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Review

Establishing the plane of symmetry for lumen formation and bilateral brain formation in the zebrafish neural rod Clare Buckley, Jon Clarke ∗ MRC Centre for Developmental Neurobiology, King’s College London, London SE1 1UL, UK

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Article history: Available online xxx Keywords: Zebrafish Neural tube Cell division Centrosome

a b s t r a c t The lumen of the zebrafish neural tube develops precisely at the midline of the solid neural rod primordium. This process depends on cell polarisation and cell rearrangements, both of which are manifest at the midline of the neural rod. The result of this cell polarisation and cell rearrangement is an epithelial tube that has overt mirror-symmetry, such that cell morphology and apicobasal polarisation are mirrored across the midline of the neural tube. This article discusses how this mirror-symmetry is established and proposes the hypothesis that positioning the cells’ centrosomes to the midline of the neural rod is a key event in organising this process. © 2014 Published by Elsevier Ltd.

Contents 1. 2. 3. 4. 5. 6.

7. 8.

Symmetry and the neural tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Establishing the plane of symmetry in the neural rod . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mirror-symmetric divisions across the plane of the midline drive cell rearrangement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell division and mirror-symmetric polarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mirror symmetric polarity without cell division: a role for centrosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Positioning the centrosome ‘mirror’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Role of the basal lamina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Role of Cadherin based adhesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Role of polarity proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A working hypothesis for the zebrafish neural rod . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why is symmetry important? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Symmetry and the neural tube The zebrafish brain and spinal cord is built from a bilaterally symmetric neural tube. The basic symmetric shape and the balance between cell numbers on the left and right sides of the neural tube will be critical for the formation of a correctly functioning bilateral brain and spinal cord. The embryo must therefore employ mechanisms that ensure this symmetry develops correctly and is maintained during the process of neural lumen formation. Throughout our studies of zebrafish neural tube formation we have

∗ Corresponding author. Tel.: +44 20 7848 6463. E-mail address: [email protected] (J. Clarke).

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been struck by the role that symmetry may play in defining where the lumen forms within the initially solid neural rod. We originally noted that a specialised mode of cell division generated mirrorsymmetric daughters around the plane of the nascent lumen [1]. More recently we described how the plane of the nascent lumen appears to organise a mirror-symmetric microtubule cytoskeleton within individual cells that transiently span the midline of the neural rod [2]. In this article we will attempt a synthesis of our data and ideas about how symmetry is generated during lumen formation. Bilateral symmetry is the most apparent type of symmetry seen in animal body plans. To identify structures with bilateral symmetry, you need to define a plane of reflection. As pointed out by Weyl [3] in the diagram in Fig. 1, the two points P and P mean nothing in relation to each other until their symmetry is revealed by

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Please cite this article in press as: Buckley C, Clarke J. Establishing the plane of symmetry for lumen formation and bilateral brain formation in the zebrafish neural rod. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.04.008

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Fig. 1. The points P and P can only be understood as symmetric when described in relation to the dotted line which is the plane of reflection (PoR). Diagram adapted from Weyl [3].

the plane of reflection PoR [3]. Such a plane of reflection or ‘mirror’ clearly develops at the midline of the zebrafish neural rod prior to lumen formation. This is recognisable both molecularly as the proteins required to assemble the lumen surface accumulate at this plane, and also (slightly later) morphologically as an interface between cells on the left and cells on the right side of the rod (Fig. 2). We propose that, during morphogenesis of the neural tube, cells must recognise where to assemble this plane of reflection (in other words identify the midline of the neural rod) and then organise their shape, their internal cytoskeleton and their cell–cell junctions in order to arrange themselves mirror-symmetrically around that plane. This raises the following questions: how is the mirror placed in the correct location and how do cells respond to it in order to generate symmetry around it? Teleost neurulation provides a tractable model in which to investigate this question. Recent work in our lab has led us to believe that the establishment of a plane of reflection (mirror-symmetry) occurs at subcellular levels, is defined by integrating cell–cell and cell–tissue interactions and provides one of the keys to the establishment of bilateral symmetry at the tissue level in the zebrafish central nervous system [2].

