ABSTRACTFormation and maintenance of tissue barriers require the coordination of cell mechanics a... more ABSTRACTFormation and maintenance of tissue barriers require the coordination of cell mechanics and cell-cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to investigate how tight junctions regulate cell mechanics and epithelial morphogenesis. We found that depletion of the tight junction adaptor ZO-1 regulates cytoskeletal tension at cell-matrix and cell-cell interfaces in an ECM stiffness-regulated manner, possibly via differential organisation of the actin cytoskeleton. ZO-1 depletion inhibited junction assembly and disrupted morphogenesis in an ECM stiffness-dependent manner. Both processes were rescued by inhibition of cell contractility. Although ZO-1-deficient cells could assemble functional barriers at low tension, their tight junctions remained corrupted with strongly reduced and discontinuous recruitment of junctional components. Our results thus reveal that reciprocal regulation between ZO-1 ...
Destruction of the ventral noradrenergic pathway elicited by administration of 6-hydroxydopamine ... more Destruction of the ventral noradrenergic pathway elicited by administration of 6-hydroxydopamine (6-OHDA, 5 micrograms into each side of the ventral pons) reduced the content of norepinephrine (NE) in the anterior hypothalamus (-80%) and induced an increase in arterial blood pressure (ABP) and in heart rate. These hypertensive rats, showed hypersensitivity to the hypotensive effect of NE (0.5-2 micrograms) and clonidine (0.75-1.5 micrograms) administered into the anterior hypothalamic preoptic (AH/PO) region. Methysergide (1-2 micrograms) and, to a lesser extent, ketanserin (1-2 micrograms) administered into the anterior hypothalamic preoptic region also reduced the arterial blood pressure in these rats treated with 6-OHDA. Bilateral administration of 5,7-dihydroxytryptamine (5,7-DHT, 8 micrograms) into the median forebrain bundle decreased the content of serotonin (5-HT) in the hypothalamus (-85%) without change in arterial blood pressure but largely prevented the development of hypertension after treatment with 6-OHDA in the ventral pons. These results suggest that neurogenic hypertension is produced after the removal of NE tonic depressor activity in the anterior hypothalamus and that serotonergic mechanisms play a major role in the development of the increased arterial blood pressure in this preparation.
American Journal of Physiology-cell Physiology, Oct 1, 1995
Epithelial tight junctions form a regulated barrier that seals the paracellular space and prevent... more Epithelial tight junctions form a regulated barrier that seals the paracellular space and prevents mixing of luminal contents with the interstitium. This barrier is composed of a group of proteins including the putative "sealing" protein occludin that appears to bind directly to a cytoplasmic junction protein, ZO-1. To study the interaction and regulation of these two components when paracellular integrity is altered, we assessed protein expression and immunofluorescent (IF) localization of ZO-1 and occludin in a rat model of hepatocyte tight junction damage induced by common bile duct ligation (CBDL). Protein levels were detected in liver by immunoblotting and IF localization by 3-dimensional reconstruction of serial 0.5-micron confocal microscopic optical sections. As previously described, ZO-1 protein levels progressively increased to threefold control levels 9 days after CBDL. In contrast, occludin protein levels decreased by 50% within 2 days after CBDL and returned to control values by 9 days. In control IF sections, ZO-1 and occludin colocalized, forming thin continuous staining outlining canaliculi. After CBDL, ZO-1 staining appeared discontinuous, and a punctate pericanalicular accumulation of signal developed around junctional areas. Occludin staining was also discontinuous after CBDL, but, in contrast to ZO-1, was not punctate and remained localized either in a linear fashion along canalicular margins or in a homogeneous fashion in immediately surrounding areas. CBDL results in changes in the expression and localization of the putative tight junction sealing protein occludin in hepatocytes that are distinct from those observed for the peripheral membrane tight junction protein ZO-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Tissue morphogenesis in developmental or physiological processes is regulated by molecular and me... more Tissue morphogenesis in developmental or physiological processes is regulated by molecular and mechanical signals. While the molecular signaling cascades are increasingly well described, the mechanical signals affecting tissue shape changes have only recently been studied in greater detail. To gain more insight into the mechanochemical and biophysical basis of an epithelial spreading process (epiboly) in early