In vertebrate species, induction of the embryonic axis is initiated by the transport of maternally supplied determinants, initially localized to the vegetal pole of the egg, toward the prospective organizer in the animal region. This transport process remains incompletely understood. Here, we review studies involving embryonic manipulations, visualization, and functional analysis of the cytoskeleton and loss- and gain-of-function conditions, which provide insights in this process. Transport of dorsal determinants requires cytoskeletal reorganization of a vegetal array of microtubules, microtubule motors, and an off-center movement of the vegetal cortex with respect to the inner egg core, a so-called cortical rotation. Additional mechanisms may be used in specific systems, such as a more general animally directed movement found in the teleost embryo. Initial polarity of the microtubule movement depends on early asymmetries, which are amplified by the movement of the outermost cortex. An interplay between microtubule organization and axis specification has also been reported in other animal species. Altogether, these studies show the importance of cytoskeletal dynamic changes, such as bundling, force-inducing motor activity, and regulated cytoskeletal growth, for the intracellular transport of maternally inherited factors to their site of action in the zygote.
- dorsoventral axis
- cortical rotation
One of the main events that take place during vertebrate development is the establishment of the dorsoventral (DV) axis. This process has been studied in a variety of vertebrate species, in particular in the amphibian
It has been shown that the process of transport of dorsal determinants is dependent on the microtubule cytoskeleton in the egg cortex, specifically on the reorganization of vegetal microtubules as long tracks of parallel bundles (Figure 1, left and center). In
This chapter reviews events involved in the cytoskeletal reorganization required for the movement of determinants leading to axis induction. The outcome of microtubule reorganization in the early embryo is the induction of the dorsal axis, and we first briefly review this process in the zebrafish as well as the amphibian
2. Induction signals for axis specification
A primary event in the establishment of the dorsoventral axis in zebrafish and
A key mediator of Wnt signaling, when localized to the nuclei, β-catenin acts as a transcriptional effector to activate dorsal-specific genes such as
The intricacies of the Wnt signaling pathway and its role in vertebrate axis induction have been determined through many studies, including functional manipulation of genes through ectopic expression and knockdown or expression of dominant-negative constructs (reviewed in Refs. [11, 17]). For example, overexpression of β-catenin induces a secondary axis in
Several mutations in zebrafish have allowed for the confirmation of an endogenous role for Wnt signaling pathway in axis induction in the early embryo. An identified recessive maternal-effect mutant in
Activation of the Wnt/β-catenin signaling pathway, as well as its important role in the expression of dorsal genes, has been extensively studied in a number of cellular systems (reviewed in Refs. [11, 17]). However, the identity of the molecules thought to activate the pathway in early vertebrate embryos, referred to as dorsal determinants, remains to be fully elucidated. Wnt11 has been proposed to be a dorsal determinant in amphibian species . In
3. Transport of dorsal determinants in
Xenopus and zebrafish
3.1. Molecular mechanism underlying cortical rotation
As mentioned above, embryological manipulations showed that, both in
Failure of cortical rotation results in a ventralized mutant phenotype in the embryo. However, in embryos treated to inhibit microtubules, a cortical rotation can be artificially induced by gravity after immobilizing the embryo in a matrix and physically turning it 90°. This manipulation results in the formation of dorsal structures, albeit delayed . Under these conditions, gravity leads to a rearrangement of the heavier yolk-containing core of the embryo relative to the cortex. This is thought to increase the proximity of vegetally localized cortical signals to internal regions in the more animally located prospective dorsal region. The ability of the entire cortex to move as a whole relative to the embryonic core contrasts with the visualization of moving particles along microtubule tracks. These observations suggest that both transport along cortical microtubules and a cortical shift relative to the embryonic core contribute to the redistribution of signals involved in axis induction during the early embryonic cell cycles. We subsequently address each of these processes.
3.2. Relocalization of RNA determinants during oogenesis and early embryogenesis
The mRNA for the putative zebrafish dorsal determinant
In addition to
Positional cloning of
Consistent with the effect of maternal-effect mutations in
Genetic analysis indicates that the Hecate/Grip2a and Tokkaebi/Syntabulin products are required for the off-center, asymmetric shift of vegetally localized determinants that follows fertilization.