2. Establishing the plane of symmetry in the neural rod The neural rod of the zebrafish embryo develops from the neural keel after the cells of the neural plate have converged towards the dorsal midline of the embryo [4,5]. Throughout the plate and keel stages the neural primordium is a bilaterally symmetric structure but there is not yet a precise morphologically identifiable plane of symmetry at the midline, since, even after cells have converged at neural keel and early rod stages, cells from the left and right sides interdigitate across the midline. We suggest that at these early stages the neural primordium is bilaterally symmetric but not yet precisely mirror-symmetric. The interdigitation of cells across the

midline must be resolved by cell rearrangements before the left and right sides can separate to form a lumen via tissue cavitation. A distinct morphological plane of symmetry thus only appears in the neural rod as its cells rearrange themselves into two columns of elongated cells that meet at the midline plane in preparation for lumen formation (Fig. 3). At the same time as cells are resolving their interdigitation they are transforming into an apicobasally polarised epithelial tube. Thus cell rearrangement and apicobasal polarisation appear to be intimately linked, and this results in our ability to recognise the plane of mirror symmetry not only by the morphological interface between cells of the left and right sides of the rod, but also by the molecular assembly of polarity proteins such as Pard3 at this interface [1].

3. Mirror-symmetric divisions across the plane of the midline drive cell rearrangement One of the characteristic cell behaviours that occurs at the plane of reflection in the zebrafish neural rod is cell division, and a number of studies support the view that cell division plays a dominant role in establishing mirror symmetry and organising lumen formation in this system. Cell division is in many ways a mirror-symmetric event. An early example of this is given by Guenter Albrecht-Buehler who studied 3T3 cells dividing in culture and found that a significant proportion of daughter cells have mirror-symmetric patterns of actin bundles and moved mirror symmetrically after completing division [6]. He hypothesised that there might be a “relationship between the universally found bilateral symmetry of organisms and the mirror symmetry between certain daughter cells” [6] but he could not suggest a way in which this mirror-symmetry at the cellular level could be transferred to and maintained at the tissue level, stating that “the perfect mirror-symmetry of the mitotic spindle can hardly be expected to

Fig. 2. Four frames from a time-lapse movie showing neural rod development in the transverse plane. In A cells from left right sides of the rod interdigitate across the neural midline (arrowed) and a plane of reflection is not visible. By frame D the cells have rearranged such that left and right cells now meet at the midline rather than intersecting it. Thus by frame D the plane of reflection is visible as this morphological interface.

Please cite this article in press as: Buckley C, Clarke J. Establishing the plane of symmetry for lumen formation and bilateral brain formation in the zebrafish neural rod. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.04.008

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Fig. 3. Two frames from a time-lapse of an embryo in which a small number of cells are labelled in order to show individual cell morphologies before (A) and after (B) cell rearrangements at the midline. These cells express Pard3-GFP illustrating that after rearrangement at the midline this polarity protein is clearly polarised in cells to the point where they meet at the midline (arrowed). Dotted lines mark the left and right basal surfaces of the neural rod.

persist for long”. A more molecular analysis of polarity through cell division in Dictyostelium shows that the nascent daughters are mirror-symmetrically polarised during mitoses such that the tumour suppressor PTEN locates to the cleavage furrow and PI3 kinases locate to the opposite poles of the nascent daughters [7]. That cell division contributes to organ symmetry in the zebrafish neural tube was revealed in 1994 when Kimmel and colleagues demonstrated that the midline division in the neural rod was responsible for generating clonally related clusters of cells with striking bilateral symmetry across the midline of the developing neural tube [4]. This demonstrated that the descendants of a cell division can indeed maintain their mirror-symmetric relationship way beyond cytokinesis and contribute to the mirror-symmetry of the developing brain. 4. Cell division and mirror-symmetric polarity How might the mirror-symmetry of division be transformed into organ symmetry in the zebrafish neural rod? Almost all cell divisions are founded on inherently symmetric events, with their mirror-symmetric spindles organising the symmetric inheritance of many basic nuclear and cytoplasmic contents. It is clear that the daughters of the midline division described by Kimmel et al. [4] are morphologically mirror-symmetric around the midline plane [1,5,8]. Apicobasal polarity was monitored using the Pard3-GFP fusion protein which in some midline divisions accumulates close

