Extracellular Fluid Flow Research Articles

Space flight/bedrest immobilization and bone. Signaling cascade induced by mechanical stress

Mechanical stress to bone plays an important role in the maintenance of bone homeostasis. Mechanical stress initiates multiple signaling pathways in osteocytes and osteoblasts. Mechanical stress to bone induces fluid shear stress (FSS) caused by a change in extracellular fluid flow. FSS induces Ca(2+) influx that activates downstream CREB-AP-1-IL-11 signaling as well as nitric oxide synthesis. Interaction of integrins with matrix proteins such as periostin also activates intracellular signaling. These signaling pathways appear to enhance osteoblast differentiation and bone formation via Wnt/β-catenin signaling. The elucidation of signaling cascade in response to mechanical stress can lead to the development of new therapeutic agents.

Serotonin and ATP4 are required for Wnt signaling and cilia-driven leftward flow in Xenopus

In fish and mammalian neurula embryos midline epithelia (Kupffer's vesicle, posterior notochord/node) harbor polarized monocilia, which rotate in a clockwise manner to produce a leftward extracellular fluid flow. Flow induces asymmetric gene expression on the left side, resulting in asymmetric organ morphogenesis and placement. In frog, much earlier asymmetries were described for the monoamine serotonin and the ion pump gastric H+/K+-ATPase (ATP4). The 'ion-flux' model proposed that an ATP4-generated voltage gradient drives serotonin through gap junctions to asymmetrically enrich on one side of the embryo to break symmetry upstream of flow. Here we show that both serotonin and ATP4 were symmetrically expressed in Xenopus and required for Wnt-mediated setup of leftward flow. Flow and asymmetry were lost in embryos in which serotonin signaling was down-regulated. Serotonin was required as competence factor of Wnt signaling, which provides the instructive signal to specify the superficial mesoderm (SM), i.e. the structure which gives rise to the ciliated gastrocoel roof plate (GRP) where flow in frog occurs. Gene knock-down or pharmacological inhibition of ATP4 compromised organ situs, asymmetric gene expression and leftward flow. The GRP revealed fewer, shortened and misaligned cilia. FoxJ1, a master control gene of motile cilia, was down-regulated in the SM. Specifically, ATP4 was required for Wnt/s-catenin regulated Foxj1 induction and Wnt/PCP dependent cilia polarization. Our work confirms flow as primary mechanism of symmetry breakage in frog and argues for evolutionary conservation of symmetry breakage in vertebrates.

Serotonin Signaling Is Required for Wnt-Dependent GRP Specification and Leftward Flow in Xenopus

In vertebrates, most inner organs are asymmetrically arranged with respect to the main body axis [1]. Symmetry breakage in fish, amphibian, and mammalian embryos depends on cilia-driven leftward flow of extracellular fluid during neurulation [2-5]. Flow induces the asymmetric nodal cascade that governs asymmetric organ morphogenesis and placement [1, 6, 7]. In the frog Xenopus, an alternative laterality-generating mechanism involving asymmetric localization of serotonin at the 32-cell stage has been proposed [8]. However, no functional linkage between this early localization and flow at neurula stage has emerged. Here, we report that serotonin signaling is required for specification of the superficial mesoderm (SM), which gives rise to the ciliated gastrocoel roof plate (GRP) where flow occurs [5, 9]. Flow and asymmetry were lost in embryos in which serotonin signaling was downregulated. Serotonin, which we found uniformly distributed along the main body axes in the early embryo, was required for Wnt signaling, which provides the instructive signal to specify the GRP. Importantly, serotonin was required for Wnt-induced double-axis formation as well. Our data confirm flow as primary mechanism of symmetry breakage and suggest a general role of serotonin as competence factor for Wnt signaling during axis formation in Xenopus.

Normal Interstitial Flow is Critical for Developmental Lymphangiogenesis in the Zebrafish

The lymphatic system plays a critical role in the body's fluid and protein homeostasis, immune regulation, and dietary fat absorption. One of the major pathologies of the lymphatic system is primary lymphedema, which occurs in approximately 0.6% of live births and is caused by missing or impaired lymphatic vessels. Although there is a great need for medical intervention into diseases of the lymphatic system, very little is known about its development or how it maintains integrity over time. Recent studies have suggested that biophysical components, such as local extracellular fluid flow, may be important factors during initiation of lymphangiogenesis. We hypothesize that interstitial fluid flow functions as an important morphoregulator during developmental lymphangiogenesis. In the present study we use pharmacological agents and a mutant fish line to modulate interstitial flow. Our data confirm that a sufficient increase or decrease in interstitial flow can profoundly affect lymphatic patterning and may result in a lymphedema-like phenotype. Proper interstitial flow appears to be necessary during LEC migration for proper lymphatic development. These results support the contention that interstitial flow is an important morphoregulator of developmental lymphangiogenesis.

