Browsing by Subject "Laterality"

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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.
Delimitation of the organizer from the posterior notochord : descriptive and functional studies in mouse and African clawed frog
(2009) Andre, Philipp; Blum, Martin
During vertebrate development, gastrulation is probably the most important phase, as the future body plan is established. Thereby the three body axes anterior-posterior, dorsal-ventral and left-right are determined as well. A central role thereby is taken by the Spemann organizer, as this part of the embryo governs the above mentioned processes. The left-right axis is specified by an extracellular leftward fluid-flow, which results in asymmetric gene expression of the TGFβ factor Nodal. In mice the ciliated epithelium responsible for the fluid-flow as well as the organizer are denominated as ?node?. In contrast to that two distinct entities are thought to be responsible for organizer function and fluid-flow in zebrafish, Xenopus and rabbit embryos. In the present study, it could be shown that this also applies for mouse embryos. In order to prevent further confusion the ciliated epithelium responsible for the fluid-flow was denominated as posterior notochord (PNC) as it is in continuity with the notochord but located anterior to the organizer (?node?). The latter is characterized by the expression of the homeobox gene Goosecoid (Gsc). Gsc possesses, like the tissue of the organizer, the potential to induce an almost complete axis and became therefore famous as ?the organizer gene?. However upon knockout of Gsc in the mouse, surprisingly no gastrulation defects could be detected. Therefore the function of Gsc during gastrulation was investigated using a gain-of-function approach. The analysis of this, in the present and previous studies, indicated that Gsc acts as a switch between two modes of cell movement. Accordingly, Gsc promotes active cell migration and inhibits convergent extension movements. Furthermore it was investigated whether the monoamines adrenaline and serotonin have an influence on the cilia and thus on the leftward fluid-flow, as it was reported in rat and Xenopus experiments. Thereby it could be detected that the addition of adrenaline led to a reduction of the ciliary beat frequency (CBF) and therefore the fluid-flow was attenuated. In contrast to that the addition of serotonin or its antagonists resulted only in minor changes of CBF and thus had no measurable effect on the fluid-flow. The consequences of a malformed PNC were analyzed using embryos mutant for Brachyury (T). Thereby, it was shown that embryos homozygous for this mutation did not develop a functional PNC and thus lacked the fluid-flow. Furthermore a possible cause for the absence of asymmetric Nodal in these embryos was brought into context of an attenuated expression of Fgf8. This indicated that T possesses two distinct roles in left-right development. On the one hand it is necessary for the correct formation of a PNC and on the other hand it is probably needed to maintain the expression of Fgf8, which is a prerequisite for the transcription of Nodal. Finally it was investigated whether these functions were conserved from the African clawed frog Xenopus. Thereby, it could be shown, that Xbra, the homologous gene of T in Xenopus, was also necessary for the formation of the gastrocoel roof plate, the homologous structure of the PNC. Additionally it was observed that the absence of Xbra led to an attenuation of Nodal expression in the midline of Xenopus embryos. This implied that not only the function of Brachyury, but also the process of laterality determination is highly conserved between mammals and amphibians.
Does carrying a rider change motor and sensory laterality in horses?
(2022) Schwarz, Sophie; Marr, Isabell; Farmer, Kate; Graf, Katja; Stefanski, Volker; Krueger, Konstanze
Laterality in horses has been studied in recent decades. Although most horses are kept for riding purposes, there has been almost no research on how laterality may be affected by carrying a rider. In this study, 23 horses were tested for lateral preferences, both with and without a rider, in three different experiments. The rider gave minimal aids and rode on a long rein to allow the horse free choice. Firstly, motor laterality was assessed by observing forelimb preference when stepping over a pole. Secondly, sensory laterality was assessed by observing perceptual side preferences when the horse was confronted with (a) an unfamiliar person or (b) a novel object. After applying a generalised linear model, this preliminary study found that a rider increased the strength of motor laterality (p = 0.01) but did not affect sensory laterality (p = 0.8). This suggests that carrying a rider who is as passive as possible does not have an adverse effect on a horse’s stress levels and mental state.
