RESUMO
Histone deacetylases (HDACs) are key posttranslational modulators of the proteome. We show that expression of histone deacetylase 6 ( hdac6 ) is dynamic and appears in a tissue specific manner throughout embryonic development of the frog Xenopus laevis . Interestingly, hdac6 transcripts often associate with ciliated tissues, like the left-right organizer at neurula stage or the pronephros. In the embryonic skin, Hdac6 protein localizes to the cilia base, suggesting a functional link.
RESUMO
The vertebrate left-right axis is specified during neurulation by events occurring in a transient ciliated epithelium termed left-right organizer (LRO), which is made up of two distinct cell types. In the axial midline, central LRO (cLRO) cells project motile monocilia and generate a leftward fluid flow, which represents the mechanism of symmetry breakage. This directional fluid flow is perceived by laterally positioned sensory LRO (sLRO) cells, which harbor non-motile cilia. In sLRO cells on the left side, flow-induced signaling triggers post-transcriptional repression of the multi-pathway antagonist dand5. Subsequently, the co-expressed Tgf-ß growth factor Nodal1 is released from Dand5-mediated repression to induce left-sided gene expression. Interestingly, Xenopus sLRO cells have somitic fate, suggesting a connection between LR determination and somitogenesis. Here, we show that doublesex and mab3-related transcription factor 2 (Dmrt2), known to be involved in vertebrate somitogenesis, is required for LRO ciliogenesis and sLRO specification. In dmrt2 morphants, misexpression of the myogenic transcription factors tbx6 and myf5 at early gastrula stages preceded the misspecification of sLRO cells at neurula stages. myf5 morphant tadpoles also showed LR defects due to a failure of sLRO development. The gain of myf5 function reintroduced sLRO cells in dmrt2 morphants, demonstrating that paraxial patterning and somitogenesis are functionally linked to LR axis formation in Xenopus.
RESUMO
Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus, zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3'-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5, Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3'-UTR mRNA reporter was responsive to pkd2, suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage.
Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Xenopus laevis/genética , Peixe-Zebra/genética , Regiões 3' não Traduzidas/genética , Animais , Desenvolvimento Embrionário/genética , Camundongos , Estabilidade de RNA/genética , Xenopus laevis/embriologia , Peixe-Zebra/embriologiaRESUMO
Mucus secretion and ciliary motility are hallmarks for muco-ciliary epithelia (MCE). Both, mammalian airways as well as the less complex epidermis of Xenopus embryos show cilia-driven mucus flow to protect the organism against harmful effects by exogenous pathogens or pollutants. Four cell types set up the epidermal MCE in Xenopus. Multi-ciliated cells (MCCs) generate an anterior to posterior flow of mucus. Ion secreting cells (ISCs) are characterized by the expression of ion transporters, presumably to maintain a favorable homeostasis. The largest cell type is represented by goblet cells, which cover most of the epidermis and exhibit secretory properties. Additionally, small secretory cells (SSCs) release mucus, antibiotic compounds, and the monoamine serotonin (5-hydroxytryptamine; 5-HT). We have recently shown that serotonin regulates flow velocity by acting on ciliary beat frequency. Here, we describe the identification and functional characterization of Xenopus polka-dots (Xpod). No homologous genes or proteins were found in other vertebrates, including Xenopus tropicalis. We demonstrate that Xpod serves as an SSC-specific marker, starting to be expressed shortly after SSC specification at neurula stages. Overexpression of a tagged Xpod protein resulted in the localization of secretory granules. Notch signaling induced SSC cell fate, in contrast to its repressing effect on MCC and ISC specification. Xpod loss-of-function revealed that mucus and 5-HT release by SSCs was severely diminished, which impaired the ciliary beating of MCCs. In summary, Xpod specifically marked SSCs and was required for muco-ciliary secretion in Xenopus laevis.