Nipbl regulates organ laterality and Kupffer's vesicle development in Zebrafish

Nipbl regulates organ laterality and Kupffer's vesicle development in Zebrafish

Pkd1l1 complexes with Pkd2 on motile cilia and functions to establish the left-right axis

The internal organs of vertebrates show distinctive left-right asymmetry. Leftward extracellular fluid flow at the node (nodal flow), which is generated by the rotational movement of node cilia, is essential for left-right patterning in the mouse and other vertebrates. However, the identity of the pathways by which nodal flow is interpreted remains controversial as the molecular sensors of this process are unknown. In the current study, we show that the medaka left-right mutant abecobe (abc) is defective for left-right asymmetric expression of southpaw, lefty and charon, but not for nodal flow. We identify the abc gene as pkd1l1, the expression of which is confined to Kupffer's vesicle (KV, an organ equivalent to the node). Pkd1l1 can interact and interdependently colocalize with Pkd2 at the cilia in KV. We further demonstrate that all KV cilia contain Pkd1l1 and Pkd2 and left-right dynein, and that they are motile. These results suggest that Pkd1l1 and Pkd2 form a complex that functions as the nodal flow sensor in the motile cilia of the medaka KV. We propose a new model for the role of cilia in left-right patterning in which the KV cilia have a dual function: to generate nodal flow and to interpret it through Pkd1l1-Pkd2 complexes.

Far from solved: a perspective on what we know about early mechanisms of left-right asymmetry.

Consistent laterality is a crucial aspect of embryonic development, physiology, and behavior. While strides have been made in understanding unilaterally expressed genes and the asymmetries of organogenesis, early mechanisms are still poorly understood. One popular model centers on the structure and function of motile cilia and subsequent chiral extracellular fluid flow during gastrulation. Alternative models focus on intracellular roles of the cytoskeleton in driving asymmetries of physiological signals or asymmetric chromatid segregation, at much earlier stages. All three models trace the origin of asymmetry back to the chirality of cytoskeletal organizing centers, but significant controversy exists about how this intracellular chirality is amplified onto cell fields. Analysis of specific predictions of each model and crucial recent data on new mutants suggest that ciliary function may not be a broadly conserved, initiating event in left-right patterning. Many questions about embryonic left-right asymmetry remain open, offering fascinating avenues for further research in cell, developmental, and evolutionary biology.

Cellular-level near-wall unsteadiness of high-hematocrit erythrocyte flow using confocal μPIV

In hemodynamics, the inherent intermittency of two-phase cellular-level flow has received little attention. Unsteadiness is reported and quantified for the first time in the literature using a combination of fluorescent dye labeling, time-resolved scanning confocal microscopy, and micro-particle image velocimetry (μPIV). The near-wall red blood cell (RBC) motion of physiologic high-hematocrit blood in a rectangular microchannel was investigated under pressure-driven flow. Intermittent flow was associated with (1) the stretching of RBCs as they passed through RBC clusters with twisting motions; (2) external flow through local obstacles; and (3) transitionary rouleaux formations. Velocity profiles are presented for these cases. Unsteady flow clustered in local regions. Extra-cellular fluid flow generated by individual RBCs was examined using submicron fluorescent microspheres. The capabilities of confocal μPIV post-processing were verified using synthetic raw PIV data for validation. Cellular interactions and oscillating velocity profiles are presented, and 3D data are made available for computational model validation.

