Duplicate rac and cdc42 genes in the Helobdella genome
In Metazoa, Rho family small GTPases are divided into six subfamilies, including Rac, CDC42, MTL, Rho, RhoU and RhoBTB (Boureux et al., 2007
). There are 11 putative Rho family small GTPase genes in the annotated H. robusta
genome, including two rac
orthologs and surprisingly, two cdc42
orthologs (). Among other animals that have been surveyed, cdc42
gene duplications have been observed in various deuterostomes (hemichordate, teleost fish, and opossum), but not in other vertebrates, echinoderms or ascidians, nor in any of other taxa including cnidarian, sponge, fluke, nematode or fly (Boureux et al., 2007
). The other seven genes encode one mtl
ortholog, three putative rho
orthologs, two rhoU
orthologs and one rhoBTB
ortholog. We were able to clone and sequence the rac
orthologs from both H. robusta
and from H.
sp. (Austin) cDNAs, thereby confirming their identity. We have therefore designated these eight genes as Hro-
and Hau-rac1, -rac2, -cdc42a
. The cdc42a
cDNA sequences in Hau
were identical at the nucleotide level, as were their respective cdc42b
Figure 2 Molecular sequence analysis of Helobdella Rac/CDC42 small GTPases. (A) Unrooted NJ tree of Rac/CDC42 small GTPases from five representative metazoan species. Bootstrap analysis (1,000 repeats) shows well-supported Rac (red), CDC42 (blue) and MTL (green) (more ...)
The amino acid sequences of the hypothetical Helobdella CDC42a and CDC42b proteins are 90% identical overall, and align closely with those of other metazoans and yeast (). In particular, their N terminal 84 amino acids are identical to those of the C. elegans protein, and it was the N terminal sequence of the C. elegans protein that was used to generate the commercial polyclonal antibody used here. Similarly, Hro-RAC1 and Hro-RAC2 are 95% identical between themselves, 69-71% identical overall in pairwise combination with the Hro-CDC42 proteins, and 81% identical to the Hro-CDC42 proteins in the N terminal region, with primarily conservative substitutions. The extensive similarities between the two rac genes and between the two cdc42 genes in Helobdella suggest that they may represent fairly recent gene duplication events. This interpretation is supported by the fact that we found no evidence for duplication of either rac or cdc42 genes in two other lophotrochozoan genomes, (Capitella sp. I and Lottia gigantea) . To examine the expression and function of these Rho GTPases in the unequal blast cell divisions, we focused on the embryos of H. sp. (Austin), which is more readily maintained in laboratory culture.
Within the N teloblast lineage, Hau-cdc42a is expressed preferentially in ns blast cells
Given the extensive conservation of amino acid sequences among the four Helobdella RAC and CDC42 peptide sequences, immunostaining is not expected to distinguish between these four proteins. At the nucleotide level, however, the two cdc42 homologs differ substantially from the two rac homologs (maximum 68% identity between Hau-cdc42a and Hau-rac1), and Hau-cdc42a and Hau-cdc42b are only 76 % identical overall. Thus, we anticipated that in situ hybridization should permit us to distinguish the expression of these genes during stages 7-8, during which the blast cells arise from the asymmetric cell divisions of the teloblasts.
Semi-quantitative RT-PCR (sqRT-PCR) analysis () revealed that Hau-cdc42a transcript is in higher abundance than Hau-cdc42b until late in development, but its level fluctuates over the course of development. The Hau-cdc42b transcript is present in early cleavage stages and in late development, but is almost undetectable during stages 6-8. Hau-cdc42b amplicon was not clearly visible at most stages until after 33 rounds of PCR amplification whereas Hau-cdc42a was readily detectable at 28 cycles (). Hence, our sqRT-PCR data suggested that, of these two genes, Hau-cdc42a is more likely to be involved in the phase of segmentation represented by the production and unequal early divisions of the blast cells.
