Rho signaling pathway and apical constriction in the early lens placode

Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho-ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan-Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49:368-379, 2011.     

A cell polarity protein aPKCλ is required for eye lens formation and growth

The organisation of individual cells into a functional three-dimensional tissue is still a major question in developmental biology. Modulation of epithelial cell shape is a critical driving force in forming tissues. This is well illustrated in the eye lens where epithelial cells elongate extensively during their differentiation into fibre cells. It is at the lens equator that epithelial cells elongate along their apical-basal axis. During this process the elongating epithelial cells and their earliest fibre cell derivatives remain anchored at their apical tips, forming a discrete region or modiolus, which we term the lens fulcrum. How this is achieved has received scant attention and is little understood. Here, we show that conditional depletion of aPKCλ, a central effector of the PAR polarity complex, disrupts the apical junctions in elongating epithelial cells so that the lens fulcrum fails to form. This results in disorganised fibre cell alignment that then causes cataract. Interestingly, aPKCλ depletion also promotes epithelial-mesenchymal transition of the lens epithelial cells, reducing their proliferation, leading ultimately to a small lens and microphthalmia. These observations indicate that aPKCλ, a regulator of polarity and apical junctions, is required for development of a lens that is the correct size and shape.     

Segregation of lens and olfactory precursors from a common territory: cell sorting and reciprocity of Dlx5 and Pax6 expression

Cranial placodes are focal regions of columnar epithelium next to the neural tube that contribute to sensory ganglia and organs in the vertebrate head, including the olfactory epithelium and the crystalline lens of the eye. Using focal dye labelling within the presumptive placode domain, we show that lens and nasal precursors arise from a common territory surrounding the anterior neural plate. They then segregate over time and converge to their final positions in discrete placodes by apparently directed movements. Since these events closely parallel the separation of eye and antennal primordia (containing olfactory sensory cells) from a common imaginal disc in Drosophila, we investigated whether the vertebrate homologues of Distalless (Dll) and Eyeless (Ey), which determine antennal and eye identity in the fly, play a role in segregation of lens and nasal precursors in the chick. Dlx5 and Pax6 are initially co-expressed by future lens and olfactory cells. As soon as presumptive lens cells acquire columnar morphology all Dlx family members are down-regulated in the placode, while Pax6 is lost in the olfactory region. Lens precursor cells that express ectopic Dlx5 never acquire lens-specific gene expression and are excluded from the lens placode to cluster in the head ectoderm. These results suggest that the loss of Dlx5 is required for cells to adopt a lens fate and that the balance of Pax6 and Dlx expression regulates cell sorting into appropriate placodal domains.     

KIBRA suppresses apical exocytosis through inhibition of aPKC kinase activity in epithelial cells

Epithelial cells possess apical-basolateral polarity and form tight junctions (TJs) at the apical-lateral border, separating apical and basolateral membrane domains. The PAR3-aPKC-PAR6 complex plays a central role in TJ formation and apical domain development during tissue morphogenesis. Inactivation and overactivation of aPKC kinase activity disrupts membrane polarity. The mechanism that suppresses active aPKC is unknown. KIBRA, an upstream regulator of the Hippo pathway, regulates tissue size in Drosophila and can bind to aPKC. However, the relationship between KIBRA and the PAR3-aPKC-PAR6 complex remains unknown. We report that KIBRA binds to the PAR3-aPKC-PAR6 complex and localizes at TJs and apical domains in epithelial tissues and cells. The knockdown of KIBRA causes expansion of the apical domain in MDCK three-dimensional cysts and suppresses the formation of apical-containing vacuoles through enhanced de novo apical exocytosis. These phenotypes are restored by inhibition of aPKC. In addition, KIBRA directly inhibits the kinase activity of aPKC in vitro. These results strongly support the notion that KIBRA regulates epithelial cell polarity by suppressing apical exocytosis through direct inhibition of aPKC kinase activity in the PAR3-aPKC-PAR6 complex.     