The Nodal Inhibitor Coco Is a Critical Target of Leftward Flow in Xenopus

Vertebrate laterality, which is manifested by asymmetrically placed organs [1], depends on asymmetric activation of the Nodal signaling cascade in the left lateral plate mesoderm [2]. In fish, amphibians, and mammals, a cilia-driven leftward flow of extracellular fluid acts upstream of the Nodal cascade [3-6]. The direct target of flow has remained elusive. In Xenopus, flow occurs at the gastrocoel roof plate (GRP) in the dorsal midline of the embryo [4, 7]. The GRP is bordered by a second, bilaterally symmetrical Nodal expression domain [8]. Here we identify the Nodal inhibitor Coco as a critical target of flow. Coco and Xenopus Nodal-related 1 (Xnr1) are coexpressed in the lateralmost ciliated GRP cells. Coco becomes downregulated on the left side of the GRP as a direct readout of flow. Ablation of flow prevented Coco repression, whereas Xnr1 expression was independent of flow. Loss of flow-induced laterality defects were rescued by knockdown of Coco on the left side. Parallel knockdown of Coco and Xnr1 in GRP cells restored laterality defects in flow-impaired embryos, demonstrating that Coco acted through GRP-expressed Xnr1. Coco thus acts as a critical target of flow, suggesting that symmetry is broken by flow-mediated left-asymmetric release of Nodal repression at the midline.

Consistent left-right asymmetry cannot be established by late organizers in Xenopus unless the late organizer is a conjoined twin.

How embryos consistently orient asymmetries of the left-right (LR) axis is an intriguing question, as no macroscopic environmental cues reliably distinguish left from right. Especially unclear are the events coordinating LR patterning with the establishment of the dorsoventral (DV) axes and midline determination in early embryos. In frog embryos, consistent physiological and molecular asymmetries manifest by the second cell cleavage; however, models based on extracellular fluid flow at the node predict correct de novo asymmetry orientation during neurulation. We addressed these issues in Xenopus embryos by manipulating the timing and location of dorsal organizer induction: the primary dorsal organizer was ablated by UV irradiation, and a new organizer was induced at various locations, either early, by mechanical rotation, or late, by injection of lithium chloride (at 32 cells) or of the transcription factor XSiamois (which functions after mid-blastula transition). These embryos were then analyzed for the position of three asymmetric organs. Whereas organizers rescued before cleavage properly oriented the LR axis 90% of the time, organizers induced in any position at any time after the 32-cell stage exhibited randomized laterality. Late organizers were unable to correctly orient the LR axis even when placed back in their endogenous location. Strikingly, conjoined twins produced by late induction of ectopic organizers did have normal asymmetry. These data reveal that although correct LR orientation must occur no later than early cleavage stages in singleton embryos, a novel instructive influence from an early organizer can impose normal asymmetry upon late organizers in the same cell field.

Flow on the right side of the gastrocoel roof plate is dispensable for symmetry breakage in the frog Xenopus laevis

Leftward flow of extracellular fluid breaks the bilateral symmetry of most vertebrate embryos, manifested by the ensuing asymmetric induction of Nodal signaling in the left lateral plate mesoderm (LPM). Flow is generated by rotational beating of polarized monocilia at the posterior notochord (PNC; mammals), Kupffer's vesicle (KV; teleost fish) and the gastrocoel roof plate (GRP; amphibians). To manipulate flow in a defined way we cloned dynein heavy chain genes dnah5, 9 and 11 in Xenopus. dnah9 expression was closely related to motile cilia from neurulation onwards. Morphant tadpoles showed impaired epidermal ciliary beating. Leftward flow at the GRP was absent, resulting in embryos with loss of asymmetric marker gene expression. Remarkably, unilateral knockdown on the right side of the GRP did not affect laterality, while left-sided ablation of flow abolished marker gene expression. Thus, flow was required exclusively on the left side of the GRP to break symmetry in the frog. Our data suggest that the substrate of flow is generated within the GRP and not at its margin, disqualifying Nodal as a candidate morphogen.

Effect of hydrocephalus on rat brain extracellular compartment

BackgroundThe cerebral cortex may be compressed in hydrocephalus and some experiments suggest that movement of extracellular substances through the cortex is impaired. We hypothesized that the extracellular compartment is reduced in size and that the composition of the extracellular compartment changes in rat brains with kaolin-induced hydrocephalus.MethodsWe studied neonatal (newborn) onset hydrocephalus for 1 or 3 weeks, juvenile (3 weeks) onset hydrocephalus for 3–4 weeks or 9 months, and young adult (10 weeks) onset hydrocephalus for 2 weeks, after kaolin injection. Freeze substitution electron microscopy was used to measure the size of the extracellular compartment. Western blotting and immunohistochemistry with quantitative image densitometry was used to study the extracellular matrix constituents, phosphacan, neurocan, NG2, decorin, biglycan, and laminin.ResultsThe extracellular space in cortical layer 1 was reduced significantly from 16.5 to 9.6% in adult rats with 2 weeks duration hydrocephalus. Western blot and immunohistochemistry showed that neurocan increased only in the periventricular white matter following neonatal induction and 3 weeks duration hydrocephalus. The same rats showed mild decorin increases in white matter and around cortical neurons. Juvenile and adult onset hydrocephalus was associated with no significant changes.ConclusionWe conclude that compositional changes in the extracellular compartment are negligible in cerebral cortex of hydrocephalic rats at various ages. Therefore, the functional change related to extracellular fluid flow should be reversible.

Microfluidic devices for studies of primary cilium mediated cellular response to dynamic flow conditions

We present the first microfabricated microfluidic devices designed specifically for studies of primary cilium mediated cellular response to dynamic flow. The primary cilium functions as a mechano-sensor in renal tubular epithelium, sensing the extracellular fluid flow. Malfunction of cilia has been implicated in e.g. polycystic kidney disease and other pathological conditions. Bending of the primary cilium by fluid flow has been shown to give rise to an intracellular calcium signal, however little is known about the sensitivity to flow duration, magnitude and direction. This paper presents a novel method for studying cilia forming cells in asymmetric microfluidic environments. The microfluidic devices presented here were designed for a dynamic control of the local fluid flow on a cellular level, and thus, enables studies of cellular responses to an amplitude, frequency and direction controlled cilium movement.

Cilia-Driven Leftward Flow Determines Laterality in Xenopus

Determination of the vertebrate left-right body axis during embryogenesis results in asymmetric development and placement of most inner organs. Although the asymmetric Nodal cascade is conserved in all vertebrates, the mechanism of symmetry breakage has remained controversial. In mammalian and fish embryos, a cilia-driven leftward flow of extracellular fluid is required for initiation of the Nodal cascade. This flow is localized at the posterior notochord ("node") and Kupffer's vesicle, respectively. In frog and chick embryos, however, molecular asymmetries are required earlier, from cleavage stages through gastrulation. The validity of a cilia-based mechanism for all vertebrates therefore has been questioned. Here we show that a cilia-driven leftward flow precedes asymmetric nodal expression in the frog Xenopus. Motile monocilia emerged on the gastrocoel roof plate during neurulation and lengthened and polarized from an initially central position to the posterior pole of cells. Concomitantly, a robust leftward fluid flow developed from stage 15 onward, significantly before asymmetric nodal transcription started in the left-lateral-plate mesoderm at stage 19. Injection of 1.5% methylcellulose into the archenteron prevented leftward flow and resulted in laterality defects, demonstrating that the flow itself was required for asymmetric gene expression and organ placement.

Local Disassembly of Actin Stress Fibers Induced by Selected Release of Intracellular Tension in Osteoblastic Cell

In adherent cells, such as osteoblasts and endothelial cells, the actin stress fiber structure is dynamically reorganized under changes in the surrounding mechanical environment such as cyclic stretch deformation or extracellular fluid flow. Although many studies have been conducted to clarify the biochemical signaling pathways in the reorganization process, details of the reorganization mechanism are still not clearly understood. In addition, to known biochemical mediators, intracellular tension has been proposed as a candidate mechanical mediator for stress fiber reorganization. In previous studies, it was reported that the release of intracellular tension by contracting the cell body induced the disassembly of the stress fiber as an initial phase of the reorganization process. However, in these experimental systems, deformation or force was applied to the entire cell body, so that it was difficult to directly discuss the relationship between the individual mechanical condition of stress fibers and the disassembling phenomenon. In this study, we have designed a novel experimental system by which local contraction was applied to a single osteoblast-like cell and intracellular tension in the targeted stress fiber was selectively released. The dynamic change in stress fiber structure was observed in the EGFP-tagged actin expressed osteoblast-like cell MC3T3-E1. The results indicated that only tension-released stress fibers were selectively disassembled and disappeared in a single cell. This result suggests that the existence of intracellular tension is essential for the dynamical stability of the stress fibers in osteoblast-like cells.

