Zebrafish Neural Tube Morphogenesis Requires Scribble-Dependent Oriented Cell Divisions

How control of subcellular events in single cells determines morphogenesis on the scale of the tissue is largely unresolved. The stereotyped cross-midline mitoses of progenitors in the zebrafish neural keel [1–4] provide a unique experimental paradigm for defining the role and control of single-cell orientation for tissue-level morphogenesis in vivo. We show here that the coordinated orientation of individual progenitor cell division in the neural keel is the cellular determinant required for morphogenesis into a neural tube epithelium with a single straight lumen. We find that Scribble is required for oriented cell division and that its function in this process is independent of canonical apicobasal and planar polarity pathways. We identify a role for Scribble in controlling clustering of α-catenin foci in dividing progenitors. Loss of either Scrib or N-cadherin results in abnormally oriented mitoses, reduced cross-midline cell divisions, and similar neural tube defects. We propose that Scribble-dependent nascent cell-cell adhesion clusters between neuroepithelial progenitors contribute to define orientation of their cell division. Finally, our data demonstrate that while oriented mitoses of individual cells determine neural tube architecture, the tissue can in turn feed back on its constituent cells to define their polarization and cell division orientation to ensure robust tissue morphogenesis

Mammalian Inscuteable Regulates Spindle Orientation and Cell Fate in the Developing Retina

During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome

Mammalian Inscuteable Regulates Spindle Orientation and Cell Fate in the Developing Retina

During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome

Molecular dissection of Wnt3a-Frizzled8 interaction reveals essential and modulatory determinants of Wnt signaling activity

Background: Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The molecular basis of Wnt binding to Frizzled and LRP5/6 co-receptors has long been unknown due to the lack of structural data on Wnt ligands. Only recently, the crystal structure of the Wnt8-Frizzled8-cysteine-rich-domain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed. Results: Here, we present an extensive structure-function analysis of mouse Wnt3a in vitro and in vivo. We provide evidence for the essential role of serine 209, glycine 210 (site 1) and tryptophan 333 (site 2) in Fz binding. Importantly, we discovered that valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRD binding site (site 3) partly abolished Wnt binding. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, increasing amounts of soluble Frizzled8-CRD protein modulated Wnt3a signaling in a biphasic manner. Conclusions: We propose a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated high mobility state as a possible modulatory mechanism in Wnt signaling gradients

Stability of person-specific blood-based infrared molecular fingerprints opens up prospects for health monitoring

Health state transitions are reflected in characteristic changes in the molecular composition of biofluids. Detecting these changes in parallel, across a broad spectrum of molecular species, could contribute to the detection of abnormal physiologies. Fingerprinting of biofluids by infrared vibrational spectroscopy offers that capacity. Whether its potential for health monitoring can indeed be exploited critically depends on how stable infrared molecular fingerprints (IMFs) of individuals prove to be over time. Here we report a proof-of-concept study that addresses this question. Using Fourier-transform infrared spectroscopy, we have fingerprinted blood serum and plasma samples from 31 healthy, non-symptomatic individuals, who were sampled up to 13 times over a period of 7 weeks and again after 6 months. The measurements were performed directly on liquid serum and plasma samples, yielding a time- and cost-effective workflow and a high degree of reproducibility. The resulting IMFs were found to be highly stable over clinically relevant time scales. Single measurements yielded a multiplicity of person-specific spectral markers, allowing individual molecular phenotypes to be detected and followed over time. This previously unknown temporal stability of individual biochemical fingerprints forms the basis for future applications of blood-based infrared spectral fingerprinting as a multiomics-based mode of health monitoring