Regulation of neurocoel morphogenesis by Pard6 gamma b

The Par3/Par6/aPKC protein complex plays a key role in the establishment and maintenance of apicobasal polarity, a cellular characteristic essential for tissue and organ morphogenesis, differentiation and homeostasis. During a forward genetic screen for liver and pancreas mutants, we identified a pard6γb mutant, representing the first known pard6 mutant in a vertebrate organism. pard6γb mutants exhibit defects in epithelial tissue development as well as multiple lumens in the neural tube. Analyses of the cells lining the neural tube cavity, or neurocoel, in wildtype and pard6γb mutant embryos show that lack of Pard6γb function leads to defects in mitotic spindle orientation during neurulation. We also found that the PB1 (aPKC-binding) and CRIB (Cdc-42-binding) domains and the KPLG amino acid sequence within the PDZ domain (Pals1-and Crumbs binding) are not required for Pard6γb localization but are essential for its function in neurocoel morphogenesis. Apical membranes are reduced, but not completely absent, in mutants lacking the zygotic, or both the maternal and zygotic, function of pard6γb, leading us to examine the localization and function of the three additional zebrafish Pard6 proteins. We found that Pard6α, but not Pard6β or Pard6γa, could partially rescue the pard6γb^s441 mutant phenotypes. Altogether, these data indicate a previously unappreciated functional diversity and complexity within the vertebrate pard6 gene family.     

Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis

BACKGROUND: The Par-3/Par-6/aPKC complex is a key regulator of cell polarity in a number of systems. In Drosophila, this complex acts at the zonula adherens (adherens junctions) to establish epithelial polarity and helps to orient the mitotic spindle during asymmetric neuroblast divisions. In MDCKII cells, this complex localizes to the zonula occludens (tight junctions) and appears to regulate epithelial polarity. However, the in vivo role of this complex during vertebrate embryogenesis is not known, due to the lack of relevant mutations. RESULTS: We have positionally cloned the zebrafish heart and soul (has) mutation, which affects the morphogenesis of several embryonic tissues, and show that it encodes atypical protein kinase C lambda (aPKC lambda). We find that loss of aPKC lambda affects the formation and maintenance of the zonula adherens in the polarized epithelia of the retina, neural tube, and digestive tract, leading to novel phenotypes, such as the formation of multiple lumens in the developing intestine. In addition, has mutants display defects in gut looping and endodermal organ morphogenesis that appear to be independent of the defects in epithelial polarity. Finally, we show that loss of aPKC lambda leads to defects in spindle orientation during progenitor cell divisions in the neural retina. CONCLUSIONS: Our results show that aPKC lambda is required for the formation and maintenance of the zonula adherens during early epithelial development in vertebrates and demonstrate a previously undescribed yet critical role for this protein in organ morphogenesis. Furthermore, our studies identify the first genetic locus regulating the orientation of cell division in vertebrates.     

The Par-PrkC Polarity Complex Is Required for Cilia Growth in Zebrafish Photoreceptors

Specification and development of the apical membrane in epithelial cells requires the function of polarity proteins, including Pard3 and an atypical protein kinase C (PrkC). Many epithelial cells possess microtubule-based organelles, known as cilia, that project from their apical surface and the membrane surrounding the cilium is contiguous with the apical cell membrane. Although cilia formation in cultured cells required Pard3, the in vivo requirement for Pard3 in cilia development remains unknown. The vertebrate photoreceptor outer segment represents a highly specialized cilia structure in which to identify factors necessary for apical and ciliary membrane formation. Pard3 and PrkC localized to distinct domains within vertebrate photoreceptors. Using partial morpholino knockdown, photo-morpholinos, and pharmacological approaches, the function of Pard3 and PrkC were found to be required for the formation of both the apical and ciliary membrane of vertebrate photoreceptors. Inhibition of Pard3 or PrkC activity significantly reduced the size of photoreceptor outer segments and resulted in mislocalization of rhodopsin. Suppression of Pard3 or PrkC also led to a reduction in cilia size and cilia number in Kupffer’s Vesicle, which resulted in left-right asymmetry defects. Thus, the Par-PrkC complex functions in cilia formation in vivo and this likely reflects a general role in specifying non-ciliary and ciliary compartments of the apical domain.     

oko meduzy mutations affect neuronal patterning in the zebrafish retina and reveal cell-cell interactions of the retinal neuroepithelial sheet

Mutations of the oko meduzy (ome) locus cause drastic neuronal patterning defect in the zebrafish retina. The precise, stratified appearance of the wild-type retina is absent in the mutants. Despite the lack of lamination, at least seven retinal cell types differentiate in oko meduzy. The ome phenotype is already expressed in the retinal neuroepithelium affecting morphology of the neuroepithelial cells. Our experiments indicate that previously unknown cell-cell interactions are involved in development of the retinal neuroepithelial sheet. In genetically mosaic animals, cell-cell interactions are sufficient to rescue the phenotype of oko meduzy retinal neuroepithelial cells. These cell-cell interactions may play a critical role in the patterning events that lead to differentiation of distinct neuronal laminae in the vertebrate retina.     