Lens specification is the ground state of all sensory placodes, from which FGF promotes olfactory identity

The sense organs of the vertebrate head comprise structures as varied as the eye, inner ear, and olfactory epithelium. In the early embryo, these assorted structures share a common developmental origin within the preplacodal region and acquire specific characteristics only later. Here we demonstrate a fundamental similarity in placodal precursors: in the chick all are specified as lens prior to acquiring features of specific sensory or neurogenic placodes. Lens specification becomes progressively restricted in the head ectoderm, initially by FGF and subsequently by signals derived from migrating neural crest cells. We show that FGF8 from the anterior neural ridge is both necessary and sufficient to promote olfactory fate in adjacent ectoderm. Our results reveal that placode precursors share a common ground state as lens and progressive restriction allows the full range of placodal derivatives to form.     

Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling

Cell polarity in Drosophila epithelia, oocytes and neuroblasts is controlled by the evolutionarily conserved PAR/aPKC complex, which consists of the serine-threonine protein kinase aPKC and the PDZ-domain proteins Bazooka (Baz) and PAR-6. The PAR/aPKC complex is required for the separation of apical and basolateral plasma membrane domains, for the asymmetric localization of cell fate determinants and for the proper orientation of the mitotic spindle. How the complex exerts these different functions is not known. We show that the lipid phosphatase PTEN directly binds to Baz in vitro and in vivo, and colocalizes with Baz in the apical cortex of epithelia and neuroblasts. PTEN is an important regulator of phosphoinositide turnover that antagonizes the activity of PI3-kinase. We show that Pten mutant ovaries and embryos lacking maternal and zygotic Pten function display phenotypes consistent with a function for PTEN in the organization of the actin cytoskeleton. In freshly laid eggs, the germ plasm determinants oskar mRNA and Vasa are not localized properly to the posterior cytocortex and pole cells do not form. In addition, the actin-dependent posterior movement of nuclei during early cleavage divisions does not occur and the synchrony of nuclear divisions at syncytial blastoderm stages is lost. Pten mutant embryos also show severe defects during cellularization. Our data provide evidence for a link between the PAR/aPKC complex, the actin cytoskeleton and PI3-kinase signaling mediated by PTEN.     

Apical accumulation of MARCKS in neural plate cells during neurulation in the chick embryo

Background

The neural tube is formed by morphogenetic movements largely dependent on cytoskeletal dynamics. Actin and many of its associated proteins have been proposed as important mediators of neurulation. For instance, mice deficient in MARCKS, an actin cross-linking membrane-associated protein that is regulated by PKC and other kinases, present severe developmental defects, including failure of cranial neural tube closure.

Results

To determine the distribution of MARCKS, and its possible relationships with actin during neurulation, chick embryos were transversely sectioned and double labeled with an anti-MARCKS polyclonal antibody and phalloidin. In the neural plate, MARCKS was found ubiquitously distributed at the periphery of the cells, being conspicuously accumulated in the apical cell region, in close proximity to the apical actin meshwork. This asymmetric distribution was particularly noticeable during the bending process. After the closure of the neural tube, the apically accumulated MARCKS disappeared, and this cell region became analogous to the other peripheral cell zones in its MARCKS content. Actin did not display analogous variations, remaining highly concentrated at the cell subapical territory. The transient apical accumulation of MARCKS was found throughout the neural tube axis. The analysis of another epithelial bending movement, during the formation of the lens vesicle, revealed an identical phenomenon.

Conclusions

MARCKS is transiently accumulated at the apical region of neural plate and lens placode cells during processes of bending. This asymmetric subcellular distribution of MARCKS starts before the onset of neural plate bending. These results suggest possible upstream regulatory actions of MARCKS on some functions of the actin subapical meshwork.     