Additional studies have shown that, as in
Interestingly, the RNA for the
Altogether, these studies indicate key roles for RNA localization pathways during oogenesis leading to the localization of factors required for axis induction to the vegetal pole of the egg. Initially localized to the vegetal pole through the action of the mitochondrial cloud during oogenesis, after fertilization and egg activation these factors exhibit an off-center shift dependent on the function of vegetally localized factors, such as Grip2a and the kinesin motor adaptor protein Syntabulin.
3.3. Reorganization of microtubules during cortical rotation
At least in the case of
In the early
A morphologically apparent cortical rotation, observed through changes in the position of an outer cortex relative to an inner core as observed in
A cortical rotation-like process is also consistent with differences in the changes in RNP particle distribution at different cortical depths, as visualized by fluorescent in situ hybridization, since RNPs located at the outermost cortex undergo a spatial shift with respect to more internally located RNPs (Figure 3E) . To understand the basis of transport for differentially localizing factors at the zebrafish vegetal-most embryonic cortex, double fluorescence in situ hybridization (FISH) was used to detect pairs of RNAs for factors involved in axis induction (
Thus, both in amphibians and teleost, an array of aligned microtubules is associated with the movement of RNA molecules and the vegetal cortex itself with respect to the inner egg core, which altogether mediates the transport of dorsal determinants toward the prospective dorsal site.
3.4. Long-range vs. short-range transport
The second step involves a long-range transport along the mediolateral region of the embryo to the base of the blastomeres by a mechanism that is neither restricted to the dorsal side nor dependent on Grip2a function [6, 26]. The presence of such a second transport mechanism can be inferred by the observation that
3.5. Other factors involved in vegetal microtubule reorganization
Additional factors have been identified to be important for dorsal axis induction. A mutation in the maternal-effect mutant
Studies have also revealed that an ubiquitin ligase,
The mRNA for
Thus, a variety of factors are required for the reorganization of vegetal cortex microtubules leading to dorsal determinant transport. In some cases, these factors are important for general processes essential for the microtubule reorganization, such as in the case of
3.6. Mechanism of microtubule alignment during cortical rotation
Even though it has been shown that microtubule-dependent cortical rotation is important for axis formation, the molecular mechanisms underlying the organization and orientation of cortical microtubule have not been fully elucidated. The process of cortical rotation is highly conserved, and it likely requires the embryo to use a significant amount of energy. Weaver and Kimelman  asked the question that if dorsal determinants can travel along microtubules, then what is the purpose of the cortical rotation? As described above, cortical rotation might directly contribute to the overall animally directed movement of the dorsalizing activity. However, studies have also suggested that cortical rotation might serve to facilitate aligning the polymerizing microtubules into parallel bundles and orienting their plus ends toward the dorsal side. One favored model for the orientation of the microtubule array is a positive feedback mechanism where initial random asymmetry in microtubule growth is amplified by continuous movement of the cortex [31, 58].
Microtubules that form the vegetal microtubule array appear to arise from several sources . Some are nucleated by the centriole of the sperm, which acts as a minus-end microtubule-organizing center, others extend toward the periphery from unknown sources deep in the cytoplasm and bend into the vegetal shear zone, and, finally, some arrays appear to polymerize spontaneously in the vegetal shear zone [49, 50]. As the vegetal microtubule array begins to form, it becomes progressively stabilized by movement of the cortex during cortical rotation, which provides an amplifying loop for microtubule alignment . The precise manner by which this cortical movement contributes to microtubule alignment and stabilization is not fully understood. Suggested mechanisms, described below, include a combing process mediated by cortically anchored kinesin-related proteins [54, 70] or the stabilization of microtubules by membrane compartments such as the endoplasmic reticulum and vesicles .
Vegetal microtubules originally appear with their plus ends in a random orientation yet subsequently become aligned in parallel arrays with plus ends directed toward the dorsal side (Figure 4) (reviewed in Ref. ; see also Ref. ). Marrari and colleagues suggested how microtubules could become aligned through cortical motor proteins and the process of the cortical rotation  (reviewed in Ref. ). They proposed that cortically anchored plus-end-directed motor proteins, such as kinesins, move toward microtubule plus ends, generating a cortical displacement with respect to the inner core [47, 54, 71]. The attachment of plus ends to the moving cortex mediates aligning of microtubules in the same direction. Thus, the movement and action of these kinesin-related proteins could potentially align the microtubules as well as generate the pulling force that is needed to translocate the cortex relative to the cytoplasm [54, 70]. This positive feedback loop also allows amplifying an original small asymmetry into the observed prominent array of parallel microtubule bundles.
