Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo

The peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12-13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types. The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.     

Development of Endothelial Paracellular Clefts and their Tight Junctions in the Pial Microvessels of the Rat

The microvessels of the pia mater lack an investment with astrocyte processes but nonetheless have a high transendothelial electrical resistance which has caused them to be regarded as part of the blood-brain barrier. This high resistance is known to be acquired in the perinatal period. The aim of our study was to relate the known physiological changes with differentiation of the endothelial paracellular clefts and especially of their tight junctions which provide the basis for the high transendothelial resistance of blood-brain barrier vessels. Tight junctions of endothelial cell paracellular clefts in pial microvessels were examined by transmission electron microscopy using goniometric tilting to reveal and measure membrane separations at tight junctions in fetal, postnatal and adult rats. These tight junctional membrane separations narrowed over the period (E16:6.3 nm, D1:6.4 nm, D7:5.4 nm) and differentiated into two groups by the adult stage: one with a membrane separation of 2.8 nm and the staining characteristics of non-brain endothelial junctions, and the other with no detectable membrane separation and the staining characteristic of blood-brain barrier endothelial junctions. This patchy and incomplete differentiation of pial tight junctions into a blood-brain barrier-like form could result either from non-uniform exposure to inductive signals or to local variation in responsiveness to such agents. Although these changes in junction organization may be related to the known increase in pial transendothelial resistance in the perinatal period, we have not yet identified any sharply defined structural change which coincides with this physiological event.     

A new angle on blood–CNS interfaces: A role for connexins?

Neuronal signaling in the CNS depends on the microenvironment around synapses and axons. To prevent fluctuations in blood composition affecting the interstitial fluid and CSF, two barriers, the blood–brain barrier (BBB) and blood–CSF barrier (BCSFB), are interposed between the blood and the brain/CSF compartment. Brain capillary endothelial cells (ECs) constitute the BBB whereas choroid plexus epithelial (CPE) cells form the BCSFB. The anatomical basis of these barriers is located at the level of an intercellular junctional complex that impedes paracellular diffusion. Tight and adherens junctions are known as the principal constituents of this junctional complex. Transmembrane connexins (Cxs) are the prime building blocks of plasma membrane hemichannels that combine to form intercellular gap junctions (GJ). Although Cxs co-exist within the junctional complex, their influence on tight/adherens junctions and their role in barrier function of BBB ECs and CPE has been mostly ignored. Here, we review current knowledge on the role of Cxs in the BBB, BCSFB and other interfaces that subside within the CNS. We conclude that Cxs are a rather unexplored but promising target for influencing CNS barrier function.     

Evidence for the effectiveness of the blood--CSF barrier in the fetal rat choroid plexus. A freeze-fracture and peroxidase diffusion study

The blood--cerebrospinal fluid (CSF) barrier in the choroid plexus is principally constituted of apical junctional complexes between epithelial cells. The effectiveness of this barrier was studied during the fetal development in the rat. Choroid plexuses from fetuses (14th and 18th embryonic day) and newborn (1 and 6 day old) rats were examined after intravascular administration of a proteic tracer (horseradish peroxidase) and investigated by freeze-fracture. From the 14th day of fetal life, apical junctions were seen to constitute a barrier that prevents the passage of peroxidase from blood to CSF; the tight junctions were morphologically similar to those of the mature animals; the junctional fibrils appeared continuous on complementary replicas. These data suggest that, from the 14th day of fetal development, the blood--CSF barrier is both morphologically and physiologically mature.     

