, tau::GFP) or 0.1 µg/µl
(nLacZ), either with or without 5 µg/µl rhodamine dextran amine (RDA,
Molecular Probes), the final concentration of mRNAs in the teloblast was
estimated to be 4 ng/µl or 1 ng/µl, as the injected volume is estimated
to be 1% of that of the N teloblast.
Morpholino injection
Antisense morpholino oligomer (AS MO, Gene Tools) complementary to the
start codon and seven downstream codons of nGFP mRNA, designated as AS-nGFP MO
(5'-CCTTACGCTTCTTCTTTGGAGCCAT-3'; anti-start codon
underlined), was injected at a concentration of 0.1 mM in the needle (1 µM
in the teloblast) if not otherwise indicated. AS-nGFP MO was used as a
4-mismatch control morpholino to nCFP and nYFP mRNA, which are similar in this
region (5'-ATGGCaCCgAAGAAGAAGaGgAAGG-3'; mismatches are
in lowercase and the start codon is underlined). The sequence of AS-nLacZ MO
is 5'-TACGCTTCTTCTTTGGAGCAGTCAT-3' with the anti-start
codon underlined. Injections procedures were as for mRNA injection. At least
20 embryos were injected for each experimental time point and all experiments
were performed in triplicate at minimum.
Time-lapse fluorescence microscopy and imaging processing
Injected embryos were allowed to develop to desired stages, mounted in HL
saline then examined and photographed using a Nikon E800 epifluorescence
microscope equipped with a cooled CCD camera (Princeton Instruments),
controlled by Metamorph software (UIC). Confocal time-lapse images were
obtained under Zeiss 510 Axioplan microscope at a time interval of 1 hour
(40-60 planes at 0.5 µm steps, using 63 x objectivewater
immersion NA 0.90). To image segmental ganglia, stage 10 embryos were fixed
(4% formaldehyde in 1 x PBS), washed (1 x PBS), then dissected.
The germinal plates were mounted and observed in 1 x PBS.
Confocal image stacks of ns/nf clones and stage 9-10 ganglia were deconvolved and reconstructed using Imaris 4.0 (Bitplane AG), or Volocity 3.0 (Improvision); nuclei were annotated and measured in Volocity. Epifluorescence images were deconvolved (2-D), then reconstructed in Metamorph. Images were exported as movies and single snapshots, and further processed with Photoshop 5.0 (Adobe) to prepare figures. Spindle images were processed and converted into time-lapse movies using Metamorph.
In situ hybridization, X-gal staining and Hoechst staining
Antisense GFP probe was made using T7 MEGAscript kit (Ambion) and
hybridizations were as previously described
(Song et al., 2002). X-gal
staining was as previously described (Liu
et al., 1998
), or as modified from a protocol in Xenopus
(Sive et al., 2000
) (details
upon request). Embryos were counterstained and mounted for observations as
previously described (Shain et al.,
2000
).
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Results |
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N teloblasts in stage 7 embryos (33 hours AZD) were injected with RDA
and nGFP mRNA, After
48 hours of subsequent development, the pattern of
nGFP-labeled nuclei was as expected from previous studies
(Zackson, 1984
;
Bissen and Weisblat, 1989
) (not
shown). In other experiments, we allowed injected embryos to develop for 143
hours post-injection. As at earlier stages, the distribution of marked cells
was indistinguishable from when teloblasts were with injected with RDA only.
The nGFP was still readily detectable, and cell nuclei in the labeled clone
were observed with excellent resolution. We counted
100 nuclei in the
anteriormost labeled hemiganglion and the N-derived peripheral neurons (nz1-3)
were observed at their stereotyped positions
(Fig. 2A,B). In a third set of
embryos, both N and P teloblasts were injected with nGFP mRNA and examined at
100 hours post-injection; neural precursor cells had migrated medially
from the p bandlet (Fig. 2C) as
reported previously for Theromyzon
(Torrence and Stuart, 1986
)
and Helobdella (Braun and Stent,
1989
). Thus, nGFP showed excellent perdurance and did not
noticeably perturb development (Fig.
2D).
|
To further refine the mRNA injections technique in Helobdella, we used it to test the efficacy of antisense knockdown reagents. RNAi did not yield reproducible knockdowns (data not shown), but antisense morpholino oligomer (AS MO) injections did. For this purpose, teloblasts were injected with nGFP mRNA and then after 3 hours, with AS MO complementary to 5' sequence of nGFP mRNA (AS-nGFP MO; see Materials and methods for details). We observed a significant and long-lasting knockdown of nGFP fluorescence in the primary blast cells and their clones that received AS-nGFP MO, relative to the cells produced prior to the second injection (Fig. 3A,B); in similar experiments, AS MO knockdown of nLacZ expression was also obtained (data not shown). Using quantitative fluorescence measurements, we estimate a knockdown of about 50% (Fig. 3C). No knockdown occurred when the target mRNA bore a mismatched target sequence or when the second injection comprised RDA only (data not shown).
|
Examining these embryos (Fig.
