Axes determination in the frog Xenopus laevis : the function of the goosecoid, myo1d and dmrt2

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.Während der frühen Embryogenese generieren embryonale Musterbildungsprozesse entlang der Kopf-Rumpf- (anteroposterior, AP), Rücken-Bauch- (dorsoventral, DV) und links-rechts (LR) Körperachse den grundlegenden Bauplan der Bilateria. Hierbei ist vor allem die Ausbildung der LR-Achse auffallend: sie besticht durch eine äußerlich sichtbare Symmetrie entlang der AP-Achse, wohingegen die asymmetrische Formgebung und Position der inneren Organe in der sekundären Leibeshöhle äußerlich nicht zu erkennen ist. Die Ausbildung der drei Körperachsen wird durch die Aktivität zahlreicher Gene während der Gastrulation und Neurulation reguliert. Ziel dieser Arbeit war es, die Rolle der hoch konservierten Gene goosecoid (gsc), myosin1d (myo1d) und doublesex-and mab3 related transcription factor 2 (dmrt2) während der Ausbildung der Körperachsen in Xenopus laevis näher zu untersuchen. Das erste Kapitel dieser Arbeit befasst sich mit der frühen Funktion des Homöobox-Transkriptionsfaktors Goosecoid während der Ausbildung der AP- und DV-Achse. Gsc wirkt als autoregulatorischer transkriptioneller Repressor, wird im Spemann-Organisator, dem Signalzentrum der primären Achseninduktion exprimiert und steuert die embryonale Musterbildung. Es reprimiert ventrale Signalwege im dorsalen Gewebe, separiert das Kopf- vom Chordamesoderm und reguliert Zellbewegungen im Zuge der Gastrulation und Neurulation. Die frühe Funktion von gsc im Spemann-Organisator war bislang enigmatisch, da der Funktionsverlust von gsc die frühe embryonale Entwicklung nicht beeinträchtigte. Durch gezielte Überexpression von gsc in der dorsalen Mittellinie von Xenopus Embryonen wurde hier die frühe Funktion von gsc näher charakterisiert. Gsc agierte sowohl zell- als auch nicht-zell-autonom als Repressor planarer Zellpolarität (planar cell polarity, PCP). In der frühen Gastrula separierte Gsc durch die Repression des mesodermalen T-box Gen Transkriptionsfaktors T (Tbxt) das Kopf- vom Chordamesoderm. Dies ermöglichte das migrieren des Kopfmesoderms und beschränkte die durch Tbxt-induzierte PCP-vermittelte mediolaterale Interkalation auf das elongierende Notochord des Embryos. Diese Funktion von Gsc scheint sich im Zuge der Evolution durch die Etablierung zweier neuer, für Vertebraten spezifische Domänen etabliert zu haben. Das zweite Kapitel befasst sich mit der Determinierung der LR-Körperachse in Xenopus, die als letzte der drei Körperachsen festgelegt wird. Diese wird durch einen Cilien-basierten nach links-gerichteten Flüssigkeitsstrom innerhalb des sog. links-rechts Organisators (LRO) in der Neurula initiiert. Die lateralen, linken sensorischen Zellen des LROs (sLRO) perzipieren den Flüssigkeitsstrom und translatieren dieses Signal in die Induktion der hoch konservierten Nodal Kaskade auf der linken Seite. Welche Funktion das unkonventionelle, Aktin-assoziierte Motorprotein Myo1d und der Transkriptionsfaktor Dmrt2 bei diesem Prozess einnimmt, wurde im Rahmen dieser Arbeit untersucht. Die Analyse von myo1d war hierbei evolutionär von großer Bedeutung, da das homologe Gene myo31df in Drosophila die Entstehung der LR-Achse, unabhängig eines links-gerichteten Flüssigkeitsstrom und einer asymmetrischen Gen-Kaskade reguliert. Die Manipulation von myo1d in Xenopus demonstrierte, dass die Funktion von Myo1d konserviert ist und auch in Vertebraten für den Symmetriebruch benötigt wird. Durch Interaktion mit dem PCP Signalweg trägt Myo1d über die Polarisierung und Ausbildung der Cilien zum links-gerichteten Flüssigkeitsstrom und somit zur Lateralitätsdeterminierung in Xenopus bei. Durch den Einfluss von Myo1d auf die PCP in Drosophila und Xenopus stellt Myo1d eine direkte Verbindung zwischen dem ancestralen Mechanismus und des in Vertebraten neu-evolvierten Flüssigkeitsstrom zum Bruch der bilateralen Symmetrie dar. Studien aus dem Zebrabärbling identifizierten Dmrt2 als einen weiteren Faktor, der sowohl für die Somitogenese als auch für die Ausbildung der LR-Körperachse benötigt wird. Ein Zusammenhang zwischen diesen Prozessen ist ein lang bekanntes Phänomen, dessen Ursache bisher nicht geklärt wurde. Aufgrund der Integration der sLRO Zellen in das paraxiale presomitische Mesoderm, dem Vorläufergewebe der Somiten, stellte sich die Frage, ob dies eine Verbindung zwischen diesen zwei Prozessen erklären könnte. Die Untersuchung von Xenopus Embryonen nach Manipulation von dmrt2 zeigte, dass die Spezifizierung des paraxialen Mesoderms in der frühen Gastrula für die Ausbildung der sLRO Zellen ausschlaggebend ist. Über die Induktion des myogenen Transkriptionsfaktors myf5 reguliert Dmrt2 die Spezifizierung des paraxialen Mesoderms und ins Besondere der sLRO Zellen in Xenopus. Dies demonstrierte zum ersten Mal experimentell eine direkte Verbindung zwischen der frühen Somitogenese und der Lateralitätsdeterminierung und liefert eine erste Erklärung wie diese Prozesse zusammenhängen