Figure 3 Expression of the Helobdella cdc42 and rac genes. (A, B) sqRT-PCR reveals distinct temporal dynamics of Hau-cdc42a and Hau-cdc42b expression. (A) For Hau-cdc42a, a clear band was observed for every developmental stage after 28 cycles of amplification (more ...)
In situ hybridization experiments were consistent with the sqRT-PCR results. In stage 7-8 embryos, Hau-cdc42a probes yielded strong staining in the germinal bands and fainter but detectable staining of teloblasts under conditions where Hau-cdc42b probes of similar length and concentration gave no staining above background (). Parallel in situ hybridization reactions revealed that Hau-rac1 and Hau-rac2 are present in somewhat complementary distributions; Hau-rac1 is present in the germinal bands, but not in teloblasts, while Hau-rac2 is more evident in teloblasts than in germinal bands. Probes for all three genes, Hau-cdc42a, Hau-rac1 and Hau-rac2 also stained cells between the germinal bands in an area known as the micromere cap (). The broad expression of these genes is in keeping with their many roles in cells.
Careful inspection of embryos stained for Hau-cdc42a revealed that Hau-cdc42a mRNA is more abundant in ns than in nf cells (). Moreover, the alternating Hro-cdc42a levels in ns and nf blast cells were evident by ~6 hours clonal age (). In n blast cells younger than ~6 hours clonal age, the pattern of Hro-cdc42a expression was variable and adjacent cells often contained similar levels of Hro-cdc42a (). This observation suggests that at least some aspects of ns and nf blast cell identity arise after the primary blast cells are born from the N teloblast. We did not observe an alternating staining pattern for Hau-cdc42b, Hau-rac1 or Hau-rac2. Thus, subsequent analyses focused on Hau-cdc42a.
Figure 4 Differential expression of Hau-cdc42a in nf and ns blast cells. (A) In situ hybridization showing the portion of n bandlet proximal to the N teloblast in stage 7 embryos. (A) Alternating pattern of Hro-cdc42a mRNA levels, elevated in ns cells (red arrows) (more ...)
Immunostaining suggests that Hau-CDC42A protein is also elevated in ns blast cells
As discussed above, immunostaining with the commercially available antibody, raised to the highly conserved N terminal portion of the CDC42 ortholog from C. elegans, cannot give conclusive evidence that the observed Hau-cdc42a mRNA is being translated, because this antibody is not expected to distinguish Hau-CDC42A from Hau-CDC42B, Hau-RAC1, Hau-RAC2 and possibly other Rho GTPases. Consistent with this prediction, we found the antibody recognizes a protein band of the appropriate molecular weight for CDC42 and Rac proteins (about 21 kD) in embryonic western blots (). This band is present at all stages examined, as is another band of about equal intensity in the range of 70 to 80 kD. We speculate that this higher MW band may correspond to the RhoBTB family member.
Figure 5 Immunostaining for Hau-CDC42A. (A) Western blot analysis of Helobdella embryos with commercial CDC42 antibody detects two prominent bands at all stages of development, one at about 21 kD, corresponding to the predicted MW of the Hau-CDC42A, Hau-CDC42B, (more ...)
In a second test of the antibody, we injected individual teloblasts (stage 7) with yfp::Hau-cdc42a mRNA transcribed in vitro. Immunostaining the injected embryos 2 days later revealed preferential staining of the column of blast cells arising from the injected teloblast (), as well as a general staining of the embryo that we attribute to endogenous Rho GTPases.
Together these results indicate that the available antibody does recognize Hau-CDC42 in the embryo, and presumably other Rho GTPases. Thus, in conjunction with the in situ results, which suggest differential expression between nf and ns blast cells only for Hau-cdc42a, the onset of CDC42-like immunoreactivity in the n bandlet should permit us to infer the earliest point at which Hau-CDC42A protein might be accumulating in cells of interest.