Junctions in the central nervous system of the cat

Summary The leptomeningeal tissue of the choroid plexuses and of the brain surfaces have been studied by means of the freeze-etching technique. The pia-arachnoid membrane and the subdural neurothel represent the morphological barrier between the extracerebral tissue and the cerebrospinal compartment. The freeze-etch findings indicate that the arachnoid and neurothelial cells are coupled by extensive zonulae occludentes which seem to represent the structural basis of the barrier mechanism provided by these cell layers. Furthermore, it became evident that gap junctions of considerable structural heterogeneity occur on the pial and arachnoid cells of the interstitial choroidal compartment and of the free brain surfaces. The structural heterogeneity of the nexuses is taken as an indication of the plasticity of the leptomeningeal tissue. The different morphological characteristics of the nexal formations are discussed with respect to their probable functional meaning.This investigation was supported by the Deutsche Forschungsgemeinschaft SFB 114 (Bionach).     

Characterization of glial fibrillary acidic protein and astroglial architecture in the brain of a continuously growing fish, the rainbow trout

Unlike mammals, some fish, including carp and trout, have a continuously growing brain. The glial architecture of teleost brain has been intensively studied in the carp and few data exist on trout brain. In this study, using immunoblotting we characterized the topographic distribution of glial fibrillary acidic protein (GFAP) in larval and adult rainbow trout brain and studied by immunohistochemistry the distribution and morphology of GFAP-immunoreactive cell systems in the rainbow trout hindbrain and spinal cord. Immunoblotting yielded a double band with an apparent molecular weight of 50-52 kDa in the spinal cord homogenate in the trout larval and adult stages. In the adult hindbrain and forebrain, our antibody cross reacted also with a second band at a higher molecular weight (90 kDa). Because the forebrain contained this band alone the two brain regions might contain two distinct isoforms. Conversely, the larval total brain homogenate contained the heavy 90 kDa band alone. Hence the heavy band might be a GFAP protein dimer or vimentin/GFAP copolymer reflecting nerve fiber growth and elongation, or the two isoforms might indicate two distinct astroglial cell types as recently proposed in the zebrafish. In sections from trout hindbrain and spinal cord the antibody detected a GFAP-immunoreactive glial fiber system observed in the raphe and in the glial septa separating the nerve tracts. These radial glia fibers thickened toward the pial surface, where they formed glial end feet. The antibody also labeled perivascular glia around blood vessels in the white matter, and the ependymoglial plexus surrounding the ventricular surface in the grey matter. Last, it labeled round astrocytes. The GFAP-immunoreactive glial systems had similar distribution patterns in the adult and larval spinal cord suggesting early differentiation.     

Development of an apical plasma membrane domain and tight junctions during histogenesis of the mammalian pancreas

The role of tight junctions (zonula occludens) in the formation of apical plasma membrane (PM) domains was investigated in the embryonic rat pancreas. In the present study, lectin-rhodamine (WGA-TRITC and RCAII-TRITC) and lectin-gold (WGA-Au and RCAII-Au) conjugates were used to monitor apical PM domain formation and freeze-fracture analysis was used to monitor tight junction formation in the pancreatic epithelium of embryonic, neonatal, and adult rats. Fluorescent and TEM analysis of WGA and RCAII binding indicated that an apical PM domain is formed as early as Day 13 of gestation in the pancreatic epithelium. While apical WGA binding remained into adult life, RCAII binding was lost by 1 day after birth. In contrast, tight junctions were not observed until Day 14 of gestation. At this time, tight junctions were found to be incomplete in formation and typically consisted of linear arrays of IMPs or discontinuous arrays of sealing strands (focal adherens). Continuous tight junctions were not completely formed until Day 15 of gestation. Continued development of tight junctions during gestation was characterized by (1) an increase in the number of sealing strands and (2) a more parallel arrangement of sealing strands within each junctional complex. By 8 weeks after birth, tight junctions were more loosely organized and contained fewer sealing strands as compared to that observed in the fetus. These results suggest that lateral diffusion of apical PM glycoconjugates may be restricted even in the absence of complete tight junctional complexes during development of the rat pancreas.     

Cell to cell relationships in the seminiferous epithelium in the mouse embryo

Summary Germ cells and Sertoli cells in embryonic mouse testes (day 14 to 20 of gestation) were examined by sectioning and freeze-fracture. Intercellular cytoplasmic bridges between the germ cells are observed in day 14 and older embryos. Membrane specializations with dense fuzzy material similar to the socalled desmosome-like structures are found between Sertoli cells and germ cells. A cell contact area with dense opposed membranes is also found between adjacent germ cells. Asymmetrical dense fuzzy lining of both Sertoli and germ cell membranes is noted. Pinocytotic pits or caveolae are frequently found in the Sertoli cell membrane. Between adjacent Sertoli cells, gap junctions of various sizes and focal meshworks of the occluding junctions are found. Most of the occluding junctional particles are located in the center of the grooves in the E face, and are similar to those in postnatal and adult Sertoli cell junctions. In addition, on both fractured faces there are ridges and grooves devoid of particles which are continuous with occluding junctions with particles, suggesting an initial stage in the formation of occluding junctions of the Sertoli cells. Particles gathered at the site of desmosome-like structures are present on the P face of the Sertoli cell.This work is supported by the Japanese Ministry of Education     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions

In the central nervous system (CNS) of pupal Calliphora, dramatic alterations occur in the perineurial and glial gap junctions. Having formed macular plaques by late larval stages, in early pupae cell migration causes the EF intramembranous junctional particles to disaggregate and move apart into linear and then disorganised arrays as shown by freeze-fracture. After nerve and glial cell reorganisation into the adult pattern, the gap junctions begin to reform in the late pupae, again seemingly by particle migration into linear arrays and clusters. Ultimately the particles form numerous macular plaques between both perineurial and glial cells. Statistical analyses support the contention that these are performed EF particles which undergo translateral movement from macular larval junctions into the disaggregated particles of early pupae and that the same particles appear to undergo realignment and reclustering in late pupae to form the mature gap junctions of adults. This is the first report to indicate breakdown and reformation of gap junctions in vivo involving reutilisation of the same intramembranous particles. Perineurial “tight” junctions are not to be found in early pupal stages and their absence can be correlated with the free entry of ionic lanthanum into the CNS observed during that period. In late pupae, when the tight junctional moniliform ridges have apparently reformed, the entry of the tracer lanthanum becomes restricted to the level of the perineurium, penetrating no deeper. This is also the case in the adult, where the blood-brain barrier is maintained. PF particles in the form of short linear ridges and clustered particle arrays in nerve cell membranes are present throughout pupal and adult stages; their continued presence throughout the whole of development suggests some role in neuronal function, as yet unclear.     

Predominance of VH-D-JH junctions occurring at sites of short sequence homology results in limited junctional diversity in neonatal antibodies.

Sequence diversity at the junctions of Ig genes differs between newborn and adult mice in two respects: 1) fetal/newborn Ig lack N regions; and 2) these N- junctional sequences very often contain 1 to 6 nucleotides that could have been encoded by either of the two joined gene segments. We address the hypothesis that such short homologies preferentially direct recombination to that site, and we analyze the effect of such homology-directed recombination upon the neonatal Ig repertoire. We examined 546 CDR3 sequences that were generated from polymerase chain reaction-amplified DNA from fetal and newborn liver using primers from three different VH families: S107, 7183, and J558. All junctional sequences using 14 frequently occurring IgH V-D and D-J gene combinations were analyzed. In 12 of the 14 combinations analyzed, there were 1 to 3 short sequence homologies, and the junctional sequences that would be created by those homologies were observed with high frequency. The D-J junctions often had two to three predominant junctional sequences, whereas the V-D junctions had one dominant junctional sequence. The only exceptions were the VHJ558-D junctions, where homology-directed recombination using the sequence homology between VHJ558 genes and most D genes would result in an out-of-frame join, and most of our sequences were productive. This latter result further suggests that homology-directed recombination may play a role in the nonrandom VH gene usage observed in fetal and newborn mice. Thus, most neonatal IgH junctions show limited diversity, not only due to the lack of N regions, but also because of nonrandom junctional sequences. Inasmuch as the few adult N- junctions also show a high frequency of homology-directed junctional sequences, V-D-J recombination throughout life may involve pairing via short homologies, with addition of N regions obscuring its role in the formation of adult IgH junctions.     