Assuntos
Muco/metabolismo , Vesículas Secretórias/metabolismo , Serotonina/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Cílios/metabolismo , Cílios/ultraestrutura , Células Epiteliais/metabolismo , Neurulação , Receptores Notch/metabolismo , Xenopus , Proteínas de Xenopus/genéticaRESUMO
Organ left-right (LR) asymmetry is a conserved vertebrate feature, which is regulated by left-sided activation of Nodal signaling. Nodal asymmetry is established by a leftward fluid-flow generated at the ciliated LR organizer (LRO). Although the role of fibroblast growth factor (FGF) signaling pathways during mesoderm development is conserved, diverging results from different model organisms suggest a non-conserved function in LR asymmetry. Here, we demonstrate that FGF is required during gastrulation in a dual function at consecutive stages of Xenopus embryonic development. In the early gastrula, FGF is necessary for LRO precursor induction, acting in parallel with FGF-mediated mesoderm induction. During late gastrulation, the FGF/Ca2+-branch is required for specification of the flow-sensing lateral LRO cells, a function related to FGF-mediated mesoderm morphogenesis. This second function in addition requires input from the calcium channel Polycystin-2. Thus, analogous to mesoderm development, FGF activity is required in a dual role for laterality specification; namely, for generating and sensing leftward flow. Moreover, our findings in Xenopus demonstrate that FGF functions in LR development share more conserved features across vertebrate species than previously anticipated.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Cílios/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Transcrição Forkhead/metabolismo , Gástrula/metabolismo , Gastrulação/fisiologia , Mesoderma/metabolismo , Transdução de Sinais/fisiologia , Canais de Cátion TRPP/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Nodal signaling controls asymmetric organ placement during vertebrate embryogenesis. Nodal is induced by a leftward fluid flow at the ciliated left-right organizer (LRO). The mechanism of flow sensing, however, has remained elusive. pkd2 encodes the calcium channel Polycystin-2, which is required for kidney development and laterality, and may act in flow perception. Here, we have studied the role of Polycystin-2 in Xenopus and show that pkd2 is indispensable for left-right (LR) asymmetry. Knockdown of pkd2 prevented left-asymmetric nodal cascade induction in the lateral plate mesoderm. Defects were due to failure of LRO specification, morphogenesis, and, consequently, absence of leftward flow. Polycystin-2 synergizes with the unconventional nodal-type signaling molecule Xnr3 to induce the LRO precursor tissue before gastrulation, upstream of symmetry breakage. Our data uncover an unknown function of pkd2 in LR axis formation, which we propose represents an ancient role of Polycystin-2 during LRO induction in lower vertebrates.
RESUMO
Anatomical and functional asymmetries are widespread in the animal kingdom [1, 2]. In vertebrates, many visceral organs are asymmetrically placed [3]. In snails, shells and inner organs coil asymmetrically, and in Drosophila, genitalia and hindgut undergo a chiral rotation during development. The evolutionary origin of these asymmetries remains an open question [1]. Nodal signaling is widely used [4], and many, but not all, vertebrates use cilia for symmetry breaking [5]. In Drosophila, which lacks both cilia and Nodal, the unconventional myosin ID (myo1d) gene controls dextral rotation of chiral organs [6, 7]. Here, we studied the role of myo1d in left-right (LR) axis formation in Xenopus. Morpholino oligomer-mediated myo1d downregulation affected organ placement in >50% of morphant tadpoles. Induction of the left-asymmetric Nodal cascade was aberrant in >70% of cases. Expression of the flow-target gene dand5 was compromised, as was flow itself, due to shorter, fewer, and non-polarized cilia at the LR organizer. Additional phenotypes pinpointed Wnt/planar cell polarity signaling and suggested that myo1d, like in Drosophila [8], acted in the context of the planar cell polarity pathway. Indeed, convergent extension of gastrula explant cultures was inhibited in myo1d morphants, and the ATF2 reporter gene for non-canonical Wnt signaling was downregulated. Finally, genetic interference experiments demonstrated a functional interaction between the core planar cell polarity signaling gene vangl2 and myo1d in LR axis formation. Thus, our data identified myo1d as a common denominator of arthropod and chordate asymmetry, in agreement with a monophyletic origin of animal asymmetry.
