An overview of bud development in Botryllus schlosseri
The blastogenic cycle in Botryllus
is organized into stages A through D (Watanabe, 1953
; ). A single cycle takes place over 7 days at 18°C, but because 3 generations of bodies coexist, development from birth of a bud to death of a zooid requires 3 cycles or 21 days. The appearance of a bud rudiment as a thickening in the atrial wall of the primary bud is coordinated with the onset of a new blastogenic cycle (stage A-1 or day 1) at which point parent zooids open their siphons and begin to feed. The bud primordium grows into a hemisphere that narrows at the base and pinches off to create a closed vesicle. A gut rudiment is formed from a fold of the innermost layers and elaboration of three inner chambers is complete by day 7, which concludes the cycle with the “grandparental” zooid death and its resorption. Following takeover, in which the primary buds replace the former zooids, a new cycle begins as a new bud rudiment grows from the wall of the now primary bud. Organogenesis in the primary buds is completed with the initiation of heartbeat on developmental day 10 (7+3) and the opening of siphons for filter-feeding on day 15 (7+7+1) as the bud becomes a zooid. Death of the zooids occurs at the end of the cycle on day 21 (Berrill, 1941a
; Brien, 1968
Fig. 1 Diagram of cyclic blastogenesis in Botryllus schlosseri depicting stages A through D (adapted from Watanabe, 1953).
Identification and characterization of Athena cDNA
was identified by differential display in a screen for developmentally modulated Botryllus
mRNA is dramatically upregulated during takeover, the 24–36 hour period of apoptotic death and resorption of zooids at the conclusion of the blastogenic cycle, termed stages D-1 through D-4, and nearly undetectable during the rest of the cycle (Lauzon et al., 1996
). The full length cDNA sequence corresponding to the differentially expressed fragment, obtained by subcloning, 5′ primer extension, and 5′ RACE, comprises 632 nucleotides with a single open reading frame encoding 142 amino acids (; Genbank accession number DQ092483). The developmental upregulation of Athena
transcript during takeover was confirmed in several different colonies by Northern blot (), although rare message could be detected at all stages by nested PCR (data not shown). Repeated efforts to localize Athena
transcript by RNA in situ
hybridization were not successful. Tiozzo et al. (2005)
recently described expression patterns of the homeobox gene Pit-X
during embryogenesis and asexual budding in Botryllus schlosseri
using in situ hybridization. We too have successfully utilized this technique to localize several genes including cytoplasmic actin in Botryllus
(data not shown). Consequently, our lack of success in localizing Athena is likely due to low abundance of expression of the transcript. However, we could detect Athena
in surgically excised buds and dying zooids from colonies in takeover by quantitative RT-PCR (). Presence of transcript in both buds and zooids at this stage is surprising, since they appear to be undergoing opposing processes of growth and programmed cell death (Lauzon et al., 1992
Fig. 2 (A) Analysis of the cDNA and predicted protein sequence of Athena. A putative polyadenylation site in the 3′ untranslated region is underlined. Sequences corresponding to the morpholino antisense oligo and the short interfering RNA sequence are (more ...)
Potential function of Athena could not be inferred from sequence comparison, as significant homology could not be found to genes of model organisms, including the solitary ascidian Ciona intestinalis. The predicted protein lacks known conserved domains and is largely hydrophilic except for a 20 residue stretch at the N-terminus which contains several hydrophobic residues but does not appear to be a transmembrane domain by available search algorithms. A low-stringency Southern blot with DNA from a related colonial ascidian, Botrylloides simodensis, yielded a single band, suggesting a potential homologue (data not shown).
RNAi and morpholino injection and soaking
Genetic knockdowns of Athena
were induced with morpholinos (MO), double-stranded (ds) RNA and short-interfering (si) RNA, delivered by injection or soaking. Previous studies demonstrated the specific knockdown of translation by MO in Ciona intestinalis
embryos (Satou et al., 2001
). However, we wanted to establish the efficacy of knockdown technologies in mature Botryllus
since budding begins only after metamorphosis. Experiments were carried out in groups of stage-synchronized clonal replicates in order to control for natural variation in the blastogenic cycle between genotypes. RNAs and morpholinos were co-microinjected with dye into terminal vascular structures called ampullae. Distribution throughout the colony, including zooids and developing buds, occurred via the common blood circulation. In addition, juvenile colonies (1–3 zooids) were subjected to repeated rounds of soaking in RNAs; distribution of RNAs throughout the body likely occurs via the neural gland, which regulates osmolarity (Ruppert et al., 2003
Blastogenic defects were observed 3 to 12 days following injection with Athena dsRNA and short-interfering RNA (siRNA) and 2 to 7 days following injection with MOs. Regardless of the substance or route of delivery, effects were not observed within the same cycle if treatment occurred after stage A-2. This result implies that translation of Athena during (or at the conclusion of) takeover is required for correct bud development in the following cycle. Defects were rarely observed in zooids or during takeover; in very few cases, resorption of old zooids was delayed into stage A-2 (day 2), whereas it is normally completed in A-1, or day 1. Affected structures included mainly secondary buds and in some cases primary buds as well.
Knockdown phenotypes were categorized as mild, definitive and severe (). Mild defects consisted of slight delays in the development of secondary buds relative to control colonies and normal progression of the blastogenic cycle. Mildly affected buds were often smaller than control counterparts at some points during development, but appeared normal 1–2 days later. These kinds of aberrations were observed at similar frequencies in all groups, regardless of what treatment, if any, was given (, column 1), demonstrating that temporary desynchronizations in the blastogenic cycle between generations of buds is within the range of normal development.
