Browsing by Subject "Left-right axis"

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ATP4 and Wnt-signaling are required for ciliogenesis and left-right axis development of Xenopus
(2012) Walentek, Peter; Blum, Martin
The vertebrate body plan displays left-right (LR) asymmetries of organ placement superimposed on an overt bilaterally symmetrical organization. Symmetry is broken during embryogenesis, and asymmetric gene expression precedes asymmetric organ morphogenesis. The proton/potassium pump ATP4 was shown to play a role in LR-development of the frog Xenopus laevis as well as in other deuterostomes. Two opposing models of symmetry-breakage were proposed, the ?ion-flux? and the ?leftward flow? model. The former proposed that symmetry was broken by LR-asymmetric expression of the a-subunit of ATP4 during cleavage stages. The latter claimed a cilia-based leftward flow at the gastrocoel roof plate (GRP) to take center stage during neurulation, i.e. a day later in development. In the present thesis work, the role of ATP4a in symmetry-breakage was re-addressed and evidence for symmetrical expression and function of ATP4a was gathered. ATP4a was shown to be required for two Wnt-signaling dependent steps during the setup of cilia driven leftward flow at the GRP: (1) Wnt/b-catenin (b-cat) dependent expression of Foxj1 during gastrulation, and (2) Wnt/planar cell polarity (PCP) dependent posterior localization of motile cilia during neurulation. These data challenge the ?ion-flux? hypothesis and argue for a conserved ATP4- and cilia-dependent symmetry-breakage mechanism throughout the vertebrates. Furthermore, the function of Wnt-signaling components was analyzed in the context of GRP-formation: The receptor Frizzled 8 (Fz8) and b-cat were required for Foxj1 expression during gastrulation. Morphogenesis of the GRP, posterior polarization of motile cilia and expression of Xnr1 and Coco in somitic cells were all required for LR-development. Loss of non-canonical Xwnt11b-signaling perturbed these process, suggesting that non-canonical Wnt-signaling branches, in addition to Wnt/PCP, were relevant for LR-development. ATP4-mediated Wnt-signaling was also required for Foxj1 expression and motile cilia in other epithelia during Xenopus development, i.e. the skin, floor plate and the ependymal cell layer. In the floor plate b-cat was required for Foxj1 expression downstream of Hedgehog-signaling. In the skin mucociliary epithelium ATP4a and Wnt/b-cat were required downstream of Notch/Delta-mediated cell-type specification of multiciliated cells. This was also true for a new cell type of serotonergic cells described here, which was characterized morphologically, by analysis of gene expression and response to manipulations of Wnt- and Notch/Delta-signaling. In summary, the data presented in this thesis suggest a conserved function of ATP4a and Wnt-signaling in vertebrate symmetry-breakage and Foxj1-dependent ciliogenesis in Xenopus.
Axes determination in the frog Xenopus laevis : the function of the goosecoid, myo1d and dmrt2
(2020) Tingler, Melanie Bianca; Schweickert, Axel
During early embryogenesis, pattern formation processes along the head-trunk (anteroposterior, AP), belly-back (dorsoventral, DV) and left-right (LR) body axis generate the fundamental body plan of the bilateria. The formation of the LR axis is exceptional because externally our body is bilateral symmetric whereas most inner organs are shaped and positioned asymmetrically. The three body axes are basically specified during gastrulation and neurulation by a set of developmental control genes. The aim of this work was to analyze the function of the highly conserved genes, goosecoid (gsc), myosin1d (myo1d) und dmrt2 during body axis determination in Xenopus. The first chapter of this work describes the activity of the homeobox transcription factor Goosecoid during AP- and DV-axis formation. Gsc acts as an autoregulatory transcriptional repressor and importantly is expressed in the Spemann Organizer (SO) of all vertebrate embryos. The SO represents the main dorsal signaling center for primary axis induction, regulates embryonic patterning and cell movements. It is further required for AP i.e. head and trunk development. Transferring of SO or gsc misexpression to ventral half of embryos resultes in secondary axis formation i.e. siamnese twins. However, SO function of Gsc was enigmatic, as gsc mutants showed no defects on early developmental processes what challenged Gsc function in the SO. In this chapter, gsc was characterized by conducting gain of function experiments in the embryonic midline of Xenopus embryos. Gsc was able to repress planar cell polarity (PCP) in a cell- and non-cell autonomous fashion leading to neural tube closure defects. In the early gastrulae, Gsc separates the head from the trunk mesoderm by repressing the mesodermal t-box gene transcription factor T (Tbxt). This inhibition allows the migration of the head mesodermal cells whereas the trunk notochord elongates by mediolateral intercalation. Gsc activity on PCP signaling seems to be specific for vertebrates only and correlates with the presence of two novel domains. The determination of the LR body axis is discussed in the second chapter of this work. At the so called left-right organizer (LRO) a cilia-mediated leftward-fluid flow initiates the symmetry breaking event in neurulae embryos. Lateral sensory cells (sLRO) of the LRO perceive flow on the left side and translate it into the left asymmetric induction of the highly conserved Nodal cascade. If and how the unconventional, actin-associated motor protein Myosin1d (Myo1d) as well as the transcription factor Doublesex and mab-3 related 2 (Dmrt2) intervene in LR specification was analyzed in this chapter. In evolutionary terms the study of myo1d was of high interest because in Drospohila, which lacks a ciliary flow mechanism, the homologous gene, myo31df, controls LR axis determination. Manipulations of myo1d in Xenopus demonstrated that in vertebrates Myo1d is involved in the cilia-based symmetry breakage event. By interacting with the PCP signaling pathway, Myo1d ensures leftward-fluid flow by regulating ciliary outgrowth and polarization. In Drosophila and Xenopus Myo1d interacts with PCP signaling and seems to link an ancestral symmetry breaking mechanism of the fly to the newly evolved leftward-fluid flow in vertebrates. Based on studies in zebrafish, which identified Dmrt2 as another factor involved in LR development and somitogenesis, we started the analysis of dmrt2 in Xenopus. Somitogenesis and laterality determination which on first sight are functionally distinct processes were analyzed in the context of dmrt2 function. In Xenopus, flow-sensing cells are affiliated to the somitic cell lineage and therefor paraxial mesoderm specification is crucial for setting up a functional LRO. Dmrt2 specifies the paraxial mesoderm and especially the sLRO by inducing the myogenic transcription factor myf5 in early gastrulae. This demonstrated for the first time experimentally how somitogenesis and laterality determination are intertwined and describes the genesis of the Xenopus sLRO cells in more detail.