Left-right asymmetry in Xenopus laevis : functional dissection of leftward flow
(2009) Vick, Philipp; Blum, Martin
Despite their external bilateral symmetry, vertebrates have a conserved left right (LR) asymmetry of their inner organs. For all vertebrates, it is well-known that the asymmetric organogenesis is preceded by the left-sided nodal signaling cascade during embryonic development. A question which has not been settled in detail is how the first asymmetrically directed signal arises, which activates nodal only on the left side. In mice and fish embryos an extracellular leftward fluid flow ? generated by rotating cilia ? was shown to be functionally necessary for gene activation. Recently, this process has also been demonstrated in frog embryos and its mechanic inhibition caused laterality defects. This raised the question if this process is also conserved among vertebrates. The aim of this study was to analyze the mechanism of flow in the frog in the context of the known models. Thereby, its prerequisites and the exact mode of activation of the left-sided genes should be assessed. Finally, general conclusions on the symmetry breakage of vertebrates were to be drawn. Loss of function of axonemal dynein heavy chains inhibited ciliary movement, fluid flow and laterality development of the embryos. By showing that flow was only necessary on the left half of the ciliated epithelium (GRP), definite statements could be made concerning origin, identity and possibility of a transported substance. Moreover, a function for GRP morphogenesis and thus for the generation of flow were proven for the serotonin receptor 3 and the calcium channel Pkd2. These results did not confirm the hypothesis that Pkd2 causes a flow-dependent left-sided calcium signal. Consequently, this contradicted the so-called "2-cilia model" in favor of an early morphogenetic function in frog. In the course of a collaboration it could be shown, that the RNA-binding protein xBic-C has a conserved function for cilia polarization and thus for the flow in both Xenopus and mice. Additionally, up to now, a right-sided nodal inhibitory function has been assigned to the protein coco. However, the exact mechanism was unknown. By specific, combined left- and right-sided loss of function experiments with coco, nodal and the above mentioned components, it could be demonstrated that coco but not nodal is directly dependent on leftward flow. With the flow, coco was downregulated on the left side only and could thus no longer inhibit nodal there. Loss of flow or xBic-C function ? but not that of Pkd2 ? could be rescued by coco inhibition; this revealed a clear hierarchy. Taken together a sequence of conditions could be formulated: Pkd2 and the serotonin receptor 3 are obligatory for the formation of the GRP and correct flow before neurulation. xBic-C also precedes the flow and is required for cilia polarization but seemed also to have a further function. coco is downstream of the fluid flow and is downregulated as its direct consequence on the left side. nodal, in turn, is downstream of this order and is only released on the left side where it can thus act as a putative mediator to transfer the generated signal into the lateral plate mesoderm. These results are discussed in terms of evolutionary origin and conservation.