Summary of RNAi and Morpholino experiments
Buds that were deficient in organogenesis, retarded in their growth or development more than 36 hours were categorized as definitive phenotypes. We observed failure of secondary bud evagination closure at stage B-2 () as compared to stage B-2 control buds (). In addition, developmentally delayed stage A-2 primary buds () and delayed stage D-2 secondary buds () were observed. Abnormally small buds were often characterized by incomplete organogenesis. Whereas the initiation of three atrial folds delineating atrial from branchial cavities can be observed microscopically in normal C-1 secondary buds, the contents of Athena knockdown buds advanced beyond stage C-1 frequently appeared as a disorganized mass of cells (stage A-2 primary bud in ). In some cases, affected buds appeared to be hollow vesicles (stage D-2 secondary bud in ) or lacked extracorporeal vascular connection (). This definitive phenotype was observed in knockdown animals produced by injection with dsRNA, siRNA and MOs and could be reproduced by soaking juvenile colonies in dsRNA and siRNA ().
Fig. 3 Definitive (A, C, E) and severe (G, I) abnormalities in bud development caused by RNAiand antisense morpholino treatment. (A) Day 6 following injection of 10 pmols of siRNA. Secondary bud vesicle (arrow) closure failed and development has stalled out (more ...)
More potent effects were observed following injection with Athena MOs than either dsRNA or siRNA. In two instances, both primary and secondary buds entirely failed to develop on the zooids on one side of a colony (); the region of absent buds coincided with the site of MO injection in both cases. Defective developmental timing was noted in two colonies by the appearance of bud rudiments 2 to 3 days early on the wall of secondary buds at stage D-3 (, compared to normal D-3 colony in ). Defects in timing of organogenesis appeared as secondary bud heartbeat initiated three days early in stage D-3 () or in a single case as an ectopic beating heart arose in the extracorporeal vasculature of a colony (data not shown).
A significant effect (p=0.0035, chi square=8.53) was found in all classes of knockdown colonies relative to those receiving control RNA or control MOs using an ordinal logistic model. The effects produced by the method of delivery (soak versus injection) or the agent given (dsRNA, siRNA or MO) were statistically indistinguishable. Given the precedence of nonspecific defects induced by dsRNAs in vertebrates(Tuschl et al., 1999
; Caplen et al., 2000
; Oates et al., 2000
; Zhao et al., 2001
), each group was compared to its control by chi-square test; we found significant differences between Athena
RNAi versus control RNAi groups (p < 0.001) and between Athena
MO versus control MO group (p=0.0097). The latter test was repeated with a combined definitive/severe category to produce a probability of 0.017 by Fisher’s Exact test. Finally, injection of Athena
MO was found to be more effective than injection of RNA (p=0.01); however, differences in the effectiveness of these two knockdown methods were not significant if data from RNA soaking was included. These analyses served to verify the efficacy of Athena
knockdown by three separate methods as well as establishing the use of knockdown techniques in Botryllus.
Analysis of knockdown animals
Inhibition of Athena transcription was verified in several colonies treated with dsRNA or siRNA by real time quantitative RT-PCR. Abundance of Athena mRNA relative to tubulin was established in a normalized cDNA library from all stages and in several normal and control stage D animals (, expressed as the difference in the cycle threshold, ΔCt). Athena cDNA could not be detected in 4 of 4 colonies treated with siRNA by injection or soaking. As expected, the lower ΔCt value obtained for the stage D control animal reflects a greater abundance of Athena transcript present during takeover relative to a normalized, pooled cDNA library.
Fig. 4 Analysis of Athena expression in siRNA knockdown animals. Buds and zooids from control and RNAi knockdown colonies were subjected to surgical excision, RNA extraction, reverse-transcription followed by DNaseI treatment, and PCR using SYBR green detection. (more ...)
Histological analysis of colonies with definitive bud defects confirmed the in vivo
observations of failed organogenesis and revealed abnormalities in cellular morphology. In sections through primary buds of a colony on day 4 following MO injection, we observed an absence of organ structures () relative to stage B-1 buds of the synchronized control colony on day 4 (). At the time of injection, the stage C secondary buds of this colony were initiating organogenesis with inward folds in the atrial epithelium; two days later, at the onset of the new blastogenic cycle, the former secondary buds (now primary buds) were smaller than those of the dye-injected control clone () and failed to grow or develop in the following days. The inner (atrial) epithelial cells of the MO-treated primary bud appear squamous (), whereas secondary bud epithelia normally become cuboidal at the initiation of organogenesis in stage C-1 (; Berrill, 1941a
; Kawamura, 1984
). With respect to the known expression of Athena
late on day 6 as takeover begins, the absence of organ structures and cuboidal morphology of the epithelium implies that this bud underwent developmental regression following MO injection. Interestingly, protrusions on either side of the bud in may represent attempts at formation of the atrial folds that give rise to the neural complex and branchial sac.
Fig. 5 Morphological analysis of bud defects induced by Athena morpholino injection. (A) Primary bud section from individual with definitive phenotype on day 4 following morpholino injection. At the time of injection, the bud in panel (A) was undergoing organogenesis (more ...)
Taken together, these observations suggest that Athena provides a signal during the death of the old generation and takeover by the next generation of zooids that is critical to development of a new set of buds. As with much work in a nonstandard model organism, the questions raised outnumber the answers; however, with the establishment of techniques for genetic knockdown by MO or RNAi in adult Botryllus colonies, perhaps the most important result here is the introduction of new tools for future work.