Microtubules induce self-organization of polarized PAR domains in Caenorhabditis elegans zygotes

A hallmark of polarized cells is the segregation of the PAR polarity regulators into asymmetric domains at the cell cortex. Antagonistic interactions involving two conserved kinases, atypical protein kinase C (aPKC) and PAR-1, have been implicated in polarity maintenance, but the mechanisms that initiate the formation of asymmetric PAR domains are not understood. Here, we describe one pathway used by the sperm-donated centrosome to polarize the PAR proteins in Caenorhabditis elegans zygotes. Before polarization, cortical aPKC excludes PAR-1 kinase and its binding partner PAR-2 by phosphorylation. During symmetry breaking, microtubules nucleated by the centrosome locally protect PAR-2 from phosphorylation by aPKC, allowing PAR-2 and PAR-1 to access the cortex nearest the centrosome. Cortical PAR-1 phosphorylates PAR-3, causing the PAR-3-aPKC complex to leave the cortex. Our findings illustrate how microtubules, independently of actin dynamics, stimulate the self-organization of PAR proteins by providing local protection against a global barrier imposed by aPKC.     

pitx3 defines an equivalence domain for lens and anterior pituitary placode

Hedgehog signaling is required for formation and patterning of the anterior pituitary gland. However, the role of Hedgehog in pituitary precursor cell specification and subsequent placode formation is not well understood. We analyzed pituitary precursor cell lineages and find that pitx3 and distal-less3b (dlx3b) expression domains define lens and pituitary precursor positions. We show that pitx3 is required for pituitary pre-placode formation and cell specification, whereas dlx3b and dlx4b are required to restrict pituitary placode size. In smoothened mutant embryos that cannot transduce Hedgehog signals, median pituitary precursors are mis-specified and form an ectopic lens. Moreover, overexpression of sonic hedgehog (shh) blocks lens formation, and derivatives of lens precursors express genes characteristic of pituitary cells. However, overexpression of shh does not increase median pituitary placode size nor does it upregulate patched (ptc) expression in pituitary precursors during early somitogenesis. Our study suggests that by the end of gastrulation, pitx3-expressing cells constitute an equivalence domain of cells that can form either pituitary or lens, and that a non-Hedgehog signal initially specifies this placodal field. During mid-somitogenesis, Hedgehog then acts on the established median placode as a necessary and sufficient signal to specify pituitary cell types.     

Distribution of F-actin during mouse facial morphogenesis and its perturbation with cytochalasin D using whole embryo culture.

Histological and experimental studies were performed in mouse embryos to elucidate possible roles of actin filaments in the nasal epithelium during facial morphogenesis. C57BL/6 mouse embryos (8.5-11.5 days of gestation) were fixed and frozen sections were stained with rhodamine-phalloidin. Before formation of the nasal placode, there was no specific localization of F-actin. After the nasal placode was formed, intense staining of F-actin was observed at the apical side of the placode. Conversely, it was located at the basal side of the epithelium of developing nasal prominences. By using the whole embryo culture system, perturbation experiments were conducted with cytochalasin D (CD), which inhibits the polymerization of actin filaments. When day-10 embryos were exposed to CD at several concentrations for 24 hr, fusion of nasal prominences was inhibited in a dose-dependent manner. Treatment with a high dose of CD for 2 hr also prevented the same development irreversibly. In contrast, when day-9 embryos were exposed to CD at several concentrations for 24 hr, invagination of the nasal placode was not perturbed at all. The results suggest that apical F-actin plays an essential role in maintaining the close apposition state of the nasal prominences and in the following fusion. During the invagination stage, F-actin might be important in maintaining the epithelial structure, but is not crucial to the initiation of placode invagination.     

The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila

Cell polarity is critical for epithelial structure and function. Adherens junctions (AJs) often direct this polarity, but we previously found that Bazooka (Baz) acts upstream of AJs as epithelial polarity is first established in Drosophila. This prompted us to ask how Baz is positioned and how downstream polarity is elaborated. Surprisingly, we found that Baz localizes to an apical domain below its typical binding partners atypical protein kinase C (aPKC) and partitioning defective (PAR)-6 as the Drosophila epithelium first forms. In fact, Baz positioning is independent of aPKC and PAR-6 relying instead on cytoskeletal cues, including an apical scaffold and dynein-mediated basal-to-apical transport. AJ assembly is closely coupled to Baz positioning, whereas aPKC and PAR-6 are positioned separately. This forms a stratified apical domain with Baz and AJs localizing basal to aPKC and PAR-6, and we identify specific mechanisms that keep these proteins apart. These results reveal key steps in the assembly of the apical domain in Drosophila.     