Assuntos
Padronização Corporal/genética , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese/genética , Miosinas/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Animais , Polaridade Celular/genética , Gástrula/embriologia , Miosinas/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
Goosecoid (Gsc) expression marks the primary embryonic organizer in vertebrates and beyond. While functions have been assigned during later embryogenesis, the role of Gsc in the organizer has remained enigmatic. Using conditional gain-of-function approaches in Xenopus and mouse to maintain Gsc expression in the organizer and along the axial midline, neural tube closure defects (NTDs) arose and dorsal extension was compromised. Both phenotypes represent convergent extension (CE) defects, arising from impaired Wnt/planar cell polarity (PCP) signaling. Dvl2 recruitment to the cell membrane was inhibited by Gsc in Xenopus animal cap assays and key Wnt/PCP factors (RhoA, Vangl2, Prickle, Wnt11) rescued Gsc-mediated NTDs. Re-evaluation of endogenous Gsc functions in MO-mediated gene knockdown frog and knockout mouse embryos unearthed PCP/CE-related phenotypes as well, including cartilage defects in Xenopus and misalignment of inner ear hair cells in mouse. Our results assign a novel function to Gsc as an inhibitor of Wnt/PCP-mediated CE. We propose that in the organizer Gsc represses CE as well: Gsc-expressing prechordal cells, which leave the organizer first, migrate and do not undergo CE like the Gsc-negative notochordal cells, which subsequently emerge from the organizer. In this model, Gsc provides a switch between cell migration and CE, i.e. cell intercalation.
Assuntos
Proteína Goosecoid/metabolismo , Organizadores Embrionários/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Polaridade Celular , Proteínas Desgrenhadas/metabolismo , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Desenvolvimento Embrionário , Genes Reporter , Proteína Goosecoid/deficiência , Proteína Goosecoid/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Transdução de Sinais , Proteínas de Xenopus/genéticaRESUMO
Conjoined twins fused at the thorax display an enigmatic left-right defect: although left twins are normal, laterality is disturbed in one-half of right twins [1-3]. Molecularly, this randomization corresponds to a lack of asymmetric Nodal cascade induction in right twins [4]. We studied leftward flow [5, 6] at the left-right organizer (LRO) [7, 8] in thoracopagus twins in Xenopus, which displayed a duplicated, fused, and ciliated LRO. Cilia were motile and produced a leftward flow from the right LRO margin of the right to the left margin of the left twin. Motility was required for correct laterality in left twins, as knockdown of dynein motor dnah9 prevented Nodal cascade induction. Nodal was rescued by parallel knockdown of the inhibitor dand5 [9, 10] on the left side of the left twin. Lack of Nodal induction in the right twin, despite the presence of flow, was due to insufficient suppression of dand5. Knockdown of dand5 at the center of the fused LRO resulted in asymmetric Nodal cascade induction in the right twin as well. Manipulation of leftward flow and dand5 in a targeted and sided manner induced the Nodal cascade in a predictable manner, in the left twin, the right one, both, or neither. Laterality in conjoined twins thus was determined by cilia-driven leftward fluid flow like in single embryos, which solves a century-old riddle, as the phenomenon was already studied by some of the founders of experimental embryology, including Dareste [11], Fol and Warynsky [12], and Spemann and Falkenberg [13] (reviewed in [14]).
Assuntos
Padronização Corporal , Gêmeos Unidos/embriologia , Xenopus laevis , Proteínas de Anfíbios/genética , Animais , Cílios/fisiologia , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intercelular/genética , Gêmeos Unidos/patologiaRESUMO
Conjoined twins occur at low frequency in all vertebrates including humans. Many twins fused at the chest or abdomen display a very peculiar laterality defect: while the left twin is normal with respect to asymmetric organ morphogenesis and placement (situs solitus), the organ situs is randomized in right twins. Although this phenomenon has fascinated already some of the founders of experimental embryology in the 19th and early 20th century, such as Dareste, Fol, Warynsky and Spemann, its embryological basis has remained enigmatic. Here we summarize historical experiments and interpretations as well as current models, argue that the frog Xenopus is the only vertebrate model organism to tackle the issue, and outline suitable experiments to address the question of twin laterality in the context of cilia-based symmetry breakage.