zebrafish development, we studied cell-cell junction formation and actomyosin network dynamics at the boundary between surface layer epithelial cells (EVL) and the yolk syncytial layer (YSL). During zebrafish epiboly, the cell mass sitting on top of the yolk cell spreads to engulf the yolk cell by the end of gastrulation. It has been previously shown that an actomyosin ring residing within the YSL pulls on the EVL tissue through a cable-constriction and a flow-friction motor, thereby dragging the tissue vegetal wards. Pulling forces are likely transmitted from the YSL actomyosin ring to EVL cells; however, the nature and formation of the junctional structure mediating this process has not been well described so far. Therefore, our main aim was to determine the nature, dynamics and potential function of the EVL-YSL junction during this epithelial tissue spreading. Specifically, we show that the EVL-YSL junction is a mechanosensitive structure, predominantly made of tight junction (TJ) proteins. The process of TJ mechanosensation depends on the retrograde flow of non-junctional, phase-separated Zonula Occludens-1 (ZO-1) protein clusters towards the EVL-YSL boundary. Interestingly, we could demonstrate that ZO-1 is present in a non-junctional pool on the surface of the yolk cell, and ZO-1 undergoes a phase separation process that likely renders the protein responsive to flows. These flows are directed towards the junction and mediate proper tension-dependent recruitment of ZO-1. Upon reaching the EVL-YSL junction ZO-1 gets incorporated into the junctional pool mediated through its direct actin-binding domain. When the non-junctional pool and/or ZO-1 direct actin binding is absent, TJs fail in their proper mechanosensitive responses resulting in slower tissue spreading. We could further demonstrate that depletion of ZO proteins within the YSL results in diminished actomyosin ring formation. This suggests that a mechanochemical feedback loop is at work during zebrafish epiboly: ZO proteins help in proper actomyosin ring formation and actomyosin contractility and flows positively influence ZO-1 junctional recruitment. Finally, such a mesoscale polarization process mediated through the flow of phase-separated protein clusters might have implications for other processes such as immunological synapse formation, C. elegans zygote polarization and wound healing. A lot of people supported me during my journey of PhD and I am very grateful to all of them. I will start acknowledging people in a more or less chronological order. First, I would like to thank my supervisor Carl-Philipp Heisenberg for giving me the opportunity to work on this project in his lab. I very much appreciate the freedom that I had in order to develop my own ideas of how to tackle problems and also for his continuous support during my project. When I initially started working with zebrafish as model system, Hitoshi Morita and Martin Behrndt thought me the basic steps and principles of zebrafish handling and for this I would like to thank both of them. Further, I would like to thank Philipp Schmalhorst and Daniel Capek for their help with establishing the CRISPR/Cas9 system in the Heisenberg lab and for their support in the troubleshooting associated with it. Kornelija Pranjic-Ferscha, Deborah Kurtzemann and Verena Mayer supported me big time in fin clipping and genotyping of the many generated zebrafish mutants-thank you so much for that. Furthermore, Kornelija Pranjic-Ferscha also supported me in executing the qRT-PCRs and in cloning constructs such as Occludin-A-mNeonGreen. I am grateful to Shayan Shamipour, Robert Hauschild and Christoph Sommer for their continuous support in data analysis aspects. In addition, Shayan Shamipour and Alexandra Schauer helped me with some experiments in the revision period, which I am very grateful for (Shayan supported me with imaging retrograde flow in caMypt and caRhoA samples and with quantification of the junctional integration efficiency; Alexandra was doing in situs of papc and supported me in imaging of Phalloidin staining of ΔC, ΔABR constructs). I would like to thank Nicoletta Petridou and Diana Pinheiro for their invaluable critical feedback and fruitful discussions throughout my PhD studies-the continuous exchange of scientific questions and ideas was integral for my progress. Before I started my PhD at IST, Daria Siekhaus and Aparna Ratheesh initially introduced me into the scientific field; this included invaluable skill sets such as how to think critically and how to address scientific questions. Besides helping me in building up such skills, their mentorship was also critical for my scientific development, which I am tremendously grateful for. I would like to further thank Daria Siekhaus, my internal committee member, for the frequent meetings we had to discuss the progress of my project. Thanks also to my external committee member Antonio Jacinto for his input at certain milestones along my PhD and to our collaborators at UCL, Karl Matter, Masazumi Tada and Maria Balda, for their valuable input and feedback on the topic of tight junctions. I would like to thank Oliver Beutel and Alf Honigmann from MPI-CBG Dresden for the exchange on unpublished data and fruitful discussions about the phase separation of Zonula Occludens proteins. Along with all the previously mentioned people, I am grateful to the current and former members of the Heisenberg group, not only for the countless discussion on various scientific and nonscientific topics, but also for the supportive atmosphere:
International Journal of Developmental Neuroscience, Nov 16, 2006
Introduction and methods: Olfactory epithelium (OE) consists of multiple cell types such as olfac... more Introduction and methods: Olfactory epithelium (OE) consists of multiple cell types such as olfactory neurons, supporting cells, gland cells and basal cells and is derived from nasal placodes. Six1, a member of Six homeobox gene family, is first expressed in the nasal placodes and continues to be expressed throughout the OE development in mice. To understand how Six1 is involved in OE development, we analyzed Six1-deficient homozygous mice (Six1) by in situ hybridization with probes of various olfactory neuron marker genes as well as by immunohistochemistry. We also assessed the effects of Six1 on neural differentiation using P19EC cells. Results: Six1 showed defects in differentiation of olfactory neurons, supporting cells and gland cells. The expression of proneural marker Mash1 was unchanged, while those of Ngn1, NeuroD and Lhx2 were severely reduced at E10.5. Olfactory neuron specific marker protein OMP was missing in the Six1 at E16.5. The expression of Six1 was unaltered in the olfactory epithelium of Mash1 mice, indicating that Six1 and Mash1 regulate OE differentiation independently. The expressions of Hes1 and Hes5 in OE were augmented in the Six1 at E10.5. No supporting cells were observed in Six1 at E18.5. The expression of Ngn1, Mash1 and Hes1 upon neural differentiation induction of P19 cells was altered when Six1 was constitutively overexpressed. Discussion: Six1 is involved in differentiation of olfactory neuron and supporting cells in OE through regulation of Ngn1, NeuroD, Lhx2, Hes1 and Hes5.
The polarized distribution of Na ؉ ,K ؉-ATPase plays a paramount physiological role, because eith... more The polarized distribution of Na ؉ ,K ؉-ATPase plays a paramount physiological role, because either directly or through coupling with co-and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca 2؉ , K ؉ , Cl ؊ , and CO 3 H ؊ across the whole epithelium. We report here that the -subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na ؉ ,K ؉-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na ؉ ,K ؉-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with  1-subunit from the dog kidney (CHO-). 4) This may be attributed to the adhesive property of the  1-subunit, because an aggregation assay using CHO (mock-transfected) and CHO- cells shows that the expression of dog  1-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of  1-subunit forces CHO- cells to coexpress endogenous ␣-subunit. Together, our results indicate that MDCK cells express Na ؉ ,K ؉-ATPase at a given border provided the contacting cell expresses the dog  1-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na ؉ ,K ؉-ATPase in epithelial cells.
Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the par... more Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the paracellular route. To avoid breakdown of the epithelial barrier, this process requires coordinated opening and closing of tight junctions, the most apical intercellular junctions in epithelia. To determine the function of epithelial tight junction proteins in this process, we analyzed neutrophil migration across monolayers formed by stably transfected epithelial cells expressing wild-type and mutant occludin, a membrane protein of tight junctions with four transmembrane domains and both termini in the cytosol. We found that expression of mutants with a modified N-terminal cytoplasmic domain up-regulated migration, whereas deletion of the C-terminal cytoplasmic domain did not have an effect. The N-terminal cytosolic domain was also found to be important for the linear arrangement of occludin within tight junctions but not for the permeability barrier. Moreover, expression of mutant occludin bearing a mutation in one of the two extracellular domains inhibited neutrophil migration. The effects of transfected occludin mutants on neutrophil migration did not correlate with their effects on selective paracellular permeability and transepithelial electrical resistance. Hence, specific domains and functional properties of occludin modulate transepithelial migration of neutrophils.
Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High co... more Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High concentrations of ethanol trigger mucosal hyperregeneration, disrupt cell adhesion, and increase the sensitivity to carcinogens. Most of these effects are thought to be mediated by acetaldehyde, a genotoxic metabolite produced from ethanol by alcohol dehydrogenases. Here, we studied the role of low ethanol concentrations, more likely to mimic those found in the intestine in vivo, and used intestinal cells lacking alcohol dehydrogenase to identify the acetaldehyde-independent biological effects of ethanol. Under these conditions, ethanol did not stimulate the proliferation of nonconfluent cells, but significantly increased maximal cell density. Incorporation of phosphatidylethanol, produced from ethanol by phospholipase D, was instrumental to this effect. Phosphatidylethanol accumulation induced claudin-1 endocytosis and disrupted the claudin-1/ZO-1 association. The resulting nuclear translocation of ZONAB was shown to mediate the cell density increase in ethanol-treated cells. In vivo, incorporation of phosphatidylethanol and nuclear translocation of ZONAB correlated with increased proliferation in the colonic epithelium of ethanol-fed mice and in adenomas of chronic alcoholics. Our results show that phosphatidylethanol accumulation after chronic ethanol exposure disrupts signals that normally restrict proliferation in highly confluent intestinal cells, thus facilitating abnormal intestinal cell proliferation. (Mol Cancer Res 2007;5(11):1147–57)
Stereotactic injection of acetytcholine (0.5-2/~g) into the lateral septal region of the rat brai... more Stereotactic injection of acetytcholine (0.5-2/~g) into the lateral septal region of the rat brain produces a long-lasting sympatheticmediated increase of the arterial blood pressure. This effect is mediated by muscarinic receptors since 1 ~g atropine abolishes this response. In this same brain region, TRH (0.5-4/~g) did not elicit any significant change in the arterial blood pressure, but potentiated the effect of acetylcholine. This phenomenon is apparently due to an increase of the number of muscarinic receptors in the lateral septal area of the rat brain.
Cells from transporting epithelia have two basic properties: 1) a plasma membrane divided into an... more Cells from transporting epithelia have two basic properties: 1) a plasma membrane divided into an apical and a basolateral domain that confers vectoriality, and 2) tight junctions that transform epithelia into diffusion barriers. The expression of both properties depends on extracellular Ca2+.
ABSTRACTFormation and maintenance of tissue barriers require the coordination of cell mechanics a... more ABSTRACTFormation and maintenance of tissue barriers require the coordination of cell mechanics and cell-cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to investigate how tight junctions regulate cell mechanics and epithelial morphogenesis. We found that depletion of the tight junction adaptor ZO-1 regulates cytoskeletal tension at cell-matrix and cell-cell interfaces in an ECM stiffness-regulated manner, possibly via differential organisation of the actin cytoskeleton. ZO-1 depletion inhibited junction assembly and disrupted morphogenesis in an ECM stiffness-dependent manner. Both processes were rescued by inhibition of cell contractility. Although ZO-1-deficient cells could assemble functional barriers at low tension, their tight junctions remained corrupted with strongly reduced and discontinuous recruitment of junctional components. Our results thus reveal that reciprocal regulation between ZO-1 ...
Destruction of the ventral noradrenergic pathway elicited by administration of 6-hydroxydopamine ... more Destruction of the ventral noradrenergic pathway elicited by administration of 6-hydroxydopamine (6-OHDA, 5 micrograms into each side of the ventral pons) reduced the content of norepinephrine (NE) in the anterior hypothalamus (-80%) and induced an increase in arterial blood pressure (ABP) and in heart rate. These hypertensive rats, showed hypersensitivity to the hypotensive effect of NE (0.5-2 micrograms) and clonidine (0.75-1.5 micrograms) administered into the anterior hypothalamic preoptic (AH/PO) region. Methysergide (1-2 micrograms) and, to a lesser extent, ketanserin (1-2 micrograms) administered into the anterior hypothalamic preoptic region also reduced the arterial blood pressure in these rats treated with 6-OHDA. Bilateral administration of 5,7-dihydroxytryptamine (5,7-DHT, 8 micrograms) into the median forebrain bundle decreased the content of serotonin (5-HT) in the hypothalamus (-85%) without change in arterial blood pressure but largely prevented the development of hypertension after treatment with 6-OHDA in the ventral pons. These results suggest that neurogenic hypertension is produced after the removal of NE tonic depressor activity in the anterior hypothalamus and that serotonergic mechanisms play a major role in the development of the increased arterial blood pressure in this preparation.