Funktionelle Analyse der Gene Brachyury, Goosecoid und Myosin1d für die frühe Musterbildung und Etablierung der Körperachsen während der Embryogenese des Krallenfroschs Xenopus laevis
(2019) Kurz, Sabrina; Blum, Martin
During a fundamental phase of vertebrate embryonic development, gastrulation, the reorganization of the future body plan, is determined. This process controls the embryonic patterning of the antero-posterior (AP) and dorso-ventral (DV) axes, as well as the internal left-right (LR) axis. The establishment of the body axes is initially controlled by the dorsally localized Spemann organizer (SO), whereby the determination of the LR axis takes place the latest. The formation of laterality during subsequent neurulation is executed by a highly conserved mechanism of symmetry breakage within fishes, amphibians and mammals. An extracellular, monocilia-driven fluid-flow („Flow") eventually defines the laterality of the embryo, by left-asymmetric gene expression of the morphogen Nodal. Responsible for the Flow is a ciliated epithelium in the postero-dorsal region of the embryo, which undergoes morphological development and correct positioning during gastrulation and is termed GRP („gastrocoel roof plate") in the frog. Ultimately, this conserved tissue describes the left-right organizer (LRO) and guarantees proper organ situs. The origin and function of the LRO can be traced back to an epithelial organized cell structure on the dorsal surface of the gastrula, characterized by the expression of the canonical Wnt target gene and cilia marker foxj1. Following the specification of this Superficial Mesoderm (SM), the future cells of the LRO invaginate in a directed fashion during gastrulation. The patterns of cellular movement require a restructuring of the actin-cytoskeleton. These migrations, executing the future body plan, are ensured by the PCP- („planar cell polarity”) signaling pathway. This non-canonical Wnt pathway guarantees polarized alignment and migration of cells along the body axes, namely convergent extension (CE). As an organizer gene, the expression of Goosecoid (Gsc) characterizes SO and its’ properties. Contrary to the assumption that a Gsc loss of function thus inhibits gastrulation, no visible impairment was detected in the Knock-Out mouse and Knock-Down in Xenopus. Gain-of-function in the frog, which analyzed the role of Gsc during gastrulation finally demonstrated the homeobox-gene exerting a function in regulating cell movements. Overexpression resulted in impaired CE of dorsal tissue due to defective localization of nuclear proteins of the PCP signaling pathway. Gsc-induced malformations could be compensated by co-injections of associated components. In conclusion, a new function as an inhibitor of PCP-dependent CE during gastrulation was suggested. The morphogenetic movements of CE are responsible for the AP-elongation and LRO-positioning. Both, mechanical forces and cilia-based Flow functionally interact for that matter. In the invertebrate Drosophila, where neither Nodal nor cilia are expressed, organ asymmetry is ensured by internal chirality conducted via motor proteins, such as myosin1d, in a PCP-dependent manner. Similarly, in vertebrates such as Xenopus, myo1d mediates interactions of the actin cytoskeleton asymmetrically, that guarantee laterality of the organ system. The functional preservation of the non-canonical Wnt pathway could be demonstrated by co-injections of PCP core proteins, being able to restore disturbed LRO morphology. The obtained data clearly demonstrated the evolutionary, interspecific, regulation of axis asymmetry by myo1d. Since the strict spatio-temporal regulation of cell movements is fundamental for patterning, CE is propagated by another transcription factor: Brachyury (Tbxt in Xenopus). Its expression induces and controls differentiation of mesodermal cell populations, such as the notochordal cells of the dorsal midline, hence LRO. The necessity specifying progenitor cells in the SM could also be highlighted, showing to be mediated non-cell autonomously to guarantee the induction of foxj1. Additionally, the determination of both tissues is exerted by functional interactions of Tbxt with either PCP-, as well as ß-catenin-dependent Wnt signaling pathway. Loss of Brachyury affects laterality in mutants and morphants. The species-spanning Brachyury-FGF „feedback-loop" for the induction of Nodal and Foxj1 could already act in the SM and showed that function as well as the process of LR development, as suggested between Fgf8 and Brachyury, to be functionally conserved. In Xenopus, signal transduction for SM and foxj1 induction was extended by the function of the Wnt receptor frizzled-8, the ligand and Tbxt target gene wnt11b, and the ventrally acting wnt8a in Whole-Mount embryos and explant co-cultures.