Browsing by Subject "Gap junctions"

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Determination of Laterality in the Rabbit Embryo: Studies on Ciliation and Asymmetric Signal Transfer
(2007) Feistel, Kerstin; Blum, Martin
The midline of the vertebrate embryo plays a pivotal role in the regulation of left-right (LR) asymmetry. In mammals recent interest has focused on a structure situated at the caudal part of the notochord, the posterior notochord (PNC), which is homologous to Kupffer?s vesicle (KV) in fish and the gastrocoel roof plate (GRP) in frog. Despite highly diverging embryonic architecture, the PNC/KV/GRP is the site where motile monocilia set up a directional fluid flow, an event indispensable for the generation of LR asymmetry. Signals created at the PNC/KV/GRP need to be transferred to the periphery of the embryo, where they initiate the left-specifying program in the left lateral plate mesoderm (LPM). In this study morphogenesis and ciliogenesis of the notochordal plate as well as the signaling processes between midline and LPM were studied in the rabbit embryo. Rabbit development progresses through a flat blastodisc phase and represents the typical mode of mammalian embryogenesis. Transcription of ciliary marker genes, the first sign of beginning ciliogenesis, initiated in Hensen?s node and persisted in the nascent notochord. Cilia emerged on cells leaving Hensen?s node anteriorly to form the notochordal plate. Cilia lengthened to about 5µm and polarized from an initially central position to the posterior pole of cells. Electron microscopic analysis revealed 9+0 and 9+2 cilia and a novel 9+4 axoneme intermingled in a salt-and-pepper-like fashion. These data showed that the ciliogenic gene program essential for laterality determination is conserved at the midline of the rabbit embryo. The present study also provided evidence that initiation as well as repression of the Nodal cascade crucially depended on communication between midline and lateral plate (LP). Separation of LP tissue from the midline before, during and after the 2 somite stage demonstrated that signals from the PNC induced and maintained the competence of LPM to express Nodal. Signals from the midline were necessary after the 2 somite stage to maintain a right-sided identity, i.e. absence of Nodal expression. Gap-junction-dependent intercellular communication (GJC) was shown to play a central role in this process. Previously, GJC had been involved in LR axis determination in cleavage stage frog embryos and early blastodisc stages in chick. This study for the first time demonstrates the role of GJC in mammalian embryos. GJs regulate the signaling between midline and periphery: permeable gap junctions were required specifically at the 2 somite stage to repress Nodal induction in the right LPM, whereas closed GJs were a prerequisite for Nodal signaling on the left side. Establishment of the right-sided fate depended on FGF8, the signaling of which was regulated by the opening status of GJs. A 3-step model is proposed for symmetry breakage and induction of the LR signaling cascade in vertebrates: (1) Nodal protein synthesized at the lateral edges of the PNC diffuses bilaterally and confers competence for the induction of the Nodal cascade to the LPM, (2) at the same time the left-specific cascade is actively repressed by action of the GJC/FGF8 module, and (3) following the onset of leftward flow at the PNC repression gets released specifically on the left side at the 2 somite stage, presumably by transient inhibition of GJC. This model not only is consistent with the presented data, but also with published work in other model organisms.
The role of serotonin and gap junctions in left-right development of Xenopus laevis
(2011) Beyer, Tina; Blum, Martin
In vertebrates, the correct determination of the left-right (LR) axis is essential for accurate placement of the inner organs, such that the heart points to the left, lung lobation differs between left and right side, spleen and stomach are located on the left,liver on the right body side and the gut coils asymmetrically. Disturbance of this organization can lead to severe impairments of organ function. In the African clawed frog Xenopus laevis, already in four-day old tadpoles asymmetric organ arrangement is visible. This coordinated organ development strictly requires prior Nodal cascade activity in the left lateral plate mesoderm (LPM) in all model organisms examined so far. The initial symmetry breaking event necessary for unilateral induction of Nodal signaling is still under debate. In X. laevis, two models, namely 'ion-flux' and 'cilia-driven leftward fluid flow', were discussed in this context. Leftward flow was first described in the mouse model and later on in fish and rabbit, whereas the 'ion-flux' hypothesis is supported by data derived from chick development. In the present work it was intended to enlighten this putative discrepancy by re-investigating the function of two 'ion-flux' components in context of leftward flow in the model organism X. laevis. First, a link between cell-cell communication via gap junctional communication (GJC)and LR axis establishment was analyzed by using heptanol for general inhibition of channel conductance on the one hand, and single knock-down (KD) of specific subunits on the other hand. Both treatments resulted in absence of the left-sided Nodal cascade. The KD led to shorter GRP cilia when compared to wildtype embryos and loss of bilateral Nodal expression at the GRP margin, respectively. Furthermore, heptanol treatments of stages in which the GRP already has been fully developed also resulted in laterality defects, thus implying a second function of GJC most likely for the signal transfer to the left side. These results indicated a role of GJC in leftward flow establishment and/or post-flow in neurula stages rather than a function in early cleavage stages for LR determination. Second, the early signaling function of the neurotransmitter serotonin (5-HT) was inhibited by over-expression of either a frog or a human receptor ligand binding domain(LBD). In addition, specific KD of a receptor class 3 subunit was performed. Both applications resulted in impaired left-sided marker gene expression and disturbed GRP morphogenesis. Remarkably, marker gene expression of the superficial mesoderm(SM) which gives rise to the GRP during development, was reduced in 5-HT signaling impaired embryos. Very importantly, receptor 3 specific 5-HT signaling was shown to represent a necessary competence factor required for Wnt-dependent axis development in the frog double axis induction assay. Besides the new function of 5-HT during early development, it was further shown that the expression of the SM marker Foxj1 (a master regulator of motile cilia) depended on maternal factors. Based on the work presented here, the following model is proposed: (1) Foxj1 expression is induced maternally, followed by (2) zygotic refinement in post-MBT stages, i.e. inhibition on the ventral and maintenance on the dorsal side. In the organizer region, an interplay of Wnt and 5-HT signaling is required for dorsal development.(3) Cilia driven leftward flow initiate an unknown downstream signal which is transferred to the left LPM. Both events, leftward flow and transfer, require active GJC. (4) A to date unknown signal gets transferred towards the left LPM in a GJC-dependent process and induces the Nodal cascade activity, a prerequisite for proper organ arrangement. Taken together, the data presented in this study indicate that the directed fluid flow in neurula embryos represent the decisive step for symmetry breakage with the 'ion-flux' components being involved in correct flow function.