Non-invasive welfare evaluations in horses : the usefulness of laterality
(2020) Marr, Isabell; Stefanski, Volker
Animal welfare is becoming increasingly important, especially for animal owners. In particular, prey animals such as horses generally suffer in silence. To improve the evaluation of animal welfare in practice and to simplify it in scientific research, new welfare indicators are needed that are easier to assess, less time consuming, and repeatable. Therefore, this thesis aimed to investigate laterality as a welfare indicator that could meet these requirements. Domestic horses were used as model organisms as they display sensory and motor laterality on individual and/or population level; their sensory organs are placed laterally, non-invasive stress hormone analysis is already well-established, as a highly social animal that evolved on open ranges it is prone to suffering from inappropriate human management regimes, and it displays various stress responses. In STUDY it could be demonstrated that a shift to an enhanced preference for the right brain hemisphere was in line with increased stress hormone concentrations in faeces. Sensory laterality and motor laterality measured while grazing, shifted to the left when natural needs were restricted by a change from group to individual housing. Sensory laterality changed immediately after the change of housing conditions, whereas the motor laterality changed with a time delay of one week. STUDY 2 demonstrated that motor laterality measured as initial forelimb use correlated with the cognitive bias (welfare indicator). Right-sided horses were faster to approach an ambiguous stimulus and therefore displayed a positive cognitive bias. But neither motor laterality, measured through grazing stance, nor sensory laterality were related to cognitive bias. STUDY 3 demonstrated that a preference for left side sensory organ use is not only evident in negative contexts but also in positive contexts, because the horses also preferred their left side during affiliative interactions that are assumed to induce positive emotions. This study demonstrated that not only the direction of shift in laterality, but also the context of the shift, should be recorded to reliably identify poor or good welfare. Therefore, it is recommended that additional stress parameters be applied to reliably evaluate animal welfare. STUDY 4 investigated whether the sampling and analysis of faecal stress hormones and immunoglobulin A could be simplified by applying a novel conservation method. Often it is not possible to immediately freeze the faecal samples and/or the transportation to the lab is lengthy. The study demonstrated that faecal samples can be dried a closed system such as an air-tight tube containing silica gel. The samples were dried within 24 hours, as fast as in controlled air-drying conditions at room temperature. The new and simpler drying method prevented the stress hormones (glucocorticoid metabolites) from enzymatic degradation and conserved them, demonstrated by the fact that the detectable concentration remained unchanged. In contrast, immunoglobulin A showed a reduction in the detectable concentration. Therefore, if possible, the conservation of faecal samples should be avoided when immunoglobulin A is to be analysed, although it would be possible to apply an extrapolation to attain fairly reliable results. This new drying method will simplify research on wild horses into the type of stressors they are confronted with, the impact of natural stressors, and effect of stressors on, for example, their laterality in comparison with domestic horses. STUDY 5 investigated whether the strength of laterality provides insight into basal physiological and immunological status, stress response, stress reactivity, or cognitive bias. Only a correlation between age and the strength of laterality was found, with strength of laterality increasing with age. However, age could explain only 30 per cent of the inter-individual variation in strength of laterality. The results demonstrated that the strength of laterality is not a reliable indicator of animal welfare. The direction of laterality may be of greater importance. Altogether, it was demonstrated that laterality is a promising, reliable, repeatable, and objective indictor of animal welfare, which is quick and easy to asses, and inexpensive. Like other well-established welfare and stress indicators, laterality has its limitations. Therefore, it is recommended that other welfare indicator should be simultaneously assessed and changes in laterality recorded, as different traits and personalities result in a high inter-individual variation in base laterality indices. Possible influences and correlations between emotional processing and cerebral lateralization are discussed. Nonetheless, further research is needed to establish a more reliable measurement of motor laterality, and to better understand the relationship between emotional processing and lateralization, as well as possible influencing factors.
Studies of human genetic diseases and developmental processes with the frog Xenopus laevis
(2020) Ott, Tim; Blum, Martin
Next generation sequencing is a driving force behind the identification of genes and alleles that are suspected to cause human genetic diseases. In silico tools are routinely used in the clinical everyday life to characterize unknown genotypes. However, these tools have a limited predictive accuracy and can only provide a first-line assessment. Especially un- or less studied genes require in every case predictive in vivo model systems that allow conclusions about disease associations. Classically, mice and zebrafish are utilized for such research, which concomitantly deepens the understanding of the involved developmental processes. In this collection of studies, the African clawed frog Xenopus laevis was used to explore and promote its suitability for the analysis of potential human disease genes, variants and their associated developmental processes. The first chapters covers potential candidate genes for primary ciliary dyskinesia (PCD). The second chapter addresses if an actin based motor protein and a novel metzincin peptidase, encoded by myosin ID (MYO1D) and leishmanolysin like peptidase (LMLN2)/tout-de-travers (TDT), respectively, are potentially causative for PCD independent laterality defects. The third chapter deals with two candidates for neurodevelopmental disorders, namely hyaluronan mediated motility receptor (HMMR) and progesterone immunomodulatory binding factor 1 (PIBF1).