Vertebrate Left-Right Asymmetry: What Can Nodal Cascade Gene Expression Patterns Tell Us?

Laterality of inner organs is a wide-spread characteristic of vertebrates and beyond. It is ultimately controlled by the left-asymmetric activation of the Nodal signaling cascade in the lateral plate mesoderm of the neurula stage embryo, which results from a cilia-driven leftward flow of extracellular fluids at the left-right organizer. This scenario is widely accepted for laterality determination in wildtype specimens. Deviations from this norm come in different flavors. At the level of organ morphogenesis, laterality may be inverted (situs inversus) or non-concordant with respect to the main body axis (situs ambiguus or heterotaxia). At the level of Nodal cascade gene activation, expression may be inverted, bilaterally induced, or absent. In a given genetic situation, patterns may be randomized or predominantly lacking laterality (absence or bilateral activation). We propose that the distributions of patterns observed may be indicative of the underlying molecular defects, with randomizations being primarily caused by defects in the flow-generating ciliary set-up, and symmetrical patterns being the result of impaired flow sensing, on the left, the right, or both sides. This prediction, the reasoning of which is detailed in this review, pinpoints functions of genes whose role in laterality determination have remained obscure

An Early Function of Polycystin-2 for Left-Right Organizer Induction in Xenopus

Summary: Nodal signaling controls asymmetric organ placement during vertebrate embryogenesis. Nodal is induced by a leftward fluid flow at the ciliated left-right organizer (LRO). The mechanism of flow sensing, however, has remained elusive. pkd2 encodes the calcium channel Polycystin-2, which is required for kidney development and laterality, and may act in flow perception. Here, we have studied the role of Polycystin-2 in Xenopus and show that pkd2 is indispensable for left-right (LR) asymmetry. Knockdown of pkd2 prevented left-asymmetric nodal cascade induction in the lateral plate mesoderm. Defects were due to failure of LRO specification, morphogenesis, and, consequently, absence of leftward flow. Polycystin-2 synergizes with the unconventional nodal-type signaling molecule Xnr3 to induce the LRO precursor tissue before gastrulation, upstream of symmetry breakage. Our data uncover an unknown function of pkd2 in LR axis formation, which we propose represents an ancient role of Polycystin-2 during LRO induction in lower vertebrates. : Zoology; Evolutionary Developmental Biology; Developmental Biology Subject Areas: Zoology, Evolutionary Developmental Biology, Developmental Biolog

A Conserved Role of the Unconventional Myosin 1d in Laterality Determination

Anatomical and functional asymmetries are widespread in the animal kingdom [ 1, 2 ]. In vertebrates, many visceral organs are asymmetrically placed [ 3 ]. In snails, shells and inner organs coil asymmetrically, and in Drosophila, genitalia and hindgut undergo a chiral rotation during development. The evolutionary origin of these asymmetries remains an open question [ 1 ]. Nodal signaling is widely used [ 4 ], and many, but not all, vertebrates use cilia for symmetry breaking [ 5 ]. In Drosophila, which lacks both cilia and Nodal, the unconventional myosin ID (myo1d) gene controls dextral rotation of chiral organs [ 6, 7 ]. Here, we studied the role of myo1d in left-right (LR) axis formation in Xenopus. Morpholino oligomer-mediated myo1d downregulation affected organ placement in \u3e50% of morphant tadpoles. Induction of the left-asymmetric Nodal cascade was aberrant in \u3e70% of cases. Expression of the flow-target gene dand5 was compromised, as was flow itself, due to shorter, fewer, and non-polarized cilia at the LR organizer. Additional phenotypes pinpointed Wnt/planar cell polarity signaling and suggested that myo1d, like in Drosophila [ 8 ], acted in the context of the planar cell polarity pathway. Indeed, convergent extension of gastrula explant cultures was inhibited in myo1d morphants, and the ATF2 reporter gene for non-canonical Wnt signaling was downregulated. Finally, genetic interference experiments demonstrated a functional interaction between the core planar cell polarity signaling gene vangl2 and myo1d in LR axis formation. Thus, our data identified myo1d as a common denominator of arthropod and chordate asymmetry, in agreement with a monophyletic origin of animal asymmetry

Bicc1 and Dicer regulate left-right patterning through post-transcriptional control of the Nodal inhibitor Dand5

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus, zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3'-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5, Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3'-UTR mRNA reporter was responsive to pkd2, suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage