Grandparental stem cell divisions in leech segmentation. (A) Asymmetric cell divisions in N teloblast lineage. Schematic depiction of cell lineage from the N teloblasts to segmental ganglia in Helobdella. The posterior growth zone comprises 4 pairs of ectodermal teloblasts (N is labeled) and 1 pair of mesodermal teloblasts (not shown). Each teloblast undergoes highly asymmetric divisions every 1.5 h, forming columns of segmental founder cells (primary blast cells) that coalesce first ipsilaterally and then along the ventral midline into the germinal plate, from which segmental tissues differentiate (only the ganglia are shown). Developmental events in primary blast cell clones can be timed in terms of “clonal age” (cl.ag., in hours on the scale at left), i.e., the time since the birth of the primary blast cell that founded the clone in question. In the N lineage, the primary blast cells comprise two types, ns (red) and nf (green) in exact alternation; ns and nf cells differ in terms of the timing (ns, 44 h cl.ag.; nf, 40 h cl.ag.) and extent (see Figure 6) of their asymmetric, obliquely longitudinal first mitoses. These divisions yield ns.a/ns.p (red/pink) and nf.a/nf.p (green/light green) daughter cell pairs, which eventually contribute distinct sets of neurons to the differentiated ganglia (140 h cl.ag.). Anterior is up in all figures if not otherwise noted. (B) Cell lineage diagrams contrasting parental and grandparental stem cell divisions in the teloblast lineages. Each stem cell division gives a new generation of teloblast (Tb-x) and a segmental founder cell (a or b). In standard (parental) divisions, each generation of teloblast gives rise to the same type of blast cell; in grandparental divisions, odd numbered generations give rise to one type of blast cell (a) and even numbered generation give rise to another (b).

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 (A, row 1); normalizing relative to 18S revealed that Hau-cdc42a is relatively abundant, and that its levels fluctuate within a four fold range over the course of embryonic development (B). In contrast, the Hau-cdc42b amplicon was barely detectable after 28 amplification cycles (A, row 2), and only became readily visible with five additional cycles (A, row 3). Hau-cdc42b declines to very low levels in stages 6 through mid 8, and increases in later stages (B). (C) In situ hybridization of embryos at early stage 8 is consistent with sqRT-PCR data; Hau-cdc42a transcripts are evident in the germinal bands and to a lesser extent in the teloblasts (arrows) while probe for Hau-cdc42b gives essentially no staining. Under identical conditions Hau-rac1 shows expression in the germinal bands at lower levels than Hau-cdc42a while Hau-rac2 transcripts are seen primarily in teloblasts (arrows). Scale bar: 80 microns.

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, Hau-RAC1 and Hau-RAC2; see Figure 2B) and another between 60-80 kD, which does not correlate with any known Rho GTPase from the genome. Minor bands are visible at stages 2 and 7. (B, C) Deep (B) and superficial (C) focal planes of an embryo in which the left N teloblast (NL) was injected with synthetic Hau-cdc42a mRNA. The embryo injected embryo was fixed and immunostained with the cross-reactive antibody roughly 2 days later. The n bandlet (arrow) derived from the injected teloblast stains more strongly, indicating that the antibody recognizes Hau-CDC42A. Scale bar: 50 microns.

Localization of YFP fusion proteins. YFP::Hau-CDC42A (A, B) and YFP::Hau-CDC42A^Q61L (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 to both anterior and posterior cortices (long and short arrows, respectively) of all n blast cells. Low levels of YFP::Hau-CDC42A^Q61L localize to posterior but not anterior cortices in ns (C) and nf (E) during interphase, and segregate to the smaller posterior daughter cells in mitosis (D, F). In (B, D. F), gray-scale images were exponentially transformed to enhance differences in fluorescence intensity. Time zero marks the initiation of cytokinesis. Note that the highest YFP intensity was detected at progressing cytokinetic furrows (arrowheads). (G, H) In more posterior cells with higher expression levels, YFP::Hau-CDC42A^Q61L localizes to multiple cortical foci (arrows) during interphase (G) and these are inherited by both daughters in the equalized mitoses (H). Scale bar, 10 microns.