American Journal of Physiology-cell Physiology, Oct 1, 1995
Epithelial tight junctions form a regulated barrier that seals the paracellular space and prevent... more Epithelial tight junctions form a regulated barrier that seals the paracellular space and prevents mixing of luminal contents with the interstitium. This barrier is composed of a group of proteins including the putative "sealing" protein occludin that appears to bind directly to a cytoplasmic junction protein, ZO-1. To study the interaction and regulation of these two components when paracellular integrity is altered, we assessed protein expression and immunofluorescent (IF) localization of ZO-1 and occludin in a rat model of hepatocyte tight junction damage induced by common bile duct ligation (CBDL). Protein levels were detected in liver by immunoblotting and IF localization by 3-dimensional reconstruction of serial 0.5-micron confocal microscopic optical sections. As previously described, ZO-1 protein levels progressively increased to threefold control levels 9 days after CBDL. In contrast, occludin protein levels decreased by 50% within 2 days after CBDL and returned to control values by 9 days. In control IF sections, ZO-1 and occludin colocalized, forming thin continuous staining outlining canaliculi. After CBDL, ZO-1 staining appeared discontinuous, and a punctate pericanalicular accumulation of signal developed around junctional areas. Occludin staining was also discontinuous after CBDL, but, in contrast to ZO-1, was not punctate and remained localized either in a linear fashion along canalicular margins or in a homogeneous fashion in immediately surrounding areas. CBDL results in changes in the expression and localization of the putative tight junction sealing protein occludin in hepatocytes that are distinct from those observed for the peripheral membrane tight junction protein ZO-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Tissue morphogenesis in developmental or physiological processes is regulated by molecular and me... more Tissue morphogenesis in developmental or physiological processes is regulated by molecular and mechanical signals. While the molecular signaling cascades are increasingly well described, the mechanical signals affecting tissue shape changes have only recently been studied in greater detail. To gain more insight into the mechanochemical and biophysical basis of an epithelial spreading process (epiboly) in early zebrafish development, we studied cell-cell junction formation and actomyosin network dynamics at the boundary between surface layer epithelial cells (EVL) and the yolk syncytial layer (YSL). During zebrafish epiboly, the cell mass sitting on top of the yolk cell spreads to engulf the yolk cell by the end of gastrulation. It has been previously shown that an actomyosin ring residing within the YSL pulls on the EVL tissue through a cable-constriction and a flow-friction motor, thereby dragging the tissue vegetal wards. Pulling forces are likely transmitted from the YSL actomyosin ring to EVL cells; however, the nature and formation of the junctional structure mediating this process has not been well described so far. Therefore, our main aim was to determine the nature, dynamics and potential function of the EVL-YSL junction during this epithelial tissue spreading. Specifically, we show that the EVL-YSL junction is a mechanosensitive structure, predominantly made of tight junction (TJ) proteins. The process of TJ mechanosensation depends on the retrograde flow of non-junctional, phase-separated Zonula Occludens-1 (ZO-1) protein clusters towards the EVL-YSL boundary. Interestingly, we could demonstrate that ZO-1 is present in a non-junctional pool on the surface of the yolk cell, and ZO-1 undergoes a phase separation process that likely renders the protein responsive to flows. These flows are directed towards the junction and mediate proper tension-dependent recruitment of ZO-1. Upon reaching the EVL-YSL junction ZO-1 gets incorporated into the junctional pool mediated through its direct actin-binding domain. When the non-junctional pool and/or ZO-1 direct actin binding is absent, TJs fail in their proper mechanosensitive responses resulting in slower tissue spreading. We could further demonstrate that depletion of ZO proteins within the YSL results in diminished actomyosin ring formation. This suggests that a mechanochemical feedback loop is at work during zebrafish epiboly: ZO proteins help in proper actomyosin ring formation and actomyosin contractility and flows positively influence ZO-1 junctional recruitment. Finally, such a mesoscale polarization process mediated through the flow of phase-separated protein clusters might have implications for other processes such as immunological synapse formation, C. elegans zygote polarization and wound healing. A lot of people supported