Almut Köhler

Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases

Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. We identified the cytoplasmic tail of Xcad-11 as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function.

Development of sexually dimorphic vasotocinergic system in the bed nucleus of stria terminalis in chickens.

The bed nucleus of stria terminalis (BnST) of the domestic fowl contains two groups of parvicellular vasotocinergic neurons that are sexually dimorphic. In adult cockerels, arginine vasotocin (AVT) synthesis is well expressed in the dorsolateral and ventromedial portions of the BnST, whereas in corresponding brain areas of hens, AVT synthesis is completely lacking. In the present study, in situ hybridization and immunocytochemical methods were used to compare the ontogeny of sexually dimorphic AVT gene expression in the BnST of male and female chickens from day 12 of embryonic development (E12) until the onset of sexual maturation. By E12, both parvicellular groups of AVT‐immunoreactive (AVT‐ir) perikarya in the developing BnST can be distinguished in some males, whereas in females their presence is questionable. A quantitative analysis, beginning at E14, showed that the parvicellular dorsolateral portion of the BnST of male embryos had more AVT perikarya compared with females. In contrast, no evident sex difference in distribution pattern and number of AVT mRNA containing neurons in this BnST portion was observable by in situ hybridization at E15. At E18, as well as on the first and second days posthatch (D1 and D2), no differences in the number of AVT synthesizing cells and intensity of immunoreactive staining in male versus female chickens were found. Between D2 and D7, the number of AVT‐ir cells in the BnST declined rapidly in both sexes until it disappeared completely in females before D35. In males, another increase in sexually dimorphic AVT‐ir cells and innervation of the lateral septum was associated with the onset of puberty and fully matched a pattern observed in adult fowls. These results demonstrate that the sexually dimorphic part of the AVT system undergoes sexual differentiation during early stages of ontogeny. J. Comp. Neurol. 408:46–60, 1999.

Sex dimorphism in the avian arginine vasotocin system with special emphasis to the bed nucleus of the stria terminalis.

The avian neuropeptide arginine vasotocin (AVT) originally characterized as the antidiuretic hormone (, Endocrinol. 66, 860-871) is produced by neurosecretory cells within the brain. Numerous neuroanatomical studies that employed immunocytochemical and in situ hybridization techniques revealed such cells in the following anatomical brain locations: (a) preoptic area including supraoptic nucleus; (b) paraventricular nucleus; (c) the bed nucleus of the stria terminalis (BnST) (, J. Hirnforsch. 27, 559-566;, J. Neuroendcrinol. 5, 281-288;, Cell Tiss. Res. 287, 69-77;, J. Comp. Neurol. 369, 141-157). The BnST which influences reproduction and sexual behavior shows sex differences in morphology, steroid responsiveness and synthesis of neuropeptides including AVT (, Brain Res. 657, 171-184). AVT is the main endocrine regulator of fluid balance in avian species and, in addition, is involved in oviposition in these species. Our recent studies clearly demonstrated that AVT secretion after osmotic stimulation is sexually dimorphic. In order to investigate whether AVT is expressed and synthesized in the BnST in a sexually dimorphic manner we have used in situ hybridization technique and immunocytochemistry to analyze AVT gene expressing neurons in the parvocellular (small-celled nulei) BnST of adult male and female chickens. In cocks, AVT peptide-containing neurons were detected in the parvocellular BnST and the lateral septal area, whereas no AVT immunoreactive neurons were detected in the corresponding regions of the hen. Even after osmotic stimulation AVT gene expression in neurons of the parvocellular BnST of hens was not upregulated (, Cell Tiss. Res. 287, 69-77). These results demonstrate: (a) AVT gene expression in the BnST of chickens; and (b) a strong sexual dimorphism in this region. Furthermore, AVT synthesis is regulated on the transcriptional level independent from osmotic stimuli. Thus, sex steroids might be the main regulator of AVT gene expression in the BnST. In this paper we not only review the sexual dimorphic vasotocinergic system in the BnST, we also focus on the ontogeny of sex differences and the role of gonadal hormones in organization and retention of these differences.