With these caveats in mind, we examined the pattern of immunostaining in the n bandlets of normal embryos. Immunostaining was seen at uniformly low levels in all primary blast cells proximal to the teloblast, but increased specifically in ns cells that were older than about 26 hours cl.ag. As a result, protein levels alternated (higher in ns cells and lower in nf cells) beginning with undivided (primary) blast cells (26 hours cl.ag.) and continued at least through their first mitoses (roughly 44 hrs. cl.ag.; ). These observations were made on embryos fixed at early stage 8 (roughly 96 hours AZD). We also observed the pattern of elevated CDC42-like immunoreactivity in undivided n blast cells of embryos fixed at mid stage 8 older absolute ages (roughly 120 hours AZD, ), indicating that this pattern does not result from segment-specific differences in expression, but instead represents a consistent difference between the nf and ns blast cells, corresponding to the observed difference in Hau-cdc42a transcript levels.
Thus, while the cross-reactivity of the antibody means that we cannot be sure that the observed immunostaining represents Hau-CDC42 protein, our results are consistent with the hypothesis that this pattern reflects the accumulation of Hau-CDC42A in ns blast cells starting at approximately 26 hours cl.ag., i.e., with a delay of about 20 hours after the appearance of Hau-cdc42a mRNA. Together, the in situ and immunostaining results suggest that differences in Hau-cdc42a transcription, transcript processing and/or stability between the ns and nf blast cells result in corresponding differences in Hau-CDC42A protein levels, and that the mRNA differences are present as a pre-pattern in the n bandlet.
Posteriorly localized CDC42A activity regulates asymmetric divisions of nf and ns cells
Injecting N teloblasts with yfp::Hau-cdc42a mRNA resulted in the expression of YFP::Hau-CDC42A protein at high levels compared to the native protein as judged by immunostaining (), yet this had no obvious effects on the production of n blast cells by the teloblast (). To look more carefully for functional consequences of what appears to be quantitative differences in Hau-CDC42A expression between ns and nf blast cells, we looked for changes in n blast cell division patterns resulting from the expression of YFP::Hau-CDC42A. For this purpose, N teloblasts were injected with a mixture of yfp::Hau-cdc42a and ngfp mRNAs to visualize the YFP::Hau-CDC42A protein distribution in living cells, and the nuclei of fixed cells inheriting the injected mRNAs, respectively. Injections were uniformly carried out at early stage 7 (about 33 hours AZD) and the injected embryos were observed at early stage 8, roughly 2 days after injection.
A priori, we anticipated that nf cells with elevated Hau-CDC42A levels might divide more like ns cells (i.e., later and/or less asymmetrically) but no such defects were observed. In all embryos (n=74) injected with a mixture of yfp::Hau-cdc42a
mRNAs, the ns and nf blast cells exhibited their normal, differentially asymmetric cell divisions (; compare with of Zhang and Weisblat, 2005
), as did the injected teloblasts (not shown). The nuclear volume ratios calculated for the products of both nf and ns blast cell divisions () were all within the range of previously measured values (Vns.p
= 0.51 +/− 0.05 and Vnf.p
= 0.23 +/− 0.03; Zhang and Weisblat, 2005
Figure 6 Expression of constitutively active Hau-CDC42A above threshold level equalizes the nf and ns blast cell divisions. (A-C) Nuclear volume ratios were obtained (Vpost. cell/Vant. cell; see text for details) for daughter cell pairs arising from the mitoses (more ...)
Figure 7 Localization of YFP fusion proteins. YFP::Hau-CDC42A (A, B) and YFP::Hau-CDC42AQ61L (C-F) in interphase cells (A, C, E) and during mitoses (B, D, F) show selected frames from time-lapse videos; elapsed time indicated in minutes. YFP::Hau-CDC42A localizes (more ...)