Assuntos
Cílios/enzimologia , Gêmeos Unidos/embriologia , Xenopus/embriologia , Animais , Humanos , Modelos Animais , Xenopus/genéticaRESUMO
Laterality of inner organs is a wide-spread characteristic of vertebrates and beyond. It is ultimately controlled by the left-asymmetric activation of the Nodal signaling cascade in the lateral plate mesoderm of the neurula stage embryo, which results from a cilia-driven leftward flow of extracellular fluids at the left-right organizer. This scenario is widely accepted for laterality determination in wildtype specimens. Deviations from this norm come in different flavors. At the level of organ morphogenesis, laterality may be inverted (situs inversus) or non-concordant with respect to the main body axis (situs ambiguus or heterotaxia). At the level of Nodal cascade gene activation, expression may be inverted, bilaterally induced, or absent. In a given genetic situation, patterns may be randomized or predominantly lacking laterality (absence or bilateral activation). We propose that the distributions of patterns observed may be indicative of the underlying molecular defects, with randomizations being primarily caused by defects in the flow-generating ciliary set-up, and symmetrical patterns being the result of impaired flow sensing, on the left, the right, or both sides. This prediction, the reasoning of which is detailed in this review, pinpoints functions of genes whose role in laterality determination have remained obscure.
RESUMO
BACKGROUND: Left-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet. RESULTS: Here we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos. CONCLUSIONS: Based on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.
Assuntos
Embrião não Mamífero/citologia , Ouriços-do-Mar/embriologia , Animais , Dineínas do Axonema/metabolismo , Padronização Corporal , Cílios/metabolismo , Embrião não Mamífero/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Gastrulação , Ligantes da Sinalização Nodal/metabolismo , Ouriços-do-Mar/citologia , Ouriços-do-Mar/metabolismo , Gravação em VídeoRESUMO
During gastrulation and neurulation, foxj1 expression requires ATP4a-dependent Wnt/ß-catenin signaling for ciliation of the gastrocoel roof plate (Walentek et al. Cell Rep. 1 (2012) 516-527.) and the mucociliary epidermis (Walentek et al. Dev. Biol. (2015)) of Xenopus laevis embryos. These data suggested that ATP4a and Wnt/ß-catenin signaling regulate foxj1 throughout Xenopus development. Here we analyzed whether foxj1 expression was also ATP4a-dependent in other ciliated tissues of the developing Xenopus embryo and tadpole. We found that in the floor plate of the neural tube ATP4a-dependent canonical Wnt signaling was required for foxj1 expression, downstream of or in parallel to Hedgehog signaling. In the developing tadpole brain, ATP4-function was a prerequisite for the establishment of cerebrospinal fluid flow. Furthermore, we describe foxj1 expression and the presence of multiciliated cells in the developing tadpole gastrointestinal tract. Our work argues for a general requirement of ATP4-dependent Wnt/ß-catenin signaling for foxj1 expression and motile ciliogenesis throughout Xenopus development.
RESUMO
Proton pump inhibitors (PPIs), which target gastric H(+)/K(+)ATPase (ATP4), are among the most commonly prescribed drugs. PPIs are used to treat ulcers and as a preventative measure against gastroesophageal reflux disease in hospitalized patients. PPI treatment correlates with an increased risk for airway infections, i.e. community- and hospital-acquired pneumonia. The cause for this correlation, however, remains elusive. The Xenopus embryonic epidermis is increasingly being used as a model to study airway-like mucociliary epithelia. Here we use this model to address how ATP4 inhibition may affect epithelial function in human airways. We demonstrate that atp4a knockdown interfered with the generation of cilia-driven extracellular fluid flow. ATP4a and canonical Wnt signaling were required in the epidermis for expression of foxj1, a transcriptional regulator of motile ciliogenesis. The ATP4/Wnt module activated foxj1 downstream of ciliated cell fate specification. In multiciliated cells (MCCs) of the epidermis, ATP4a was also necessary for normal myb expression, apical actin formation, basal body docking and alignment of basal bodies. Furthermore, ATP4-dependent Wnt/ß-catenin signaling in the epidermis was a prerequisite for foxa1-mediated specification of small secretory cells (SSCs). SSCs release serotonin and other substances into the medium, and thereby regulate ciliary beating in MCCs and protect the epithelium against infection. Pharmacological inhibition of ATP4 in the mature mucociliary epithelium also caused a loss of MCCs and led to impaired mucociliary clearance. These data strongly suggest that PPI-associated pneumonia in human patients might, at least in part, be linked to dysfunction of mucociliary epithelia of the airways.