me during my journey of PhD and I am very grateful to all of them. I will start acknowledging people in a more or less chronological order. First, I would like to thank my supervisor Carl-Philipp Heisenberg for giving me the opportunity to work on this project in his lab. I very much appreciate the freedom that I had in order to develop my own ideas of how to tackle problems and also for his continuous support during my project. When I initially started working with zebrafish as model system, Hitoshi Morita and Martin Behrndt thought me the basic steps and principles of zebrafish handling and for this I would like to thank both of them. Further, I would like to thank Philipp Schmalhorst and Daniel Capek for their help with establishing the CRISPR/Cas9 system in the Heisenberg lab and for their support in the troubleshooting associated with it. Kornelija Pranjic-Ferscha, Deborah Kurtzemann and Verena Mayer supported me big time in fin clipping and genotyping of the many generated zebrafish mutants-thank you so much for that. Furthermore, Kornelija Pranjic-Ferscha also supported me in executing the qRT-PCRs and in cloning constructs such as Occludin-A-mNeonGreen. I am grateful to Shayan Shamipour, Robert Hauschild and Christoph Sommer for their continuous support in data analysis aspects. In addition, Shayan Shamipour and Alexandra Schauer helped me with some experiments in the revision period, which I am very grateful for (Shayan supported me with imaging retrograde flow in caMypt and caRhoA samples and with quantification of the junctional integration efficiency; Alexandra was doing in situs of papc and supported me in imaging of Phalloidin staining of ΔC, ΔABR constructs). I would like to thank Nicoletta Petridou and Diana Pinheiro for their invaluable critical feedback and fruitful discussions throughout my PhD studies-the continuous exchange of scientific questions and ideas was integral for my progress. Before I started my PhD at IST, Daria Siekhaus and Aparna Ratheesh initially introduced me into the scientific field; this included invaluable skill sets such as how to think critically and how to address scientific questions. Besides helping me in building up such skills, their mentorship was also critical for my scientific development, which I am tremendously grateful for. I would like to further thank Daria Siekhaus, my internal committee member, for the frequent meetings we had to discuss the progress of my project. Thanks also to my external committee member Antonio Jacinto for his input at certain milestones along my PhD and to our collaborators at UCL, Karl Matter, Masazumi Tada and Maria Balda, for their valuable input and feedback on the topic of tight junctions. I would like to thank Oliver Beutel and Alf Honigmann from MPI-CBG Dresden for the exchange on unpublished data and fruitful discussions about the phase separation of Zonula Occludens proteins. Along with all the previously mentioned people, I am grateful to the current and former members of the Heisenberg group, not only for the countless discussion on various scientific and nonscientific topics, but also for the supportive atmosphere:
International Journal of Developmental Neuroscience, Nov 16, 2006
Introduction and methods: Olfactory epithelium (OE) consists of multiple cell types such as olfac... more Introduction and methods: Olfactory epithelium (OE) consists of multiple cell types such as olfactory neurons, supporting cells, gland cells and basal cells and is derived from nasal placodes. Six1, a member of Six homeobox gene family, is first expressed in the nasal placodes and continues to be expressed throughout the OE development in mice. To understand how Six1 is involved in OE development, we analyzed Six1-deficient homozygous mice (Six1) by in situ hybridization with probes of various olfactory neuron marker genes as well as by immunohistochemistry. We also assessed the effects of Six1 on neural differentiation using P19EC cells. Results: Six1 showed defects in differentiation of olfactory neurons, supporting cells and gland cells. The expression of proneural marker Mash1 was unchanged, while those of Ngn1, NeuroD and Lhx2 were severely reduced at E10.5. Olfactory neuron specific marker protein OMP was missing in the Six1 at E16.5. The expression of Six1 was unaltered in the olfactory epithelium of Mash1 mice, indicating that Six1 and Mash1 regulate OE differentiation independently. The expressions of Hes1 and Hes5 in OE were augmented in the Six1 at E10.5. No supporting cells were observed in Six1 at E18.5. The expression of Ngn1, Mash1 and Hes1 upon neural differentiation induction of P19 cells was altered when Six1 was constitutively overexpressed. Discussion: Six1 is involved in differentiation of olfactory neuron and supporting cells in OE through regulation of Ngn1, NeuroD, Lhx2, Hes1 and Hes5.
