Basal bodies in Xenopus
© Zhang and Mitchell. 2016
Received: 13 November 2015
Accepted: 20 January 2016
Published: 3 February 2016
Xenopus has been one of the earliest and most important vertebrate model organisms for investigating the role and structure of basal bodies. Early transmission electron microscopy studies in Xenopus revealed the fine structures of Xenopus basal bodies and their accessory structures. Subsequent investigations using multiciliated cells in the Xenopus epidermis have further revealed many important features regarding the transcriptional regulation of basal body amplification as well as the regulation of basal body/cilia polarity. Future basal body research using Xenopus is expected to focus on the application of modern genome editing techniques (CRISPR/TALEN) to characterize the components of basal body proteins and their molecular functions.
KeywordsXenopus Basal body Centriole Deuterosome Ciliogenesis Cilia Multiciliated cells
The term Xenopus refers to a collection of approximately 20 fully aquatic frog species within the genus Xenopus. In scientific classifications, they belong to Kingdom Animalia, Phylum Chordata, Class Amphibia, Order Anura, and Family Pipidae. The early Xenopus embryo, due to their large size and free development outside the mother’s body, have been one of the most important models for the investigation of early vertebrate development as well as basic biology for many years . Initial investigations using the Xenopus species, which can be dated back to the mid-late 1800s, are mainly restricted to the larger, easy-to-handle X. laevis strain. However, a genome duplication event during the evolution of X. laevis has been discovered, which indicates that X. laevis is a pseudotetraploid species with genetic redundancy . This has prevented detailed genetic studies to be performed on the Xenopus species. However, a diploid Xenopus system X. (Silurana) tropicalis with a much smaller genome size was introduced allowing detailed genetic manipulations to be performed in Xenopus [3, 4]. Recently, the full genome sequence of both X. laevis and X. tropicalis has been released to provide the basis for advanced genomic manipulations, such as CRISPR/Cas9 and/or TALEN, in addition to the traditional morpholino oligo (MO) knockdown approach .
Basic basal body structure
The basal bodies of Xenopus MCCs can be labeled by the strong presence of γ-tubulin during both live imaging and immunostaining . Several additional tubulin genes, in addition to the α-, β-, and γ-tubulin genes found in most organisms, have been identified in the Xenopus system. The δ-tubulin gene (tubd1, tubulin, delta 1) gene has been identified in X. laevis by the Stearns lab . The ε-tubulin (tube1, tubulin, epsilon 1) gene has also been identified in X. laevis for its roles in centriole duplication and microtubule organization [11, 12]. In addition, a special tubulin gene, ζ-tubulin (tubz1, tubulin, zeta 1), has also been characterized as an important component of the basal foot in MCCs. In contrast, in cycling cells, ζ-tubulin does not locate to centrioles but rather associates with the TRiC/CCT cytoplasmic chaperone complex in the cytoplasm . Interestingly, from an evolutionary point of view, the latter three tubulin families form a co-conserved module, named the ZED module. This ZED module has been independently lost in several branches of the evolution tree, such as in higher fungi, higher plants, and placenta mammals. It is also important to note that for the species that possess the ZED module, ε-tubulin gene is always present, while there is a chance of losing either δ- or ζ-tubulin, but not both . It has been proposed that the presence of the ZED module may be essential for the formation of centriolar appendages; however, further investigations will be required to resolve this question.
Additional basal body structures or accessory structures
The origins of basal body
The basal bodies in Xenopus are both converted from centrioles as well as built de novo depending on the cell type. For cells that generate a single cilium, such as cells found in the GRP and gut, basal bodies are converted from the mother centriole similar to other systems . While cycling cells contain both a mother and a daughter centriole, typically it is only the older “mother” centriole that has gone through a full cell cycle that is competent to become a basal body. Interestingly, ectopic over-expression of Foxj1, a protein that plays a crucial role during the differentiation and maintenance of ciliated cells, is able to drive basal body conversion inducing the formation of 1–2 cilia per cell when expressed in non-ciliated epithelial cells . This phenomenon suggests that, in the Xenopus skin, both the mother and daughter centrioles may maintain a certain level of basal body competency. In MCCs that generate dozens of basal bodies, the process appears to be quite distinct. Instead of nucleating from an older “mother” centriole, the vast majority of basal bodies nucleate from a structure termed the deuterosome . The regulation of this process is still poorly understood but clearly requires the key centriole duplication regulating proteins Plk4 and Cep152 [30, 31]. Remarkably, while these cells are post-mitotic, the nascent centrioles are immediately competent to become cilia-nucleating basal bodies without going through a cell cycle. As soon as centrioles are generated, they begin their migration to the apical cell surface and immediately initiate cilia formation. How this centriole-to-basal body conversion is regulated remains a mystery.