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) clusters. ML trees show a similar topology (not shown). The Helobdella genes that are further isolated and studied here are indicated by bold type and arrows. Amino acid sequences for all genes from Capitella sp. I (a polychaete worm) and Lottia gigantea (a limpet) and the Helobdella MTL homolog were recovered from JGI gene model collections. Gene model protein ID numbers assigned by JGI are used instead of generic names for these unconfirmed gene models. Abbreviated species names: Hsa (Homo sapiens), Dme (Drosophila melanogaster), Cel (Caenorhabditis elegans); Cap (Capitella sp. I); Lgi (Lottia gigantea); Hro (Helobdella robusta). (B) The first 50 N terminal amino acid residues of Cel-CDC42 align with the four Helobdella Rac/CDC42 proteins. The amino acids in this region were completely identical between the CDC42 proteins. Only ten different amino acid changes were identified between CDC42 and Rac proteins (yellow boxes).

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) is evident soon (~7 h cl.ag.) after the n blast cells are born; younger ns cells (dashed red arrows) that do not show elevated Hro-cdc42 levels lie closer to the parent teloblast (N). Black arrow points to an nf which is the latest born. (B, C) Examples of left (B) and right (C) N lineages in early stage 8 embryos show that CDC42-like immunoreactivity is higher in premitotic ns cells (red arrows) than in the intervening nf cells. The oldest ns cell that has not yet upregulated CDC42-like immunoreactivity (dashed red arrows, ~26 h cl.ag.) and the most recently divided nf cell (green arrows, ~40 h cl.ag.) are indicated in each sample. (D) The same pattern of elevated immunoreactivity in ns blast cells (red arrows) is seen in cells destined for posterior segments in embryos fixed and stained at mid stage 8. Scale bar, 10 microns.

Expression of constitutively active Hau-CDC42A above threshold level equalizes the nf and ns blast cell divisions. (A-C) Nuclear volume ratios were obtained (V_{post. cell}/V_{ant. cell}; see text for details) for daughter cell pairs arising from the mitoses of ns and nf blast cells (red and green, respectively). Nuclei of undivided, primary blast cells are shown in white. (A) Over-expression of YFP::Hau-CDC42A does not affect the differentially asymmetric ns and nf blast cell mitoses (quantified as described in text); nuclear volume rations are in distinguishable from previous measurements (Zhang and Weisblat, 2005) and the injected lineages developed normally (not shown). (B-D) Over-expression of YFP::Hau-CDC42A^G12V/Q61L equalizes both ns and nf divisions, as judged by nuclear ratios (B, C) and by cell sizes (B, dashed outlines), but only in blast cells arising several cell cycles after injection of the parent N teloblast. [In (C), the anteriormost nf clone has undergone a normal second mitosis, so the nf.p/nf.a nuclear volume ratio could not be determined for that clone.] Orientations of divisions in affected blast cells appear to be randomized [e.g., posteriormost ns mitosis in (C)]. (D) After two days of subsequent development, clones of ns and nf with normal first divisions appear to develop normally, as evidenced by overall clone morphology, further asymmetric divisions (note range of nuclear sizes in 4 anterior clones) and the formation of intersegmental segmental fissures between such clones (arrow). In contrast, clones of affected n blast cells develop abnormally, with fewer and equal mitoses, often associated with their separation from the anterior portions of the bandlet. Here, the two posterior clones contain only 5-6 cells each. The posterior clone also shows what appear to be nuclear fragments indicative of dying cells (arrowhead). Such clones cannot be distinguished as nf or ns by their morphology and the assignments indicated here are based on their sequence relative to the anterior portion of the n bandlet. (E) Some time after the transition to producing blast cells that undergo equal mitoses, the injected N teloblast itself has undergone a roughly equal division (progeny indicated by arrow and arrowhead). After one or two such divisions, the affected teloblasts cease dividing. (F) The transition from normal to equalized n blast cells after the injection of N teloblast with Hau-cdc42a^G12V/Q61L mRNA is dose dependent. Teloblasts were injected after seven n blast cells had been produced (black bar) and the number of n blast cells undergoing normal, differentially asymmetric division after injection was determined (green bar). At the lowest concentration, (0.05 μg/μl) no transition from normal to equalized mitoses was observed (denoted by jagged bar end).. Scale bar, 20 microns.