4), we concluded that the injected mRNA diffused throughout the
injected teloblast and was readily passed on to the primary blast cell
progeny. The nGFP mRNA degraded more quickly within the blast cell clones than
within the teloblast and primary blast cells, however; n blast cells produced
after the injection stained intensely during the first 24 hours
post-injection, at which time they have not yet divided, but by 72 hours
post-injection the mRNA was barely detectable within the equivalent cell
clone. Comparisons with RDA injections indicate that this reflects mRNA
breakdown and not dilution (data not shown). At 120 hours post-injection, nGFP
mRNA was readily detected in the injected teloblast and supernumerary blast
cells and faintly in the most posterior segments, but not in any of the
anterior segmental progeny. Thus, the in situ signal results indicate the
perdurance of translatable transcripts in blast cell clones until 72
hours cl.ag. Finally, nGFP protein could be readily detected throughout the
germinal plate for as long as 145 hours post-injection, i.e.
3 days after
no nGFP mRNA could be detected (compare
Fig. 2D with
Fig. 4F). We conclude that the
anteroposterior gradient of nGFP reflects gradually increasing levels of nGFP
protein and decreasing levels of nGFP mRNA inherited by successive blast cells
from the parent teloblast, coupled with declining levels of residual nGFP
protein in older blast cells clones, from which the mRNA has been degraded.
Similar gradients of expressed protein were seen with all the synthetic mRNAs
used (data not shown).
|
Ganglionic primordia separate as the nf.p clone in one neuromere
delaminates from the ns.a clone in the next (fissure formation)
(Shain et al., 1998). To
identify differences between the nf and ns clones, we sought to compare the
lineages leading from the continuous columns of primary blast cells to the
separation of ganglionic primordia. Fissure formation occurs between
45-50 hours cl.ag. in Theromyzon rude
(Shain et al., 1998
),
corresponding to
85-95 hours cl.ag. in the Austin strain of H.
robusta (Fig. 1).
For this purpose, tandem injections 90 minutes apart were used to
uniquely label single blast cell clones
(Zackson, 1982
). Labeled
embryos were then examined by time-lapse confocal microscopy, beginning at 58
hours cl.ag., by which time the first labeled clone (an ns clone) contained
two or three cells (Fig. 5A).
To identify the limits within which photo-damage did not perturb development,
we compared the patterns of labeled nuclei in embryos subjected to various
illumination paradigms with those in equivalent embryos that were imaged only
at the end of the experiment. We found that sampling embryos at a 1 hour time
interval was sufficient to capture cell divisions and movements in the N
lineage during the period of interest, and that after a 12 hour observation
period, the pattern of labeled nuclei was as in an unirradiated sibling embryo
(Fig. 5A). These results
suggested that cell divisions had occurred normally during this period in the
imaged embryos. However, imaging periods greater than 12 hours often resulted
in arrested cell division, so to follow further events in the nf and ns
lineages, we undertook multiple overlapping time-lapse imaging experiments
covering 40-86 hours cl.ag. for the ns clone and 40-82 hours cl.ag. for the nf
clone.
|
|
Second, many divisions exhibited stereotyped asymmetries, as judged by the
relative volumes of the nuclei of the daughter cells
(Fig. 7). Asymmetric divisions
of the nf and ns blast cells have been described qualitatively
(Bissen and Weisblat, 1989;
Song et al., 2002
). Here, we
measured the nuclear volume ratios for the daughter cell pairs of 26 ns and 31
nf divisions. There was no overlap between the ratios for ns and nf divisions
and little variation within each class of n blast cells
(Fig. 7B), despite the fact
that the exact clonal age and position of the dividing cells varied
significantly between embryos. We conclude that the differentially asymmetric
ns and nf divisions reflect inherent differences between the two blast cell
types. Asymmetric cell divisions occurred throughout the regions of the ns and
nf lineages studied here. After each asymmetric division, the smaller daughter
cell (as judged by nuclear volume) invariably had a longer cell cycle than its
sister, but there was not a strict correlation between nuclear volume and cell
cycle duration overall (data not shown).
|
Non-standard N teloblast progeny
The N teloblasts also contribute two sets of non-segmental cells to the
anterior micromere cap (Smith and
Weisblat, 1994). One is a clone of squamous epithelial cells
derived from micromere n', which arises from the N teloblast after it
has already made three divisions (Sandig
and Dohle, 1988
; Bissen and
Weisblat, 1989
) (Fig.
8A). The other is a small domain of apparently columnar epithelial
cells derived from one or more of the first three N teloblast progeny. To
enable comparisons with lineage maps in other spiralians, we have resolved
uncertainties regarding the fates of the cells produced prior to micromere
n'.
|
Finding that the anteriormost n blast cell is of the nf class is
paradoxical because in standard neuromeres, the ns clone lies anterior to the
nf clone (Fig. 1)
(Bissen and Weisblat, 1987).
The paradox is resolved by the realization that the anterior neuromere of the
subesophageal ganglion (R1) contains extra neurons compared with other
neuromeres (Fig. 8D). These
extra neurons arise from the nf clone (Fig.