The lens: a classical model of embryonic induction providing new insights into cell determination in early development

The lens was the first tissue in which the concept of embryonic induction was demonstrated. For many years lens induction was thought to occur at the time the optic vesicle and lens placode came in contact. Since then, studies have revealed that lens placodal progenitor cells are specified already at gastrula stages, much earlier than previously believed, and independent of optic vesicle interactions. In this review, I will focus on how individual signalling molecules, in particular BMP, FGF, Wnt and Shh, regulate the initial specification of lens placodal cells and the progressive development of lens cells. I will discuss recent work that has shed light on the combination of signalling molecules and the molecular interactions that affect lens specification and proper lens formation. I will also discuss proposed tissue interactions important for lens development. A greater knowledge of the molecular interactions during lens induction is likely to have practical benefits in understanding the causes and consequences of lens diseases. Moreover, knowledge regarding lens induction is providing fundamental important insights into inductive processes in development in general.     

PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity

The organization of the Golgi apparatus is essential for cell polarization and its maintenance. The polarity regulator PAR complex (PAR3, PAR6, and aPKC) plays critical roles in several processes of cell polarization. However, how the PAR complex participates in regulating the organization of the Golgi remains largely unknown. Here we demonstrate the functional cross-talk of the PAR complex with CLASP2, which is a microtubule plus-end–tracking protein and is involved in organizing the Golgi ribbon. CLASP2 directly interacted with PAR3 and was phosphorylated by aPKC. In epithelial cells, knockdown of either PAR3 or aPKC induced the aberrant accumulation of CLASP2 at the trans-Golgi network (TGN) concomitantly with disruption of the Golgi ribbon organization. The expression of a CLASP2 mutant that inhibited the PAR3-CLASP2 interaction disrupted the organization of the Golgi ribbon. CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185. This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2. Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization. On the basis of these observations, we propose that PAR3 and aPKC control the organization of the Golgi through CLASP2 phosphorylation.     

Equivalent progenitor cells in the zebrafish anterior preplacodal field give rise to adenohypophysis, lens, and olfactory placodes

Embryonic organizing centers secrete signaling molecules that instruct target cells about their position and future identity. Information about cell position in relation to sources of instructive signals and about precursor cell lineages is key to our understanding of developmental processes that restrict cell potency and determine cell fate. We review adenohypophysis, lens, and olfactory placode formation and how gene expression patterns, cell positions, and cell fates in the anterior neural plate and anterior placodal field correlate in zebrafish and other vertebrates. Single cell lineage analysis in zebrafish suggests that the majority of preplacodal cells might be specified for pituitary, lens, or olfactory placode by the end of gastrulation.     

Analysis of distribution patterns of gap junctions during development of embryonic chick facial primordia and brain.

Gap junction distribution in the facial primordia of chick embryos at the time of primary palate formation was studied employing indirect immunofluorescence localization with antibodies to gap junction proteins initially identified in rat liver (27 x 10(3) Mr, connexin 32) and heart (43 x 10(3) Mr, connexin 43). Immunolocalization with antibodies to the rat liver gap junction protein (27 x 10(3) Mr) demonstrated a ubiquitous and uniform distribution in all regions of the epithelium and mesenchyme except the nasal placode. In the placodal epithelium, a unique non-random distribution was found characterized by two zones: a very heavy concentration of signal in the superficial layer of cells adjacent to the exterior surface and a region devoid of detectable signal in the interior cell layer adjacent to the mesenchyme. This pattern was seen during all stages of placode invagination that were examined. The separation of gap junctions in distinct cell layers was unique to the nasal placode, and was not found in any other region of the developing primary palate. One other tissue was found that exhibited this pattern-the developing neural epithelium of the brain and retina. These observations suggest the presence of region-specific signaling mechanisms and, possibly, an impedance of cell communication among subpopulations of cells in these structures at critical stages of development. Immunolocalization with antibodies to the 'heart' 43 x 10(3) Mr gap junction protein also revealed the presence of gap junction protein in facial primordia and neural epithelium. A non-uniform distribution of immunoreactivity was also observed for connexin 43.     