Cell differentiation of pluripotent tissue sheets immobilized on supported membranes displaying cadherin-11.

Investigating cohesive tissue sheets in controlled cultures still poses a challenge since the complex intercellular interactions are difficult to mimic in in vitro models. We used supported lipid membranes functionalized by the adhesive part of the extracellular domain of the cell adhesion molecule cadherin-11 for the immobilization of pluripotent tissue sheets, the animal cap isolated from Xenopus laevis blastula stage embryos. Cadherin-11 was bound via histidine tag to lipid membranes with chelator head groups. In the first step, quantitative functionalization of the membranes with cadherin-11 was confirmed by quartz crystal microbalance and high energy specular X-ray reflectivity. In the next step, animal cap tissue sheets induced to neural crest cell fate were cultured on the membranes functionalized with cadherin-11. The adhesion of cells within the cohesive tissue was significantly dependent on changes in lateral densities of cadherin-11. The formation of filopodia and lamellipodia in the cohesive tissue verified the viability and sustainability of the culture over several hours. The expression of the transcription factor slug in externally induced tissue demonstrated the applicability of lipid membranes displaying adhesive molecules for controlled differentiation of cohesive pluripotent tissue sheets.

PAPC and the Wnt5a/Ror2 pathway control the invagination of the otic placode in Xenopus.

BackgroundParaxial protocadherin (PAPC) plays a crucial role in morphogenetic movements during gastrulation and somitogenesis in mouse, zebrafish and Xenopus. PAPC influences cell-cell adhesion mediated by C-Cadherin. A putative direct adhesion activity of PAPC is discussed. PAPC also promotes cell elongation, tissue separation and coordinates cell mass movements. In these processes the signaling function of PAPC in activating RhoA/JNK and supporting Wnt-11/PCP by binding to frizzled 7 (fz7) is important.ResultsHere we demonstrate by loss of function experiments in Xenopus embryos that PAPC regulates another type of morphogenetic movement, the invagination of the ear placode. Knockdown of PAPC by antisense morpholinos results in deformation of the otic vesicle without altering otocyst marker expression. Depletion of PAPC could be rescued by full-length PAPC, constitutive active RhoA and by the closely related PCNS but not by classical cadherins. Also the cytoplasmic deletion mutant M-PAPC, which influences cell adhesion, does not rescue the PAPC knockdown. Interestingly, depletion of Wnt5a or Ror2 which are also expressed in the otocyst phenocopies the PAPC morphant phenotype.ConclusionsPAPC signaling via RhoA and Wnt5a/Ror2 activity are required to keep cells aligned in apical-basal orientation during invagination of the ear placode. Since neither the cytoplasmic deletion mutant M-PAPC nor a classical cadherin is able to rescue loss of PAPC we suggest that the signaling function of the protocadherin rather than its role as modulator of cell-cell adhesion is required during invagination of the ear placode.

Quantitative Determination of Lateral Concentration and Depth Profile of Histidine-Tagged Recombinant Proteins Probed by Grazing Incidence X‑ray Fluorescence

We have demonstrated that the complementary combination of grazing incidence X-ray fluorescence (GIXF) with specular X-ray reflectivity (XRR) can be used to quantitatively determine the density profiles of Ni(2)(+) ions complexed with chelator headgroups as well as S atoms in recombinant proteins anchored to lipid monolayers at the air/water interface. First, we prepared phospholipid monolayers incorporating chelator lipid anchors at different molar fractions at the air/water interface. The fine-structures perpendicular to the global plane of monolayers were characterized by XRR in the presence of Ni(2)(+) ions, yielding the thickness, roughness, and electron density of the stratified lipid monolayers. X-ray fluorescence intensities from Ni Kα core levels recorded at the incidence angles below and above the critical angle of total reflection allow for the determination of the position and lateral density of Ni(2)(+) ions associated with chelator headgroups with a high spatial accuracy (±5 Å). The coupling of histidine-tagged Xenopus cadherin 11 (Xcad-11) can also be identified by changes in the fines-structures using XRR. Although fluorescence intensities from S Kα level were much weaker than Ni Kα signals, we could detect the location of S atoms in recombinant Xcad-11 proteins.