Developmental changes in BDNF protein levels in the hamster retina and superior colliculus

Quantitative studies of ontogenetic changes in the levels of brain‐derived neurotrophic factor (BDNF) mRNA and its effector, BDNF protein, are not available for the retinal projection system. We used an electrochemiluminescence immunoassay to measure developmental changes in the tissue concentration of BDNF within the hamster retina and superior colliculus (SC). In the SC, we first detected BDNF (about 9 pg/mg tissue) on embryonic day 14 (E14). BDNF protein concentration in the SC rises about fourfold between (E14) and postnatal day 4 (P4), remains at a plateau through P15, then declines by about one‐third to attain its adult level by P18. By contrast, BDNF protein concentration in the retina remains low (about 1 pg/mg tissue) through P12, then increases 4.5‐fold to attain its adult level on P18. The developmental changes in retinal and collicular BDNF protein concentrations are temporally correlated with multiple events in the structural and functional maturation of the hamster retinal projection system. Our data suggest roles for BDNF in the cellular mechanisms underlying some of these events and are crucial to the design of experiments to examine those roles. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 173–187, 2001     

Desmosomal adhesiveness is developmentally regulated in the mouse embryo and modulated during trophectoderm migration

During embryonic development tissues remain malleable to participate in morphogenetic movements but on completion of morphogenesis they must acquire the toughness essential for independent adult life. Desmosomes are cell-cell junctions that maintain tissue integrity especially where resistance to mechanical stress is required. Desmosomes in adult tissues are termed hyper-adhesive because they adhere strongly and are experimentally resistant to extracellular calcium chelation. Wounding results in weakening of desmosomal adhesion to a calcium-dependent state, presumably to facilitate cell migration and wound closure. Since desmosomes appear early in mouse tissue development we hypothesised that initial weak adhesion would be followed by acquisition of hyper-adhesion, the opposite of what happens on wounding. We show that epidermal desmosomes are calcium-dependent until embryonic day 12 (E12) and become hyper-adhesive by E14. Similarly, trophectodermal desmosomes change from calcium-dependence to hyper-adhesiveness as blastocyst development proceeds from E3 to E4.5. In both, development of hyper-adhesion is accompanied by the appearance of a midline between the plasma membranes supporting previous evidence that hyper-adhesiveness depends on the organised arrangement of desmosomal cadherins. By contrast, adherens junctions remain calcium-dependent throughout but tight junctions become calcium-independent as desmosomes mature. Using protein kinase C (PKC) activation and PKCα-/- mice, we provide evidence suggesting that conventional PKC isoforms are involved in developmental progression to hyper-adhesiveness. We demonstrate that modulation of desmosomal adhesion by PKC can regulate migration of trophectoderm. It appears that tissue stabilisation is one of several roles played by desmosomes in animal development.     

Coupled heterocellular arrays in the brain

Gap junctions are transcellular pathways that enable a dynamic metabolic coupling and a selective exchange of biological signaling mediators. Throughout the course of the brain development these intercellular channels are assembled into regionally and temporally defined patterns. The present review summarizes the possibilities of heterocellular gap junctional pairing in the brain parenchyma, involving glial cells, neurons and neural precursors as well as it highlights on the meaningfulness of these coupled arrays to the concept of brain functional compartments.     

Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation

Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.     