Assuntos
Infecção Hospitalar/etiologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Depuração Mucociliar/efeitos dos fármacos , Pneumonia/etiologia , Inibidores da Bomba de Prótons/efeitos adversos , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Animais Geneticamente Modificados , Infecção Hospitalar/fisiopatologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Silenciamento de Genes , ATPase Trocadora de Hidrogênio-Potássio/genética , Humanos , Depuração Mucociliar/fisiologia , Pneumonia/fisiopatologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/embriologia , Mucosa Respiratória/fisiopatologia , Via de Sinalização Wnt , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Asymmetric development of the vertebrate embryo has fascinated embryologists for over a century. Much has been learned since the asymmetric Nodal signaling cascade in the left lateral plate mesoderm was detected, and began to be unraveled over the past decade or two. When and how symmetry is initially broken, however, has remained a matter of debate. Two essentially mutually exclusive models prevail. Cilia-driven leftward flow of extracellular fluids occurs in mammalian, fish and amphibian embryos. A great deal of experimental evidence indicates that this flow is indeed required for symmetry breaking. An alternative model has argued, however, that flow simply acts as an amplification step for early asymmetric cues generated by ion flux during the first cleavage divisions. In this review we critically evaluate the experimental basis of both models. Although a number of open questions persist, the available evidence is best compatible with flow-based symmetry breakage as the archetypical mode of symmetry breakage.
Assuntos
Padronização Corporal , Vertebrados/embriologia , Animais , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/embriologia , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/embriologia , Peixes/embriologia , Regulação da Expressão Gênica no Desenvolvimento , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Fatores de Determinação Direita-Esquerda/metabolismo , Mamíferos/embriologia , Mesoderma/metabolismo , Proteína Nodal/metabolismo , Organizadores Embrionários/fisiologia , Serotonina/metabolismo , Transdução de Sinais/genética , Vertebrados/anatomia & histologia , Xenopus/embriologiaRESUMO
Morphological asymmetry is a common feature of animal body plans, from shell coiling in snails to organ placement in humans. The signaling protein Nodal is key for determining this laterality. Many vertebrates, including humans, use cilia for breaking symmetry during embryonic development: rotating cilia produce a leftward flow of extracellular fluids that induces the asymmetric expression of Nodal. By contrast, Nodal asymmetry can be induced flow-independently in invertebrates. Here, we ask when and why flow evolved. We propose that flow was present at the base of the deuterostomes and that it is required to maintain organ asymmetry in otherwise perfectly bilaterally symmetrical vertebrates.
Assuntos
Evolução Biológica , Padronização Corporal , Animais , Humanos , Organogênese , Vertebrados/embriologiaRESUMO
Vertebrates display asymmetric arrangements of inner organs such as heart and stomach. The Nodal signaling cascade in the left lateral plate mesoderm in all cases directs asymmetric morphogenesis and placement during organogenesis. Mechanisms that lead up to left-asymmetric Nodal induction seem to differ between the vertebrates. Cilia produce a leftward extracellular fluid flow in zebrafish, medaka, mouse, rabbit, and Xenopus embryos during neurulation. In Xenopus, earlier asymmetric cues were described. Some, such as Rab11, apparently act in the zygote. Others were efficiently manipulated in ventral-right cells at the four-cell stage, a lineage presumably independent of the ciliated gastrocoel roof plate (GRP) during neurulation. Here, we show that one- and four-cell manipulations of Rab11 showed equal low efficiencies of left-right disturbances. We also reevaluated the lineage of the GRP. By tracing back future ciliated cells from the gastrula to the four-cell stage, we show that ventral cells contribute to ciliated sensory cells at the border of the GRP. Knockdown of the Nodal inhibitor Coco in the ventral right lineage resulted in embryos with ectopic right-sided Nodal and Pitx2c expression. Together, these experiments support a cilia-based mechanism of symmetry breakage in the frog Xenopus.