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to or on either side of the cleavage furrow and in most cells quickly accumulates on either side of the intercellular interface that is maintained between the two sisters after mitosis [1,2]. During the midline division the cell maintains its connection to the basal surface of the rod and this is inherited and maintained by the ipsilateral daughter, while the contralateral daughter stretches across to the contralateral basal surface and maintains an interface with its sibling at the midline. The nature of this sisterly interface is currently unknown, but timelapse analyses suggest the sisters adhere to one another for up to several hours before they separate as the lumen opens [1]. Throughout this period the accumulation of Pard3-GFP is maintained on either side of the interface and presumably marks the location at which adherens junctions and tight junctions are being assembled to generate the two apical surfaces of the lumen. Thus the midline division is a mirror-symmetric division that organises cell rearrangements and cell morphology as well as cell polarisation around the midline. Evidence that this mirror-symmetric midline division is a dominant morphogenetic influence during lumen formation comes from the analyses of several mutations that interfere with this division. The mirror-symmetric division appears to be regulated by a timing mechanisms that ensures that it occurs “on time” in normal embryos [9]. Therefore, when convergence of the neural plate cells to the midline is delayed, for example in mutants of the planar cell polarity pathway, the mirror-symmetric divisions still occur “on time” but now in ectopic locations lateral to the actual organ midline. This results in the formation of ectopic lumens on either side of the midline and effectively generates a double neural tube phenotype [1]. Similarly, misorientation of the mirror-symmetric division’s spindle via the abrogation of proteins such as Scribble, N-cadherin [10], Frizzled-7 [11] or regionally specific Hoxb1b [12] results in severe neural rod midline defects and lumen malformations. Since these malformations are all corrected by inhibiting cells from entering division these results demonstrate the instructive and dominant nature of the midline crossing division in neural lumen development. These results show that cell mitosis at the centre of the zebrafish neural rod is one mechanism that, by restricting apical proteins to the interface between daughters, is able to localise apical proteins to the tissue midline. In this way mitosis organises the apico-basal polarity and appropriate orientation of neuroepithelial cells. Although the midline division generates a mirror-symmetric epithelial tube, individual cells are highly polarised (i.e., asymmetric) with distinct apical and basal specialisations. The establishment of left-right mirror-symmetry in the zebrafish neural rod may be a specialised example of lumen formation in which left-right symmetry is particularly obvious. However a general and dominant role of mitosis in organising apical polarity domains and lumen formation has also been suggested in other systems. For example, the Rho GTPase Cdc42 is important in establishing epithelial apical polarity in many systems [13–15]. However, depletion of Cdc42 has also been shown to result in mitotic spindle misorientation, which, due to the consequent mislocalization of apical proteins, is thought to be partly responsible for the abrogation of lumen formation independently of its role in apical complex formation. This has been shown in both the Caco2 human intestinal epithelial cell line [16] and in Madin-Darby Canine Kidney (MDCK) cysts [17] and is thought to be mediated via Pard6B and aPKC [18,19]. A study in MDCK cells has demonstrated that aPKC localisation is dependent on Pard3 and orients spindles by phosphorylating, and therefore excluding, Pins from the apical cortex [20]. Depletion of Cdc42-specific guanine nucleotide exchange factors (GEFs) such as intersectin (ITSN2) [17] and Tuba [19] result in similar mitotic spindle and lumen morphogenesis phenotypes. Misorienting the spindle by abrogation of unrelated proteins such as syntaxin 16 [21] and LGN [17] also correlate with lumen defects,

Please cite this article in press as: Buckley C, Clarke J. Establishing the plane of symmetry for lumen formation and bilateral brain formation in the zebrafish neural rod. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.04.008

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supporting the notion that mitotic spindle orientation plays an instructive role in polarity establishment and morphogenesis.