PAR1 specifies ciliated cells in vertebrate ectoderm downstream of aPKC

Partitioning-defective 1 (PAR1) and atypical protein kinase C (aPKC) are conserved serine/threonine protein kinases implicated in the establishment of cell polarity in many species from yeast to humans. Here we investigate the roles of these protein kinases in cell fate determination in Xenopus epidermis. Early asymmetric cell divisions at blastula and gastrula stages give rise to the superficial (apical) and the deep (basal) cell layers of epidermal ectoderm. These two layers consist of cells with different intrinsic developmental potential, including superficial epidermal cells and deep ciliated cells. Our gain- and loss-of-function studies demonstrate that aPKC inhibits ciliated cell differentiation in Xenopus ectoderm and promotes superficial cell fates. We find that the crucial molecular substrate for aPKC is PAR1, which is localized in a complementary domain in superficial ectoderm cells. We show that PAR1 acts downstream of aPKC and is sufficient to stimulate ciliated cell differentiation and inhibit superficial epidermal cell fates. Our results suggest that aPKC and PAR1 function sequentially in a conserved molecular pathway that links apical-basal cell polarity to Notch signaling and cell fate determination. The observed patterning mechanism may operate in a wide range of epithelial tissues in many species.     

Delamination of cells from neurogenic placodes does not involve an epithelial-to-mesenchymal transition

Neurogenic placodes are specialized regions of embryonic ectoderm that generate the majority of the neurons of the cranial sensory ganglia. Here we examine in chick the mechanism underlying the delamination of cells from the epibranchial placodal ectoderm. We show that the placodal epithelium has a distinctive morphology, reflecting a change in cell shape, and is associated with a breach in the underlying basal lamina. Placodal cell delamination is distinct from neural crest cell delamination. In particular, exit of neuroblasts from the epithelium is not associated with the expression of Snail/Snail2 or of the Rho family GTPases required for the epithelial-to-mesenchymal transition seen in neural crest cell delamination. Indeed, cells leaving the placodes do not assume a mesenchymal morphology but migrate from the epithelium as neuronal cells. We further show that the placodal epithelium has a pseudostratified appearance. Examination of proliferation shows that the placodal epithelium is mitotically quiescent, with few phosphohistone H3-positive cells being identified. Where division does occur within the epithelium it is restricted to the apical surface. The neurogenic placodes thus represent specialized ectodermal niches that generate neuroblasts over a protracted period.     

Regional-specific alterations in cell–cell junctions,cytoskeletal networks and myosin-mediated mechanical cues coordinate collectivity of movement of epithelial cells in response to injury

This study investigates how epithelial cells moving together function to coordinate their collective movement to repair a wound. Using a lens ex vivo mock cataract surgery model we show that region-specific reorganization of cell–cell junctions, cytoskeletal networks and myosin function along apical and basal domains of an epithelium mediates the process of collective migration. An apical junctional complex composed of N-cadherin/ZO-1/myosin II linked to a cortical actin cytoskeleton network maintains integrity of the tissue during the healing process. These cells’ basal domains often preceded their apical domains in the direction of movement, where an atypical N-cadherin/ZO-1 junction, linked to an actin stress fiber network rich in phosphomyosin, was prominent in cryptic lamellipodia. These junctions joined the protruding forward-moving lamellipodia to the back end of the cell moving directly in front of it. These were the only junctions detected in cryptic lamellipodia of lens epithelia migrating in response to wounding that could transmit the protrusive forces that drive collective movement. Both integrity of the epithelium and ability to effectively heal the wound was found to depend on myosin mechanical cues.