We have shown previously that injecting teloblasts with mRNA yields a gradient of protein expression within the descendant blast cells: blast cells born soon after the injection express low levels of the protein, while those born later inherit progressively higher levels from the injected teloblast (Zhang and Weisblat, 2005
). This was also true for the YFP fusion proteins expressed in our present experiments (data not shown), but ns and nf blast cells corresponding to all axial positions in YFP::Hau-CDC42A-expressing embryos divided normally.
Imaging live embryos revealed that YFP::Hau-CDC42A was localized to the cortical cytoplasm of n blast cells in interphase and mitosis. In contrast to the situation in budding yeast (Richman et al., 2002
), this fusion protein did not show an asymmetric distribution between the anterior and posterior cortices in either nf or ns blast cells (n=16) ().
As a Rho family GTPase, Cdc42 cycles between an active GTP-bound form and an inactive GDP-bound form and the proportion of the overall Cdc42 protein in these two forms is under kinetic control by guanine nucleotide exchange factors, GTPase-activating proteins and GDP-dissociation inhibitors (Hall, 2005
). Thus, changing overall Cdc42 protein levels in the experiment described above may not have affected the levels of activated Cdc42. To affect levels of active Hau-CDC42A directly, and follow the distribution of the activated, GTP-bound form of Hau-CDC42A, we co-injected N teloblasts with a mixture of ngfp
mRNA and mRNA encoding YFP fused to Hau-CDC42AG12V
. These mutant proteins cannot efficiently hydrolyze bound GTP and thus are trapped in the active form (Hall, 2005
; Hutterer et al., 2004
). Identical results were obtained with both constructs. Thus, for simplicity they are designated hereinafter as YFP::Hau-CDCA42G12V/Q61L
In accord with the expected gradient of protein expression in blast cells after injecting the teloblast with mRNA, the first few blast cells born after the yfp::Hau-cdc42aG12V/Q61L mRNA injection (up to 7 blast cells in a single N teloblast lineage) expressed YFP::Hau-CDC42AG12V/Q61L at tracer levels. That is to say, these cells exhibited relatively low YFP fluorescence and the ns and nf cells divided with the normal timing and asymmetry, so the mutant protein was evidently not present in levels sufficient to disrupt normal cell division pattern. Consistent with our previous findings, the nf blast cell divisions were markedly unequal with nuclear volume ratios (Vnf.p/Vnf.a) of about 0.25; ns blast cell divisions were also unequal but less so, with nuclear volume ratios (Vns.p/Vns.a) of about 0.5 (). These volume ratios are within the range of those measured for control blast cells and for blast cells over-expressing wild-type Hau-CDC42A. In such cells, (11/12), YFP::Hau-CDC42AG12V/Q61L localized preferentially to the posterior cortices and was segregated to the posterior (smaller) daughters of both blast cell types(), in clear distinction to the wild-type protein.
In contrast to the first few n blast cells born after N teloblasts were injected with yfp::Hau-cdc42aG12V/Q61L, those born later exhibited higher levels of YFP fluorescence, reflecting higher levels of the translated mutant protein. Strikingly, these cells appeared to divide equally, and with unpredictable, apparently randomized spindle orientation, in 68 of 72 embryos (94%). Moreover, although the increase in YFP::Hau-CDC42aG12V/Q61L levels was presumably gradual, the transition from normal to abnormal divisions was abrupt, occurring between one blast cell and the next (). Quantification of the change in division pattern was obtained as before, by reconstructing sister cell nuclei from stacks of confocal images obtained from embryos fixed at a point where they contained 2-cell clones of both normal, asymmetric and abnormal, equalized n blast cell divisions (). Consistent with the qualitative observations, the measured nuclear volume ratios changed from normally asymmetric divisions to nuclear volume ratios that were not significantly different from unity (nuclear ratios of 0.95+/−0.04, n=8 including both nf and ns divisions).
Despite the disruption of the normal asymmetry and orientation of mitosis in n blast cells resulting from elevated levels of constitutively active YFP::Hau-CDC42AG12V/Q61L, the nominal ns and nf cells divided at their normal clonal ages of 44 hours and 40 hours, respectively. Together, these observations suggest that the cell cycle duration is controlled independently of the position and orientation of the mitotic apparatus, and that only these two latter aspects of the blast cell division are regulated by the polarized distribution of activated CDC42A.