5. Mirror symmetric polarity without cell division: a role for centrosomes The discussion above illustrates that in the zebrafish neural rod a specialised cell division has a dominant role in organising cell polarisation, lumen formation and symmetry. In other contexts mitoses and spindle orientation are also shown to play a role in lumen formation. However, this does not mean that mitosis is necessary for polarisation and lumen formation. In the context of the zebrafish neural rod, if the mirror-symmetric division is specifically prevented, the midline is still able to form [1,2,8,10,11]. This demonstrates that, although sufficient, the midline division is not necessary for organising neuroepithelial polarity. There must therefore be other underlying mechanisms that can generate mirror-symmetry around the midline of the neural rod. It is possible that such a division-independent mechanism of polarisation may also be responsible for determining that the mirror-symmetric division occurs close to the midline. So what could the divisionindependent plane of reflection be? A clue to the division-independent mechanism comes from our recent demonstration that the microtubule cytoskeleton of cells during interphase is organised mirror-symmetrically around the point at which these cells intersect the zebrafish neural rod midline. This occurs in advance of mitosis and, since this is also apparent in cells in which division has been arrested in G2-phase, is also independent of mitosis [2]. Furthermore proteins important for lumen formation, such as Pard3 and Rab11a, are able to localise to the point where division-blocked cells intersect the midline, which is where the microtubule cytoskeleton is reversed. Experimental depolymerisation of the microtubule cytoskeleton in division-blocked cells mislocalises these proteins to the basal ends of neuroepithelial cells, demonstrating that the microtubule cytoskeleton is necessary for correct apical protein trafficking independently of mitosis [2]. This work shows that cells are able to detect the point where they intersect the midline of the neural rod and traffic apical proteins to that point via a microtubule dependent mechanism. To our knowledge, this is the first example of the initiation of epithelial apical domains part way along a cell, rather than at the cell extremity. EB3 imaging demonstrates that the mirror-symmetric microtubule array is nucleated close to whichever part of the cell intersects the centre of the developing organ [2]. The most likely candidate for the Microtubule Organising Centre in zebrafish neuroepithelial cells is the centrosome. In polarised epithelial cells, the centrosome plays an important role in the organisation and nucleation of microtubules, even when microtubules become gradually organised at non-centrosomal junctional locations [22,23]. During zebrafish neural keel to rod transition, centrosomes gradually move from lateral to medial positions [24]. More recently, we have shown that this medial centrosomal movement is directed specifically to the centre of the neural rod. Thus, although many cells span the midline the movement of the centrosome stops at the point in the cells where they intersect the midline. We also showed that localisation of centrosomes to the midline occurs in advance of the midline division [2]. We therefore suggest that the correct localisation of the centrosome at the midline of the zebrafish neural rod is responsible for co-ordinating two critical aspects of lumen formation. One is the establishment of a mirror-symmetric microtubule cytoskeleton within individual cells at the point where they intersect the midline; this enables microtubule dependent Pard3 and Rab11 proteins to be delivered to the organ midline. Second the midline centrosome initiates construction of the mitotic spindle at the midline, thus positioning the dominant mirror-symmetric

division to the plane of lumen formation. The midline centrosome could thus direct the assembly of polarity proteins to the midline prior to division as well as reinforcing lumen formation at this location by nucleating the division at this position. In this way, the centrosome acts as the mirror for microtubule directed transport and for organising mirror-symmetric daughter organisation via the midline division.

6. Positioning the centrosome ‘mirror’ If centrosomal positioning is key to establishing the correct localisation of polarity proteins such as Pard3, how does the centrosome localise specifically to the midline of the neural rod? The short answer is we do not yet know the full story, but data from other systems as well as our own work demonstrates that at least three factors may be important. One is the influence of the basal lamina at the superficial surface of the neural rod, a second is the influence of Cadherin-based adhesions around the midline of the rod, and the third is the early deposition of polarity proteins such as Pard3 and aPKC around the midline.