IP 6 in Left-Right Asymmetry

All embryos start out bilaterally symmetrical, and in response to a directed flow of extracellular fluid (nodal flow), this symmetry is broken. Sarmah et al. show that in zebrafish embryos, embryo asymmetry is dependent on inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1, encoded by ipk1 ), which catalyzes the formation of inositol 1,2,3,4,5,6-phosphate (IP 6 ). Knockdown of ipk1 using three different methods resulted in accumulation of IP 4 and IP 5 (an indication of loss of IPK1 activity) and randomization of heart placement and positioning of visceral organs (based on expression of various genes normally expressed at higher levels on one side). Furthermore, brain asymmetry was also disrupted in the absence of IPK1. Loss of IPK1 did not disrupt formation of the ciliated cells of the Kupffer's vesicle (KV), which is believed to be the source of the nodal flow in zebrafish embryos, and this suggests that IP 6 may be important for downstream signaling in response to the nodal flow as opposed to generation of nodal flow. Knockdown of ipk1 resulted in loss of calcium signaling in the KV region. Thus, IPK1 activity, presumably leading to the production of IP 6 , appears to be required for calcium signaling in response to nodal flow. B. Sarmah, A. J. Latimer, B. Appel, S. R. Wente, Inositol polyphosphates regulate zebrafish left-right asymmetry. Dev. Cell 9 , 133-145 (2005). [PubMed]

Inositol Polyphosphates Regulate Zebrafish Left-Right Asymmetry

Vertebrate body plans have a conserved left-right (LR) asymmetry manifested in the position and anatomy of the heart, visceral organs, and brain. Recent studies have suggested that LR asymmetry is established by asymmetric Ca2+ signaling resulting from cilia-driven flow of extracellular fluid across the node. We report here that inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), which generates inositol hexakisphosphate, is critical for normal LR axis determination in zebrafish. Zebrafish embryos express ipk1 symmetrically during gastrulation and early segmentation. ipk1 knockdown by antisense morpholino oligonucleotide injection randomized LR-specific gene expression and organ placement, effects that were associated with reduced intracellular Ca2+ flux in cells surrounding the ciliated Kupffer's vesicle, a structure analogous to the mouse node. Our data suggest that the pathway for inositol hexakisphosphate production is a key regulator of asymmetric Ca(2+) flux during LR specification.

Molecular mechanisms of brain tumor edema

Despite their diverse histological types, most brain tumours cause brain oedema, which is a significant cause of patient morbidity and mortality. Brain tumour oedema occurs when plasma-like fluid enters the brain extracellular space through impaired capillary endothelial tight junctions in tumours. Under-expression of the tight junction proteins occludin, claudin-1 and claudin-5 are key molecular abnormalities responsible for the increased permeability of tumour endothelial tight junctions. Recent evidence suggests that the membrane water channel protein aquaporin-4 (AQP4) also plays a role in brain tumour oedema. AQP4-deficient mice show remarkably altered brain water balance after various insults, including brain tumour implantation. AQP4 expression is strongly upregulated around malignant human brain tumours in association with reduced extracellular volume, which may restrict the flow of extracellular fluid from the tumour bed into the brain parenchyma. Elimination of excess fluid leaking into brain parenchyma requires passage across three AQP4-rich barriers: a) the glia limitans externa, b) the glia limitans interna/ependyma, and c) the blood–brain barrier. Modulation of the expression and/or function of endothelial tight junction proteins and aquaporins may provide novel therapeutic options for reducing brain tumour oedema.

Left-right asymmetry: Nodal points

The striking left-right asymmetry of visceral organs is known to depend on left- and right-side-specific cascades of gene expression during early embryogenesis. Now, developmental biologists are characterizing the earliest steps in asymmetry determination that dictate the sidedness of asymmetric gene expression. The proteins and structures involved control fascinating physiological processes, such as extracellular fluid flow and membrane voltage potential and yet little is known about how their activities are coordinated to control laterality. By analogy with intercellular signalling in certain epithelial and endothelial cells, however, it is reasonable to speculate that at least three of these players, monocilia, gap junction communication and the Ca2+ channel polycystin-2, participate in a signalling pathway that propagates left-right cues through multicellular fields.

Two Populations of Node Monocilia Initiate Left-Right Asymmetry in the Mouse

The vertebrate body plan has conserved handed left-right (LR) asymmetry that is manifested in the heart, lungs, and gut. Leftward flow of extracellular fluid at the node (nodal flow) is critical for normal LR axis determination in the mouse. Nodal flow is generated by motile node cell monocilia and requires the axonemal dynein, left-right dynein (lrd). In the absence of lrd, LR determination becomes random. The cation channel polycystin-2 is also required to establish LR asymmetry. We show that lrd localizes to a centrally located subset of node monocilia, while polycystin-2 is found in all node monocilia. Asymmetric calcium signaling appears at the left margin of the node coincident with nodal flow. These observations suggest that LR asymmetry is established by an entirely ciliary mechanism: motile, lrd-containing monocilia generate nodal flow, and nonmotile polycystin-2 containing cilia sense nodal flow initiating an asymmetric calcium signal at the left border of the node.