The polarized distribution of Na ؉ ,K ؉-ATPase plays a paramount physiological role, because eith... more The polarized distribution of Na ؉ ,K ؉-ATPase plays a paramount physiological role, because either directly or through coupling with co-and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca 2؉ , K ؉ , Cl ؊ , and CO 3 H ؊ across the whole epithelium. We report here that the -subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na ؉ ,K ؉-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na ؉ ,K ؉-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with  1-subunit from the dog kidney (CHO-). 4) This may be attributed to the adhesive property of the  1-subunit, because an aggregation assay using CHO (mock-transfected) and CHO- cells shows that the expression of dog  1-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of  1-subunit forces CHO- cells to coexpress endogenous ␣-subunit. Together, our results indicate that MDCK cells express Na ؉ ,K ؉-ATPase at a given border provided the contacting cell expresses the dog  1-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na ؉ ,K ؉-ATPase in epithelial cells.
Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the par... more Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the paracellular route. To avoid breakdown of the epithelial barrier, this process requires coordinated opening and closing of tight junctions, the most apical intercellular junctions in epithelia. To determine the function of epithelial tight junction proteins in this process, we analyzed neutrophil migration across monolayers formed by stably transfected epithelial cells expressing wild-type and mutant occludin, a membrane protein of tight junctions with four transmembrane domains and both termini in the cytosol. We found that expression of mutants with a modified N-terminal cytoplasmic domain up-regulated migration, whereas deletion of the C-terminal cytoplasmic domain did not have an effect. The N-terminal cytosolic domain was also found to be important for the linear arrangement of occludin within tight junctions but not for the permeability barrier. Moreover, expression of mutant occludin bearing a mutation in one of the two extracellular domains inhibited neutrophil migration. The effects of transfected occludin mutants on neutrophil migration did not correlate with their effects on selective paracellular permeability and transepithelial electrical resistance. Hence, specific domains and functional properties of occludin modulate transepithelial migration of neutrophils.
Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High co... more Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High concentrations of ethanol trigger mucosal hyperregeneration, disrupt cell adhesion, and increase the sensitivity to carcinogens. Most of these effects are thought to be mediated by acetaldehyde, a genotoxic metabolite produced from ethanol by alcohol dehydrogenases. Here, we studied the role of low ethanol concentrations, more likely to mimic those found in the intestine in vivo, and used intestinal cells lacking alcohol dehydrogenase to identify the acetaldehyde-independent biological effects of ethanol. Under these conditions, ethanol did not stimulate the proliferation of nonconfluent cells, but significantly increased maximal cell density. Incorporation of phosphatidylethanol, produced from ethanol by phospholipase D, was instrumental to this effect. Phosphatidylethanol accumulation induced claudin-1 endocytosis and disrupted the claudin-1/ZO-1 association. The resulting nuclear translocation of ZONAB was shown to mediate the cell density increase in ethanol-treated cells. In vivo, incorporation of phosphatidylethanol and nuclear translocation of ZONAB correlated with increased proliferation in the colonic epithelium of ethanol-fed mice and in adenomas of chronic alcoholics. Our results show that phosphatidylethanol accumulation after chronic ethanol exposure disrupts signals that normally restrict proliferation in highly confluent intestinal cells, thus facilitating abnormal intestinal cell proliferation. (Mol Cancer Res 2007;5(11):1147–57)
Stereotactic injection of acetytcholine (0.5-2/~g) into the lateral septal region of the rat brai... more Stereotactic injection of acetytcholine (0.5-2/~g) into the lateral septal region of the rat brain produces a long-lasting sympatheticmediated increase of the arterial blood pressure. This effect is mediated by muscarinic receptors since 1 ~g atropine abolishes this response. In this same brain region, TRH (0.5-4/~g) did not elicit any significant change in the arterial blood pressure, but potentiated the effect of acetylcholine. This phenomenon is apparently due to an increase of the number of muscarinic receptors in the lateral septal area of the rat brain.
Cells from transporting epithelia have two basic properties: 1) a plasma membrane divided into an... more Cells from transporting epithelia have two basic properties: 1) a plasma membrane divided into an apical and a basolateral domain that confers vectoriality, and 2) tight junctions that transform epithelia into diffusion barriers. The expression of both properties depends on extracellular Ca2+.
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Papers by Maria Balda