Institut für Biologie

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dmrt2 and myf5 link early somitogenesis to left-right axis determination in Xenopus laevis
(2022) Tingler, Melanie; Brugger, Amelie; Feistel, Kerstin; Schweickert, Axel
The vertebrate left-right axis is specified during neurulation by events occurring in a transient ciliated epithelium termed left-right organizer (LRO), which is made up of two distinct cell types. In the axial midline, central LRO (cLRO) cells project motile monocilia and generate a leftward fluid flow, which represents the mechanism of symmetry breakage. This directional fluid flow is perceived by laterally positioned sensory LRO (sLRO) cells, which harbor non-motile cilia. In sLRO cells on the left side, flow-induced signaling triggers post-transcriptional repression of the multi-pathway antagonist dand5. Subsequently, the co-expressed Tgf-β growth factor Nodal1 is released from Dand5-mediated repression to induce left-sided gene expression. Interestingly, Xenopus sLRO cells have somitic fate, suggesting a connection between LR determination and somitogenesis. Here, we show that doublesex and mab3-related transcription factor 2 (Dmrt2), known to be involved in vertebrate somitogenesis, is required for LRO ciliogenesis and sLRO specification. In dmrt2 morphants, misexpression of the myogenic transcription factors tbx6 and myf5 at early gastrula stages preceded the misspecification of sLRO cells at neurula stages. myf5 morphant tadpoles also showed LR defects due to a failure of sLRO development. The gain of myf5 function reintroduced sLRO cells in dmrt2 morphants, demonstrating that paraxial patterning and somitogenesis are functionally linked to LR axis formation in Xenopus.
tsCRISPR based identification of Rab proteins required for the recycling of Drosophila TRPL ion channel
(2024) Zeger, Matthias; Stanisławczyk, Lena Sarah; Bulić,Marija; Binder, Andrea Maria; Huber, Armin
In polarized cells, the precise regulation of protein transport to and from the plasma membrane is crucial to maintain cellular function. Dysregulation of intracellular protein transport in neurons can lead to neurodegenerative diseases such as Retinitis Pigmentosa, Alzheimer’s and Parkinson’s disease. Here we used the light-dependent transport of the TRPL (transient receptor potential-like) ion channel in Drosophila photoreceptor cells to study the role of Rab proteins in TRPL recycling. TRPL is located in the rhabdomeric membrane of dark-adapted flies, but it is transported out of the rhabdomere upon light exposure and localizes at the Endoplasmatic Reticulum within 12 h. Upon subsequent dark adaptation, TRPL is recycled back to the rhabdomeric membrane within 90 min. To screen for Rab proteins involved in TRPL recycling, we established a tissue specific (ts) CRISPR/Cas9-mediated knock- out of individual Rab genes in Drosophila photoreceptors and assessed TRPL localization using an eGFP tagged TRPL protein in the intact eyes of these mutants. We observed severe TRPL recycling defects in the knockouts of Rab3, Rab4, Rab7, Rab32, and RabX2. Using immunohistochemistry, we further showed that Rab3 and RabX2 each play a significant role in TRPL recycling and also influence TRPL transport. We localized Rab3 to the late endosome in Drosophila photoreceptors and observed disruption of TRPL transport to the ER in Rab3 knock-out mutants. TRPL transport from the ER to the rhabdomere ensues from the trans-Golgi where RabX2 is located. We observed accumulated TRPL at the trans-Golgi in RabX2 knock-out mutants. In summary, our study reveals the requirement of specific Rab proteins for different steps of TRPL transport in photoreceptor cells and provides evidence for a unique retrograde recycling pathway of TRPL from the ER via the trans-Golgi

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