While the structures of centrioles and basal bodies are generally comparable, there are important structural and functional distinctions between them. For example, microtubules (part of the ciliary axoneme) directly and specifically emerge from the distal end of the basal body, whereas cytoplasmic and mitotic microtubules nucleate in all directions from the pericentriolar material surrounding the centriolar pair of the centrosome. In addition to the basal body-specific appendages detailed above, another important distinction between centrioles and basal bodies is their relationships with cell membranes. Basal bodies associate with membrane-bound vesicles as the vesicles migrate to and fuse with the apical cell membrane . This membrane association is critical to basal body function and components of the basal body-linked transition zone and is thought to regulate distinct membrane compartments. More detailed reviews on this topic, including the structural and functional differences between ciliary membrane and cell membrane, are provided in [32, 33].
The life cycle of basal body and its other functions
Identification of basal body components
To date, no studies that systematically address the protein components of Xenopus basal bodies have been identified. However, numerous proteins are known to localize to basal bodies, including many proteins that localize to centrioles in other systems, and the components seem quite comparable to other vertebrate (and non-vertebrate) centrioles. In addition, gene expression analyses in MCCs indicate the up-regulation of many centriolar components that are most certainly contained in the basal bodies of motile cilia [29, 34, 41–43]. While many centriolar components (e.g., Centrin, Poc1, HYLS, Sas6, Plk4, Cep152, and numerous others) appear similar to all centrioles, other components (e.g., Dvl2) likely represent unique features of multiciliated basal bodies.
Notable basal body findings
The ciliated epithelium of Xenopus has proven to be a particularly powerful system for the study of cilia and basal bodies (reviewed in [6, 44]). Specifically, this system has provided the first evidence of PCP signaling and fluid flow affecting cilia/basal body polarity [24, 25, 45], the first evidence of a septin-based cilia diffusion barrier , the characterization of MCC-specific transcriptional regulators [29, 41–43, 47], the first characterization of miRNA-mediated regulation of basal body duplication and ciliogenesis [48, 49], and the first molecular characterization of the basal body-generating structure the deuterosome . These and many other important discoveries were facilitated by the molecular, embryological, and imaging techniques that are available in Xenopus coupled with the fact that the ciliated epithelia develop on the external surface of the embryos rather than inside the organism. Notably, the discoveries in Xenopus have been validated in other vertebrate systems [50–53]. In addition, many human genetic defects have been authenticated and more thoroughly characterized using the tools available in Xenopus [54–56].
Strengths and future of basal body research in Xenopus
It is a very exciting time to be using Xenopus as a model system to study basal bodies. Recent advances in the detailed quantification of both protein and RNA levels across early Xenopus development stages promises to facilitate the analysis of many developmental processes including basal body formation and functions . In addition, recent advances in genome editing technologies including TALENs and CRISPR/Cas hold great potential to allow rapid analysis of genetic mutations . Specifically, the ability to couple CRISPR/Cas with homologous recombination to insert either fluorescent markers or specific mutations will greatly enhance our ability to model human disease in Xenopus. Important questions that remain to be answered are as follows: how are centriole amplification and centriole-to-basal body conversion regulated in MCCs that are no longer progressing through the cell cycle; what is the driving force of apical migration/insertion of basal bodies; how do basal bodies and their accessory structures interact with the cytoskeleton as well as with the cell cortex; and what are the similarities/differences between the basal bodies of motile and primary cilia. With these new tools in hand, the next few years will certainly lead to many new advances in our understanding of basal body formation and functions.
transmission electron microscopy
gastrocoel roof plate
microtubule organizing center
planar cell polarity
SZ and BJM contributed to writing this review. Both authors read and approved the final manuscript.
We would like to thank Sun Kim and Jennifer Mitchell for helpful comments on the manuscript. BJM was supported by NIH-NIMGS 2R01GM089970.
The authors declare that they have no competing interests.
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