6.1. Role of the basal lamina Many studies have shown the importance of extracellular matrix (ECM) proteins, such as Laminin and ␤1 Integrin, in orienting apico-basal polarity within epithelial tubes. MDCK studies have demonstrated that Laminin assembly at the basement membrane is necessary for apicobasal polarity axis organisation and is dependent on signalling by the small GTPase Rac1 [25]. ␤1 Integrin interaction with collagen has been implicated to initiate Rac1 pathway signalling [26] and this activation is thought to be specifically attributable to ␣2␤1 and ␣6␤4 Integrins [27]. Rac1 is implicated in many downstream signalling pathways, which may mediate its effects on apicobasal polarity. For example, Rac1 activates PI 3kinase/Akt1 during MDCK cyst polarity reorientation [28] and may inhibit the RhoA-RockI-MyosinII pathway [29]. However, a recent study of mice mammary gland lumen formation demonstrates that, downstream of basement membrane deposition, ␤1 Integrin signalling acts via a separate, Rac1-independent mechanism. In this case ␤1 Integrin acts through Integrin linked kinase (ILK), and is necessary for orienting mammary epithelial cell microtubules and therefore regulating apical targeting of polarity proteins such as Pard3, aPKC and ZO1. It does this via the recruitment of the microtubule protein EB1 to the basolateral surface, which anchors the plus ends of microtubules at this point and orients the trafficking of apical components [30]. The requirement for ␤1-Integrin mediated apical polarity localisation for appropriate lumen formation has been demonstrated in vivo in the mouse arterial endothelium [31] and similar molecular mechanisms exist in in vitro models of human endothelia cell lumenogenesis [32]. Recent studies have also shown that the role of the ECM in organising the epithelial polarity axis and junction location is in part due to the physical confinement and intercellular forces that the ECM mediates [33,34]. These studies suggest that ECM components are universally important in apico-basal orientation in epithelial tubes. Our work has confirmed that this is also the case in the developing zebrafish hindbrain, where knock down or mutation of Laminin1 causes an inversion of apical polarity to the basal ends of neuroepithelial cells [2]. This has subsequently been confirmed in studies of another area of the developing zebrafish brain, the developing eye field, which also showed that close contact with the basal lamina is required for polarisation [35]. These works show that ECM at the basal surface of the neural rod is responsible for directing apical polarity proteins away from the basal surface, and suggest that centrosome location

Please cite this article in press as: Buckley C, Clarke J. Establishing the plane of symmetry for lumen formation and bilateral brain formation in the zebrafish neural rod. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.04.008

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might also be influenced by basal lamina components. This has yet to be tested however. 6.2. Role of Cadherin based adhesions Even if centrosomes are responding to basal lamina components by moving to the opposite (anti-basal) ends of the cells, this does not easily explain why the centrosomes halt at the midline of the neural rod, part-way along the cells that span the midline. Our results strongly suggest that this refinement of centrosomal position is dependent on cell–cell interactions in the zone where cells from the left and right sides of the rod interdigitate across the midline [2]. By studying division-blocked cells to eliminate the polarising influence of the midline division, we showed that if cells from left and right sides are prevented from interacting, they polarise at their most ‘anti-basal’ pole and no longer initiate apical complex formation part way along their length. However cells that are allowed to converge and interdigitate accumulate ␣-Catenin puncta along their membranes throughout the interdigitation zone at the midline, suggesting that Cadherin based adhesions may be prominent in this region. Since there are strong precedents for Cadherin based junctions influencing centrosomal positioning in other systems, we hypothesise that they are instructing centrosomal position in the zebrafish neural rod [2]. Particularly relevant to the current discussion are the findings that N-Cadherin junctions [36,37] and E-Cadherin junctions [38] are instructive in centrosomal positioning in migrating cells. An important role has also been found for functional ␤-Catenin in maintaining centrosomes, microtubule networks and therefore apical polarity within neural progenitors of the mouse midbrain [39]. Even in the non-metazoan Dictyostelium, which lack Cadherins, an ␣-Catenin homolog appears to be necessary for centrosome positioning [40]. Although direct evidence for Cadherins influencing centrosomes location has not yet been sought in the zebrafish neural tube, N-Cadherin has been shown to be important for general tube morphogenesis [41–43] and nascent ␣-Catenin positive adhesion clusters have been proposed to influence the orientation of spindles during midline divisions and to be dependent on Scribble [10]. 6.3. Role of polarity proteins A further possibility is that polarity proteins such as Pard3 may play a role in positioning the centrosomes at the neural rod midline. Polarity proteins have been shown to influence centrosome positioning in several different organisms and cell types. In intestinal cells in C. elegans, the centrosome moves towards Pard3 and Pard6 accumulations, which then move together to the apical surface. If Par3 is depleted then centrosomes do not localise apically [23]. Similarly, aPKC has recently been implicated in centrosomal positioning and subsequent lumen formation in MDCK cells [33]. In migrating fibroblasts, Pard3 is thought to interact with dynein to tether MTs near cell–cell contacts and Pard3 depletion prevented centrosome orientation towards the leading edge of cells without affecting junctional markers [44]. In the zebrafish neural tube, morpholino knock down of Pard3 prevents centrosomes from localising to the apical surface [24]. Since centrosomes are likely responsible for organising the microtubule cytoskeleton required for delivery of Pard3 to the apical end of neural rod cells [2], a self-reinforcing loop of Pard3 and centrosome interactions may establish and maintain these components at the midline of the neural rod. 7. A working hypothesis for the zebrafish neural rod Clearly the formation of apical complexes and junctions that build the lumen at the midline of the zebrafish neural rod is a complicated process, encompassing an interrelated network of proteins