8E,F), which could explain observations that the subesophageal
ganglion contains more (N-derived) serotonergic neurons than expected from
four fused standard neuromeres (Lent et
al., 1991
). Although we propose that it forms the anteroventral
adhesive organ (Smith and Weisblat,
1994
), the definitive fate of the n° clone remains to be
determined. By 120 hours cl.ag., the n°-derived cells lie immediately
ventral to the first nf clone (Fig.
8G,H).
Polarity and asymmetry of blast cell divisions
To examine the regulation of the asymmetric n blast cell divisions, we
injected left N teloblasts with tau::GFP mRNA, then followed spindle dynamics
in 26 ns and 18 nf divisions.
Mitosis took about 30 minutes for both nf and ns blast cells. No differences were observed until late in mitosis; in both ns and nf, spindle assembly was evidenced by increasing fluorescence intensity at the centrosomes (Fig. 9A, 00-04 minutes; Fig. 9B, 00-05 minutes). Centrosomes in the n blast cells had separated at several hours before the onset of mitosis (Fig. 9C), and usually retained an obliquely transverse orientation with respect to the AP axis of the n bandlet as cells rounded up for mitosis (Fig. 9A, 08 minutes; Fig. 9B, 05 minutes). In three of the divisions (1 nf and 2 ns), the centrosomes were already in an anteroposterior orientation prior to the onset of mitosis. No asymmetries were evident as the spindles began to rotate following nuclear breakdown (Fig. 9A, 12-20 minutes; Fig. 9B, 05-11 minutes).
|
Differences between nf and ns blast cells appeared late in mitosis. Cytokinesis occurred with the spindles displaced towards the posterior end of the cell, with the result that the cleavage furrow was also shifted posteriorly. The posterior shift of the spindle was correlated with a diminution in fluorescence of the posterior aster relative to the anterior aster in both cell types. The intensity difference between the anterior and posterior poles was not observed until after the completion of spindle rotation. Though we could not stain for DNA in these experiments, we estimate that the change in the posterior spindle pole occurred at the onset of anaphase. The posterior displacement of the furrow was greater in nf than in ns (Fig. 9C), as was the relative diminution of the posterior spindle pole fluorescence (Fig. 9B, 22-24 minutes). Within the nf blast cells, the spindle midbody was displaced towards the posterior centrosome, which further increased the extent of the mitotic asymmetry (Fig. 9C, 10 minutes). This difference between nf and ns correlates with the differences in the nuclear volume ratios of their respective progeny (Fig. 7).
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Discussion |
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Injected mRNA is degraded more quickly in the blast cells than in the
teloblast, and more quickly within the germinal bands than in the bandlets.
Previous work has shown that blast cells accumulate transcripts more rapidly
than teloblasts (Bissen and Weisblat,
1991) and there is a dramatic prolongation of the cell cycle in
blast cells relative to teloblasts
(Zackson, 1984
;
Bissen and Weisblat, 1989
).
These features support the idea that the teloblast-to-blast cell transition in
leech is analogous to the `mid-blastula transition' in Xenopus and
Drosophila (reviewed by Yasuda
and Schubiger, 1992
).
For all the mRNAs tested, we observed graded expression of the encoded protein; the first blast cells born after injection contain low levels of the protein and those born later contain progressively higher levels. No such gradient is observed when passive tracers are used. From the in situ results and the stem cell mode by which blast cells arise, we conclude that: (1) clones founded by blast cells born immediately after the injection contain only the relatively low levels of protein expressed before the mRNA they inherit from the teloblast is degraded; and (2) blast cells born at progressively later times post-injection inherit not only the mRNA, but also inherit increasing amounts of protein expressed in the teloblast, in which the injected message is relatively stable.
Whatever the mechanism by which they are formed, the gradients of protein expression driven by injection of synthetic mRNAs means that dose effects of mutant or ectopic regulatory and signaling proteins from such injections can be determined simply by examining their effects on blast cells born at different times and by comparing their effects on teloblast divisions at various times after injection (S.O.Z. and D.A.W., unpublished). Finally, we have demonstrated the efficacy and specificity of AS MO knockdown as a means of modulating the expression of injected mRNAs. Together, these techniques provide a powerful tool for functional analysis of gene function in an animal for which standard genetic approaches are not available.
Application of the mRNA injection technique to analysis of the N teloblast lineage
In Helobdella, most ganglionic neurons arise from the N teloblasts
via two distinct classes of blast cells, ns and nf, that arise in exact
alternation. Homologous lineages have been described in both lumbricid and
tubificid oligochaetes (Storey,
1989; Arai et al.,
2001
). Therefore, this grandparental mode of stem cell division is
almost certainly ancestral to clitellate annelids. To further understand this
process, we have applied the mRNA injection technique in three ways.
In C. elegans, the asymmetric division of the zygote is driven by
a posterior shift in the mitotic spindle
(Albertson, 1984). By contrast,
it has been reported that the asymmetric divisions of Drosophila
neuroblasts involve a basal shift of the spindle midbody relative to the
spindle poles (Kaltschmidt et. al.,
2000
). Our findings show that these processes can occur together
in cells, and that they can be regulated differentially to generate the
distinct asymmetries of the alternating nf and ns blast cells in
Helobdella.
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ACKNOWLEDGMENTS |
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