Archiving of zebrafish lines can reduce animal experiments in biomedical research

The use of animals in scientific research in Europe is governed by the DIRECTIVE 2010/63/EU of 22 September 2010. This framework is built on the 3R principle with the aim to reduce, refine and replace animal experiments to the indispensable minimum and to conduct them humanely [1]. Careful experimental planning and statistical evaluation can reduce the number of animals necessary for an experiment. Refinement can, for example, include appropriate analgesia and anaesthesia, the improvement of assay procedures and non‐invasive methods. The third and most rigorous strategic line of the 3R principle is the replacement of animals altogether by in vitro methods including cell, organ and embryo culture or by in silico simulation. Since its adoption in 2010, DIRECTIVE 2010/63/EU has been implemented by national legal guidelines such as the Animal Protection Act (TierSchG—Tierschutzgesetz) in Germany. The administrative burden for scientists has increased along with the new guidelines, prompting researchers to avoid this additional workload by employing of alternative research models. Big hopes rest on artificial stem cell‐derived in vitro organ systems. However, significant technological advances are still required to faithfully reproduce organ function in vitro . In addition, many studies involving interactions at a system level or complex behavioural outcomes require the intact animal model. The zebrafish embryo is increasingly used as an alternative model system. Many aspects of biology, including disease …

Cell migration under control of Wnt‐signaling in the vertebrate embryo

During embryonic development, cell fate specification and morphogenetic movements are tightly controlled by a network of signaling cascades. Among these, Wnt‐signaling historically has predominantly been associated with cell fate decisions, however, during the last years evidence is accumulating that Wnt‐signaling pathways also play an essential role in cell migration as part of morphogenetic processes. Wnt signals control and coordinate cell polarity and proper formation of lamellipodia and filopodia in a large number of morphogenetic movements, including gastrulation, neural crest migration, and eye field formation. More recently, Wnt ligands and Wnt antagonists have been reported to function as cell guidance molecules, for example, in the migration of the anterior visceral endoderm of mouse embryos and in axon pathfinding during establishment of retinotectal projection. In this chapter, we aim to provide an overview of Wnt‐regulated cell migration processes, the different downstream signaling pathways and the newly identified receptors and coreceptors involved in Wnt‐stimulated signal transduction. Deciphering the growing complexity of the Wnt‐signaling network and its multiple roles will be the challenge of the next future.

How Cell-Cell Adhesion Contributes to Early Embryonic Development

Cell-cell adhesion is a crucial process during embryonic development from earliest stages on. Keeping cells together during morphological changes in tissue composition is essential for the integrity of the developing organism and different cell-cell adhesion molecules including cadherins, immunoglobulins, and integrins contribute to this tissue cohesion. Cadherins dominate in early Xenopus development and for this reason the review will focus on this group. Cadherins are transmembrane glycoproteins mediating a calcium-dependent cell-cell adhesion. They consist of an intracellular and a transmembrane domain together with an extracellular domain with several repeats specific for the different cadherin types. The first described cadherins had five extracellular repeats with four calcium-binding sites each for three calcium ions as a common structure (Fig. 13.1, see type I). Typically, they possess an HAV tripeptide localized to function as binding site for homophilic trans dimerization. They were named classical cadherins or type I cadherins. In other cadherins, which were identified later, the HAV tripeptide is replaced mostly by a QAV tripeptide. Still there are striking similarities in gene structure justifying the term “cadherin” for these molecules as well. Therefore, the subfamily of type II or atypical cadherins (Fig. 13.1, type II) was created for them.