Fig. 4. Diagram to illustrate how the midline division shares daughters equally across the midline and can rebalance cell numbers across the midline. In this hypothetical scenario at the keel stage, the left hand side has only 2 cells (red) while the right side has 6 cells (blue). In the rod stage all left and right cells divide at the midline. After the midline division at the late rod or tube stage the numbers of cells are now equal on left and right sides (with 8 on each side).

and signalling cascades. We propose that the overall apicobasal orientation of cells is determined by interactions with basal lamina components at the basal surface of the rod. We hypothesise these interactions direct apical components such as Cadherins, Pard3, aPKC and centrosomes away from the basal surface potentially through an integrin-ILK mechanism [30]. Forming Cadherin-based junctions in the interdigitation zone at the neural rod midline [2] might then act as the ‘seed’ for polarity establishment at the centre of the rod. This would trigger the broad accumulation of puncta of polarity proteins such as Pard3 around this point. Once Pard3 puncta accumulate, the centrosomes could then be recruited to this point, anchoring the microtubule cytoskeleton at the middle of the tissue. This centrosomal ‘mirror’ could then reinforce and refine polarity protein delivery to the midline via microtubule-mediated trafficking as well as specifying the location of the mirror-symmetric midline division [2]. 8. Why is symmetry important? This article has focussed on the mechanisms that assemble a lumen at the midline of the zebrafish neural rod and generate the mirror symmetric neural tube. The mechanisms that achieve leftright symmetry of the neural tube are rarely discussed, in fact much more attention has been directed at investigating the mechanisms that break left-right symmetry in the relatively rare regions of the neural tube that are asymmetric (reviewed in [45]). Such symmetry breaking mechanisms are imposed onto an initially symmetric neural tube, thus the original blueprint is for symmetry, which can then be modified. Left-right symmetry has an obvious importance in the neural tube, as it is the first step towards ensuring an equal distribution of progenitors and neurons on either side of the neural midline. The mirror-symmetric division of the zebrafish neural rod, in addition to organising lumen formation, has the interesting consequence of sharing daughters equally across the neural midline. Thus even if the numbers of progenitors on left and right sides are initially not equal, this imbalance will be corrected by the midline division (Fig. 4). The zebrafish neural tube provides one example of a system in which symmetry and balance must exist in order for correct function. This principle is broadly relevant throughout both natural and man-made structures. For example, the Roman architect, Vitruvius, drew parallels between the symmetry found in ideal human proportions and in the geometry that should be mirrored in architecture. His three principles for building design were firmitas (strength), utilitas (functionality) and venustas (beauty) (British library online). Whether the strength and function that symmetry allows in nature is linked to the beauty that symmetry also conveys is difficult to test but even human’s perception of beauty and mate preference appears to be significantly influenced by body symmetry [46]. Leonardo da Vinci’s famous drawing of the Vitruvian man,

Please cite this article in press as: Buckley C, Clarke J. Establishing the plane of symmetry for lumen formation and bilateral brain formation in the zebrafish neural rod. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.04.008

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Please cite this article in press as: Buckley C, Clarke J. Establishing the plane of symmetry for lumen formation and bilateral brain formation in the zebrafish neural rod. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.04.008

Establishing the plane of symmetry for lumen formation and bilateral brain formation in the zebrafish neural rod.

The lumen of the zebrafish neural tube develops precisely at the midline of the solid neural rod primordium. This process depends on cell polarisation...
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