Marrari and colleagues also investigated the role of kinesin and dynein motors in the formation of the cortical microtubule array as well as their role in the translocation of the vegetal cortex [47, 54, 71]. The function of kinesin was inhibited using an antibody against a highly conserved peptide of the kinesin motor domain, LAGSE. Anti-LAGSE antibodies block spindle elongation in semi-in vitro systems [47, 54, 71, 72] and successfully interfere with kinesin function [47, 54, 71]. The function of dynein was inhibited by microinjection of p50/dynamitin beneath the vegetal cortex . In
Inhibition of kinesin-related function results not only in expected defects in mitosis and cell cleavage but also in disruptions in the array of vegetal microtubules and cortical rotation . On the other hand, inhibition of dynein causes an inward shift in the distribution of microtubules with respect to the cortex, indicating that dynein functions to move microtubules outward, into the vegetal subcortical layer . Moreover, these experiments showed that the formation of the vegetal microtubule array (and therefore cortical rotation) is sensitive to dynein inhibition prior to array formation, but that cortical rotation remains sensitive to inhibition of kinesin function throughout the normal period of rotation . Together, these data suggest that kinesin and dynein motors have different functions during cortical rotation (Figure 5) . In this model, dynein motors anchored to internal elements generate an outward force to facilitate bringing microtubules from the inner egg core region to the vegetal cortex. Kinesins, on the other hand, are thought to act by tethering microtubule plus ends to the cortex, thus generating a pulling force on microtubule arrays, mediating the rotation of the cortex itself, and favoring further parallel alignment of microtubules within the array. It is important to note that, after the vegetal microtubule array has formed, further microtubule alignment and cortical rotation can occur independent of dynein function, but motors of the kinesin-related protein family are needed for the movement of the cortex . Thus, kinesin motor function appears to be essential for
Olson and colleagues performed experiments that would characterize microtubule plus-end dynamics in
It was previously noted that the direction of microtubule polymerization in cultured cells depends on the arrangement of elongated tubes of endoplasmic reticulum . Endoplasmic reticulum, vesicles, and tubes possess kinesin-like microtubule-associated proteins that associate with microtubules during transport and elongation, and it is possible that similar membrane organelles are attached to the vegetal cortex and facilitate kinesin-mediated anchoring of microtubules during cortical rotation . A precedent for this is the association of cortical ER with aligned microtubules in early ascidian embryos (see below) . Further studies will be required to address a potential role for membrane organelle attachment in
Studies in zebrafish are consistent with mechanisms for cortical microtubule array formation and alignment as detailed in amphibians, including the presence of early internal microtubules, increase in cortical microtubule polymerization concomitant with microtubule alignment and bulk movement of the cortex, and the aligned orientation of microtubule plus ends toward the prospective dorsal site .
Altogether, these studies suggest that the formation of the vegetal microtubule array is dependent on the orchestration of various influences, including dynein-dependent outward translocation of existing microtubules, kinesin-dependent vegetal anchoring of cortical microtubules, and microtubule polymerization at the vegetal cortex. Vegetal microtubule and cortical rotation are interdependent and enhance each other, resulting in the alignment of preexisting and new microtubules and allowing dorsal determinant transport.
4. Cortical rotation and cytoskeletal dynamics in invertebrate and protovertebrate systems
As described above, a cortical rotation process has been described in amphibians, and a related process proposed in teleosts. However, other studies have described processes of cytoskeletal reorganization that serve a similar purpose as the cortical rotation, namely, the early distribution of cellular determinants that will help pattern the egg or embryonic axis. We briefly discuss three such examples below, in ascidians (a chordate protovertebrate), the nematode
In ascidians, gastrulation and neurulation involve cellular rearrangements that are comparable to those in vertebrates, with the exception that ascidians are composed of just a few hundred cells, while vertebrate embryos contain thousands of cells . In fact, the very first classical evidence that localized determinants control cell fate specification was found in ascidians [82, 83].
The ascidian egg undergoes dramatic cytoplasmic and cortical reorganizations between fertilization and the beginning of the first cleavage, a process that has been referred to as ooplasmic segregation [83–85]. Ascidian ooplasmic segregation occurs in two major phases (Figure 1, right). The first phase occurs shortly after fertilization. The first consequence of fertilization is that a calcium wave is initiated from the site where the sperm and egg fuse . Upon fertilization, the sperm activates the stage IV oocyte, which was arrested in metaphase I of meiosis, resulting in the contraction of the egg cortex and the plasma membrane as a wave that travels across the egg in the animal to vegetal direction. It was suggested early on that an oocyte actomyosin cortical network can only contract in a general animal to vegetal direction regardless of the sperm entry site, because of it being less dense around the animal pole, in a basket-like arrangement [86, 87]. This animal-to-vegetal contraction in turn causes the segregation of cortical and subcortical components including microfilaments, mitochondria, and the cortical endoplasmic reticulum (cER) [77, 88, 89].