Given the short time windows over which time lapse observations could be made, it was not technically feasible to follow the subsequent divisions of affected n blast cells in detail. However, in embryos examined about 96 hours after injection yfp::Hau-cdc42aG12V/Q61L, affected n blast cell clones had apparently undergone very few additional, equal divisions. The resultant clones comprised isolated clusters of several equally sized nuclei. In some cases, fragmented nuclei suggested the occurrence of cell death in the affected clones (), but at least some affected cells were observed to persist, without obvious differentiation, through developmental stage 10, when normally developing segments are well-differentiated (not shown).
Imaging live embryos revealed that the equally dividing n blast cells had multiple patches of cortical YFP distributed throughout the cortex (), in contrast to the posteriorly localized signal seen in asymmetrically dividing n blast cells expressing low levels of constitutively active YFP::Hau-CDC42aG12V/Q61L. Taken together, these results suggest that Hau-CDC42A activity is normally localized to posterior cortices of nf and ns blast cells, and that over-expressing the constitutively active form of Hau-CDC42A disrupts nf and ns asymmetric mitoses once a critical threshold CDC42 activity is exceeded.
Common threshold of nf and ns in response to CDC42AG12V/Q61L over-expression
Do the normally observed differences in the extent of the asymmetry between ns and nf blast cell divisions result from quantitative differences in the amounts of activated Hau-CDC42A in the two cell types? If so, we predicted that the nuclear ratios of dividing n blast cells would shift gradually from their normal values toward unity, and also that nf cells might be more sensitive than ns cells to increasing levels of Hau-CDC42AG12V/Q61L. In fact, neither of these predictions was met. In experimental embryos, the transition from asymmetric to symmetric divisions was abrupt, as if the cells respond to increasing CDC42 activity in a switch-like, all or none manner (), and the first affected clone was equally likely to arise from an ns or nf blast cell (data not shown).
To further test the possibility that quantitative differences in intracellular levels of activated Hau-CDC42A control the differential asymmetry of nf and ns blast cell divisions, N teloblasts were injected with serially diluted yfp::Hau-cdc42aG12V/Q61L mRNA, to generate a shallower gradient of increasing YFP::Hau-CDC42AG12V/Q61L expression in the blast cells. As the mRNA concentration in the needle decreased from 0.4 to 0.2 μg/μl, the number of blast cells produced before abnormal (equal) divisions began increased to ~14 (), but the transition remained abrupt, with nuclear ratios changing to unity with neither any intermediate values nor with any bias in terms of which cell type was affected first. At lower mRNA concentrations, increasing numbers of n blast clones developed normally but there was still an abrupt transition from normal to abnormal development in the clones expressing Hau-CDC42AG12V/Q61L (). We also injected N teloblasts at different time during stage 7, thereby targeting primary blast cells at various positions along the A-P axis, with no change in the results. We therefore conclude that a slight increase in the concentrations of Hau-CDC42G12V/Q61L level above some common threshold value disrupted the normal asymmetry in both ns and nf blast cells in an “all-or-none” manner. Moreover, all ns and nf primary blast cells along A-P axis have the same threshold to the effects of Hau-CDC42AG12V/Q61L.
Further evidence for this threshold effect comes from the N teloblasts themselves. Teloblasts injected with yfp::Hau-cdc42aG12V/Q61L continued making morphologically normal primary blast cells for some time even when the blast cells produced were destined to divide evenly, but in most embryos, the injected teloblast eventually made an approximately equal division and ceased producing primary blast cells (), truncating that N lineage. This suggests that the N teloblasts also respond in an all-or-none manner to increasing levels of constitutively active Hau-CDC42A, but with a higher threshold than the primary blast cells.