Suppression of lens growth by alphaA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish

In order to study lens-retina relationships during development, we cloned the zebrafish alphaA-crystallin cDNA and its promoter region. Using a 2.8-kb fragment of the zebrafish alphaA-crystallin promoter (z(alpha)Acry), we expressed the diphtheria toxin A fragment (DTA) in zebrafish embryos in a lens-specific manner. Injection of the z(alpha)Acry-DTA plasmid into eggs at the one-or two-cell stage resulted in the formation of small eyes, in which both lens and retina were reduced in size. In the DTA-expressing lenses, their fiber structure was disorganized, indicating that normal lens development had been abrogated. The neural retina also showed abnormal development, although this tissue did not express DTA. Lamination in the retina did not develop well, and molecular markers for the outer and inner plexiform layers were either abnormally expressed or absent. However, cell type-specific markers of ganglion and bipolar cells, as well as photoreceptors, were expressed in appropriate positions, indicating that initial differentiation of these retinal subpopulations occurred in the DTA-expressing embryos. Cell proliferation also proceeded normally in these embryos, although apoptosis was enhanced. These results suggest that the differentiated lens plays a critical role in the morphogenetic organization of retinal cells during eye development in zebrafish embryos.     

Retina development in zebrafish requires the heparan sulfate proteoglycan agrin

Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.     

Inhibition of integrin-mediated adhesion and signaling disrupts retinal development

Integrins are the major family of cell adhesion receptors that mediate cell adhesion to the extracellular matrix (ECM). Integrin-mediated adhesion and signaling play essential roles in neural development. In this study, we have used echistatin, an RGD-containing short monomeric disintegrin, to investigate the role of integrin-mediated adhesion and signaling during retinal development in Xenopus. Application of echistatin to Xenopus retinal-derived XR1 glial cells inhibited the three stages of integrin-mediated adhesion: cell attachment, cell spreading, and formation of focal adhesions and stress fibers. XR1 cell attachment and spreading increased tyrosine phosphorylation of paxillin, a focal adhesion associated protein, while echistatin significantly decreased phosphorylation levels of paxillin. Application of echistatin or beta(1) integrin function blocking antibody to the embryonic Xenopus retina disrupted retinal lamination and produced rosette structures with ectopic photoreceptors in the outer retina. These results indicate that integrin-mediated cell-ECM interactions play a critical role in cell adhesion, migration, and morphogenesis during vertebrate retinal development.     

A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells

Inosine monophosphate dehydrogenase (IMPDH) is a key regulatory enzyme in the de novo synthesis of the purine base guanine. Dominant mutations in human IMPDH1 cause photoreceptor degeneration for reasons that are unknown. Here, we sought to provide some foundational information on Impdh1a in the zebrafish retina. We found that in zebrafish, gene subfunctionalization due to ancestral duplication resulted in a predominant retinal variant expressed exclusively in rod and cone photoreceptors. This variant is structurally and functionally similar to the human IMPDH1 retinal variant and shares a reduced sensitivity to GTP-mediated inhibition. We also demonstrated that Impdh1a forms prominent protein filaments in vitro and in vivo in both rod and cone photoreceptor cell bodies, synapses, and to a lesser degree, in outer segments. These filaments changed length and cellular distribution throughout the day consistent with diurnal changes in both mRNA and protein levels. The loss of Impdh1a resulted in a substantial reduction of guanine levels, although cellular morphology and cGMP levels remained normal. Our findings demonstrate a significant role for IMPDH1 in photoreceptor guanine production and provide fundamental new information on the details of this protein in the zebrafish retina.     

Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A?/?; Sema5B?/? mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.     

Stepwise polarisation of the Drosophila follicular epithelium

The function of epithelial tissues is dependent on their polarised architecture, and loss of cell polarity is a hallmark of various diseases. Here we analyse cell polarisation in the follicular epithelium of Drosophila, an epithelium that arises by a mesenchymal-epithelial transition. Although many epithelia are formed by mesenchymal precursors, it is unclear how they polarise. Here we show how lateral, apical, and adherens junction proteins act stepwise to establish polarity in the follicular epithelium. Polarisation starts with the formation of adherens junctions, whose positioning is controlled by combined activities of Par-3, β-catenin, and Discs large. Subsequently, Par-6 and aPKC localise to the apical membrane in a Par-3-dependent manner. Apical membrane specification continues by the accumulation of the Crumbs complex, which is controlled by Par-3, Par-6, and aPKC. Thus, our data elucidate the genetic mechanisms leading to the stepwise polarisation of an epithelium with a mesenchymal origin.     

In vivo electroporation of morpholinos into the adult zebrafish retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection. In all cases, resident Müller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days. Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.     

Par-1 and PP2A: Yin-Yang of Bazooka Localization

Apical basal cell polarity is a fundamental feature of all epithelial cells. Identification of the genes involved in the polarization of epithelial cells has begun to reveal the mechanisms underlying the establishment and maintenance of cell polarity. An important issue is to understand the molecular basis for localization of cell polarity proteins in the context of the developing organism. Bazooka (Baz, Drosophila homolog of Par-3) plays a crucial role in organizing cell polarity in several different tissues. In the ovarian follicle epithelium, Par-1 protein kinase regulates Baz localization to the apical cell cortex by excluding phosphorylated Baz from the lateral region. In photoreceptor cells of retinal epithelium, Baz is targeted to the adherens junction (AJ) instead of the apical domain. Our study suggests that in photoreceptors, Par-1 blocks the localization of Baz to AJ whereas protein phosphatase 2A (PP2A) promotes Baz localization by antagonizing the Par-1 effects. In this extra view, we provide a brief overview and perspective of our findings on the antagonistic function of Par-1 and PP2A in Baz localization during photoreceptor morphogenesis.     

Par-1 and PP2A: Yin-Yang of Bazooka localization

Apical basal cell polarity is a fundamental feature of all epithelial cells. Identification of the genes involved in the polarization of epithelial cells has begun to reveal the mechanisms underlying the establishment and maintenance of cell polarity. An important issue is to understand the molecular basis for localization of cell polarity proteins in the context of the developing organism. Bazooka (Baz, Drosophila homolog of Par-3) plays a crucial role in organizing cell polarity in several different tissues. In the ovarian follicle epithelium, Par-1 protein kinase regulates Baz localization to the apical cell cortex by excluding phosphorylated Baz from the lateral region. In photoreceptor cells of retinal epithelium, Baz is targeted to the adherens junction (AJ) instead of the apical domain. Our study suggests that in photoreceptors, Par-1 blocks the localization of Baz to AJ whereas protein phosphatase 2A (PP2A) promotes Baz localization by antagonizing the Par-1 effects. In this extra view, we provide a brief overview and perspective of our findings on the antagonistic function of Par-1 and PP2A in Baz localization during photoreceptor morphogenesis.     

Protocadherin‐17 function in Zebrafish retinal development

Cadherin cell adhesion molecules play crucial roles in vertebrate development including the development of the retina. Most studies have focused on examining functions of classic cadherins (e.g. N‐cadherin) in retinal development. There is little information on the function of protocadherins in the development of the vertebrate visual system. We previously showed that protocadherin‐17 mRNA was expressed in developing zebrafish retina during critical stages of the retinal development. To gain insight into protocadherin‐17 function in the formation of the retina, we analyzed eye development and differentiation of retinal cells in zebrafish embryos injected with protocadherin‐17 specific antisense morpholino oligonucleotides (MOs). Protocadherin‐17 knockdown embryos (pcdh17 morphants) had significantly reduced eyes due mainly to decreased cell proliferation. Differentiation of several retinal cell types (e.g. retinal ganglion cells) was also disrupted in the pcdh17 morphants. Phenotypic rescue was achieved by injection of protocadherin‐17 mRNA. Injection of a vivo‐protocadherin‐17 MO into one eye of embryonic zebrafish resulted in similar eye defects. Our results suggest that protocadherin‐17 plays an important role in the normal formation of the zebrafish retina. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Par-1b is required for morphogenesis and differentiation of myoepithelial cells during salivary gland development

The salivary epithelium initiates as a solid mass of epithelial cells that are organized into a primary bud that undergoes morphogenesis and differentiation to yield bilayered acini consisting of interior secretory acinar cells that are surrounded by contractile myoepithelial cells in mature salivary glands. How the primary bud transitions into acini has not been previously documented. We document here that the outer epithelial cells subsequently undergo a vertical compression as they express smooth muscle α-actin and differentiate into myoepithelial cells. The outermost layer of polarized epithelial cells assemble and organize the basal deposition of basement membrane, which requires basal positioning of the polarity protein, Par-1b. Whether Par-1b is required for the vertical compression and differentiation of the myoepithelial cells is unknown. Following manipulation of Par-1b in salivary gland organ explants, Par-1b-inhibited explants showed both a reduced vertical compression of differentiating myoepithelial cells and reduced levels of smooth muscle α-actin. Rac1 knockdown and inhibition of Rac GTPase function also inhibited branching morphogenesis. Since Rac regulates cellular morphology, we investigated a contribution for Rac in myoepithelial cell differentiation. Inhibition of Rac GTPase activity showed a similar reduction in vertical compression and smooth muscle α-actin levels while decreasing the levels of Par-1b protein and altering its basal localization in the outer cells. Inhibition of ROCK, which is required for basal positioning of Par-1b, resulted in mislocalization of Par-1b and loss of vertical cellular compression, but did not significantly alter levels of smooth muscle α-actin in these cells. Overexpression of Par-1b in the presence of Rac inhibition restored basement membrane protein levels and localization. Our results indicate that the basal localization of Par-1b in the outer epithelial cells is required for myoepithelial cell compression, and Par-1b is required for myoepithelial differentiation, regardless of its localization.     

MPP3 is recruited to the MPP5 protein scaffold at the retinal outer limiting membrane

Mutations in the human Crumbs homologue 1 (CRB1) gene are a frequent cause of various forms of retinitis pigmentosa. The CRB1-membrane-associated palmitoylated protein (MPP)5 protein complex is thought to organize an intracellular protein scaffold in the retina that is involved in maintenance of photoreceptor-Müller glia cell adhesion. This study focused on the binding characteristics and subcellular localization of MPP3, a novel member of the MPP5 protein scaffold at the outer limiting membrane (OLM), and of the DLG1 protein scaffold at the outer plexiform layer of the retina. MPP3 localized at the photoreceptor synapse and at the subapical region adjacent to adherens junctions at the OLM. Localization studies in human retinae revealed that MPP3 colocalized with MPP5 and CRB1 at the subapical region. MPP3 and MPP4 colocalized with DLG1 at the outer plexiform layer. Mouse Dlg1 formed separate complexes with Mpp3 and Mpp4 in vivo. These data implicate a role for MPP3 in photoreceptor polarity and, by association with MPP5, pinpoint MPP3 as a functional candidate gene for inherited retinopathies. The separate Mpp3/Dlg1 and Mpp4/Dlg1 complexes at the outer plexiform layer point towards additional yet unrecognized functions of these membrane associated guanylate kinase proteins.     

Fgf19 is required for zebrafish lens and retina development

Fgf signaling plays crucial roles in morphogenesis. Fgf19 is required for zebrafish forebrain development. Here, we examined the roles of Fgf19 in the formation of the lens and retina in zebrafish. Knockdown of Fgf19 caused a size reduction of the lens and the retina, failure of closure of the choroids fissure, and a progressive expansion of the retinal tissue to the midline of the forebrain. Fgf19 expressed in the nasal retina and lens was involved in cell survival but not cell proliferation during embryonic lens and retina development. Fgf19 was essential for the differentiation of lens fiber cells in the lens but not for the neuronal differentiation and lamination in the retina. Loss of nasal fate in the retina caused by the knockdown of Fgf19, expansion of nasal fate in the retina caused by the overexpression of Fgf19 and eye transplantation indicated that Fgf19 in the retina was crucial for the nasal-temporal patterning of the retina that is critical for the guidance of retinal ganglion cell axons. Knockdown of Fgf19 also caused incorrect axon pathfinding. The present findings indicate that Fgf19 positively regulates the patterning and growth of the retina, and the differentiation and growth of the lens in zebrafish.     

Alpha-actinin and actin in the outer retina: a double immunoelectron microscopic study

Actin has many diverse functions in the outer retina. To help elucidate its organization in this area, we have investigated the extent of its association with the actin cross-linking protein alpha-actinin. Ultrathin sections of chicken retina were double-immunolabelled with monospecific antibodies against actin and alpha-actinin. The highest relative amount of alpha-actinin to actin label was measured in the adherens junctions between the individual retinal pigmented epithelial (RPE) cells and between the photoreceptor and Mueller cells; in the photoreceptor myoid; and in the RPE basal microvilli. The lowest amount was in the Mueller cell microvilli, the RPE apical processes, and in the photoreceptor ellipsoid. It is likely that the areas containing the highest ratio of alpha-actinin to actin labelling are where the actin filaments are most highly cross-linked into bundles and linked to the plasma membrane by alpha-actinin. Actin filaments terminate in these areas, and, except for the myoid region, they are involved in cell-cell or cell-substrate adherens junctions.