Unfertilized eggs after the first phase of ooplasmic segregation are radially symmetrical along the animal-vegetal (A-V) axis. This symmetry is broken in the second phase of reorganization after the movement of cortical and subcortical components from the vegetal pole toward the posterior pole occurs, generating an anteroposterior asymmetry, and eggs become bilaterally symmetrical . In this second ooplasmic segregation phase, a number of cellular organelles such as the ER and mitochondria are brought toward the future posterior pole . These organelles also anchor specific RNAs, termed postplasmic/PEM, which are important for muscle determination and the specification of the posterior cell fate, in particular the germ line . Other factors involved in endoderm formation and gastrulation do not move toward the future posterior pole and instead expand their distribution to the vegetal hemisphere (see Figure 1) . Reminiscent of asymmetry development in
Ascidian embryonic polarity is directed by a posteriorly located centrosome, introduced through sperm entry in this region [77, 93, 94]. In contrast to the first phase which is driven by microfilaments, and where the sperm triggers a cortical contraction , the second phase is mediated by anchoring one of the centrosomes of the bipolar spindle to the vegetal posterior cortex, resulting in the posterior asymmetric localization of germ line-determining components. Spindle pole posterior anchoring also results in the eccentric, posteriorly located placement of the spindle, which in turn (because of the influence of the spindle midzone on furrow induction) [51, 52], results in asymmetric division [75, 77]. In this manner, the embryo generates sets of smaller posterior cells fated to become the germ line.
Thus, in both
In the nematode
Altogether, these studies show that the microtubule cytoskeleton, and in some cases the actomyosin cortex, is used to generate axis asymmetry in various organisms, although the precise details of the interactions, and whether microtubules act as tracks that mediate transport or attraction centers, are specific to different species [97, 104].
5. Relationship between axis induction and germ cell specification
As mentioned above, in addition to dorsal determinants, anuran and teleost embryos contain other vegetally localized factors, particularly RNAs that become associated with the germplasm. The germplasm, also referred to as nuage, is a maternally inherited cytoplasmic structure containing RNPs present in some animal species. Through a mechanism referred to a preformation, inherited germplasm determines the germ cell fate . Evidence for preformation mechanism for PGC induction in anurans was originally shown by the inheritance of electron-dense cytoplasm, corresponding to germplasm, into the primordial germ cells of this organism . This electron-dense cytoplasm was later shown to contain specialized mRNAs involved in germ cell specification . Similarly, RNAs involved in germ cell development in zebrafish, such as for the gene
Maternally inherited germplasm in
Recent studies in these systems have begun to suggest a functional connection between axis induction and germ cell determination. As described above, during oogenesis both dorsal determinants are transported to the vegetal pole of the egg through the mitochondrial cloud in
Conversely, factors known to be involved in dorsal axis induction also function in germ cell development. One example is maternal Syntabulin, which as mentioned above is important for vegetal microtubule array reorganization and axis induction in both zebrafish and
It is important to note, as stated above, that there is a difference with respect to cortical depth between the factors that are localized to the vegetal pole. Those that are important for microtubule reorganization, and thereby patterning the embryonic axis, namely,
These set of studies highlight commonalities between processes and factors involved in axis induction and germ cell specification. Factors such as Dead end, Grip2, and Syntabulin may form a core gene set with a current or ancestral function in both axis induction and germ cell determination.
6. Conclusion: challenges and future directions
The cytoskeleton plays an essential role in axis specification, through its role mediating the movement of maternal factors within the early zygote. Studies have shown that the reorganization of the microtubule cytoskeleton is important for the transport of factors from the vegetal pole of the embryo to the future dorsal side in both zebrafish and
We would like to thank the members of the Pelegri Lab for feedback and encouragement. This work was supported by NIH grants 5RO1GM065303 and 3RO1GM65303-10A1S1.Elaine L. Welch received additional support from NIH Training Grant 2 T32 GM007133 to the Laboratory of Genetics.
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