Assuntos
Blastômeros/metabolismo , Padronização Corporal/fisiologia , Xenopus/embriologia , Proteínas rab de Ligação ao GTP/genética , Animais , Gástrula/embriologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The embryonic skin of Xenopus tadpoles serves as an experimental model system for mucociliary epithelia (MCE) such as the human airway epithelium. MCEs are characterized by the presence of mucus-secreting goblet and multiciliated cells (MCCs). A third cell type, ion-secreting cells (ISCs), is present in the larval skin as well. Synchronized beating of MCC cilia is required for directional transport of mucus. Here we describe a novel cell type in the Xenopus laevis larval epidermis, characterized by serotonin synthesis and secretion. It is termed small secretory cell (SSC). SSCs are detectable at early tadpole stages, unlike MCCs and ISCs, which are specified at early neurulation. Subcellularly, serotonin was found in large, apically localized vesicle-like structures, which were entirely shed into the surrounding medium. Pharmacological inhibition of serotonin synthesis decreased the velocity of cilia-driven fluid flow across the skin epithelium. This effect was mediated by serotonin type 3 receptor (Htr3), which was expressed in ciliated cells. Knockdown of Htr3 compromised flow velocity by reducing the ciliary motility of MCCs. SSCs thus represent a distinct and novel entity of the frog tadpole MCE, required for ciliary beating and mucus transport across the larval skin. The identification and characterization of SSCs consolidates the value of the Xenopus embryonic skin as a model system for human MCEs, which have been known for serotonin-dependent regulation of ciliary beat frequency.
Assuntos
Cílios/fisiologia , Células Epidérmicas , Epiderme/metabolismo , Serotonina/metabolismo , Xenopus/crescimento & desenvolvimento , Animais , Separação Celular , Embrião não Mamífero , Epiderme/embriologia , Epiderme/crescimento & desenvolvimento , Íons/metabolismo , Larva , Movimento/fisiologia , Muco/metabolismo , Receptores de Serotonina/fisiologiaRESUMO
The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences.
Assuntos
Epiderme/embriologia , Epiderme/imunologia , Células Caliciformes/imunologia , Imunidade Inata/fisiologia , Xenopus/embriologia , Xenopus/microbiologia , Animais , Diferenciação Celular/fisiologia , Cílios/imunologia , Embrião não Mamífero , Epiderme/metabolismo , Glicoproteínas/análise , Glicoproteínas/metabolismo , Fator 3-alfa Nuclear de Hepatócito/fisiologia , Íons/metabolismo , Larva , Muco/química , Muco/metabolismo , Via Secretória/imunologia , Vesículas Secretórias/imunologia , Vesículas Secretórias/metabolismo , Xenopus/imunologiaRESUMO
Breakage of bilateral symmetry in amphibian embryos depends on the development of a ciliated epithelium at the gastrocoel roof during early neurulation. Motile cilia at the gastrocoel roof plate (GRP) give rise to leftward flow of extracellular fluids. Flow is required for asymmetric gene expression and organ morphogenesis. Wnt signaling has previously been involved in two steps, Wnt/ß-catenin mediated induction of Foxj1, a regulator of motile cilia, and Wnt/planar cell polarity (PCP) dependent cilia polarization to the posterior pole of cells. We have studied Wnt11b in the context of laterality determination, as this ligand was reported to activate canonical and non-canonical Wnt signaling. Wnt11b was found to be expressed in the so-called superficial mesoderm (SM), from which the GRP derives. Surprisingly, Foxj1 was only marginally affected in loss-of-function experiments, indicating that another ligand acts in this early step of laterality specification. Wnt11b was required, however, for polarization of GRP cilia and GRP morphogenesis, in line with the known function of Wnt/PCP in cilia-driven leftward flow. In addition Xnr1 and Coco expression in the lateral-most GRP cells, which sense flow and generate the first asymmetric signal, was attenuated in morphants, involving Wnt signaling in yet another process related to symmetry breakage in Xenopus.