Ultrastructural localization of lectin-binding sites and laminin-like immunoreactivity in glial cells and neurites growing out from explant cultures of the central nervous system of embryonic locusts

Summary Lectins with different sugar specificities and labeled with horseradish peroxidase or gold were used to study, at the electron-microscopic level, surface glycoconjugates of glial cells and neurites growing out from explant cultures of the central nervous system of embryonic locusts. Differential binding to differentiating glial cells and to neurites was demonstrated. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) bound to glial and neurite surfaces with different degrees of labeling. The formation of glial processes and junctional complexes was invariably accompanied by a corresponding increase of Con A- and WGA-receptors. Peanut agglutinin (PNA) failed to bind to glial cells but strongly stained the plasma membrane of neurite junctions. Lotus tetragonolobus a. (LTA) did not bind either to glial cells or to neurites. In addition, staining with an antibody against laminin showed labeling in areas of neurite outgrowth and neurite interactions; this resembled the localization of PNA receptors. These findings provide evidence for the presence of different carbohydrates at the surface of neurites and glial cells of locust. Their predominant localization in glial processes and neurite junctions suggests that these carbohydrates constitute part of a group adhesion glycoproteins that also includes laminin.     

Functional Evaluation of Low Resistance Junctions: Influence of Cell Shape and Size

Specialized junctions, which allow small molecules to move directlybetween adjacent cells in many adult and embryonic tissues,may be involved in electrical or non-electrical forms of intercellularcommunication. The ability of the junctions to mediate eitherform of communication depends on the permeability of the junctionsand on the shape, size, and arrangement of the interconnectedcells. Electrical communication depends on junctional resistanceand on non-junctional resistance which is a function of cellsurface area. Non-electrical communication, it is argued, dependson junctional permeability to small molecules and on cell volume.The dual dependency of non-electrical communication in particularis discussed in detail and some of the possible implicationsare illustrated with specific examples.     

Dye coupling and connexin expression by cortical radial glia in the early postnatal subventricular zone

In this study, we have analyzed the specific contribution of the cortical radial glia (RG) for gap junctional communication (GJC) within the postnatal subventricular zone (SVZ). To specifically target RG as source of dye‐coupling in situ, we have developed a new technique that involves direct cell loading through the processes that reach the pial surface, with a mix of gap junction permeant (Lucifer yellow, LY) and nonpermeant (rhodamine‐conjugated dextran 3 KDa, RD) fluorochromes, the latter used as a marker for direct loaded cells. Tissue sections were analyzed for identification of directly loaded (LY+RD+) and coupled cells (LY+RD–) in the SVZ. Directly loaded cells were restricted to the region underlying the pial loading surface area. Coupled cells were distributed in a bistratified manner, along the outer dorsal surface of the SVZ and aligning the ventricle, leaving the SVZ core relatively free. Blocking GJC prior to pial loading greatly reduced dye coupling. Phenotypic analysis indicated that coupling by RG excludes neuroblasts and is mostly restricted to cells of glial lineage. Notwithstanding, no corresponding restriction to specific cell phenotype was found for two connexin isotypes, Cx43 and Cx45, in the postnatal SVZ. The extensive homocellular cell coupling by RG suggests an important role in the regulation of neurogenesis and functional compartmentalization of the postnatal SVZ. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 2012     

Totally tubular: the mystery behind function and origin of the brain ventricular system

A unique feature of the vertebrate brain is the ventricular system, a series of connected cavities which are filled with cerebrospinal fluid (CSF) and surrounded by neuroepithelium. While CSF is critical for both adult brain function and embryonic brain development, neither development nor function of the brain ventricular system is fully understood. In this review, we discuss the mystery of why vertebrate brains have ventricles, and whence they originate. The brain ventricular system develops from the lumen of the neural tube, as the neuroepithelium undergoes morphogenesis. The molecular mechanisms underlying this ontogeny are described. We discuss possible functions of both adult and embryonic brain ventricles, as well as major brain defects that are associated with CSF and brain ventricular abnormalities. We conclude that vertebrates have taken advantage of their neural tube to form the essential brain ventricular system.