We use zebrafish and medaka models to investigate fundamental processes of embryonic development and to approach the molecular basis of different human disorders. This includes formation and patterning of the central nervous system, molecular mechanisms of neural degeneration, development of somites and bones, and fish models for human neurodegenerative disorders and bone diseases.
Professor Winkler studies the development of the nervous system in the model fishes, zebrafish and medaka, with powerful confocal microscopes. Because the fish nervous system can be imaged by the light microscopes, the Winkler lab is dissecting apart the critical steps in the formation of the central nervous system. Using the same tools and approaches, they are applying their expertise toward understanding how the nervous system degenerates during neural diseases.
Photoreceptor degeneration in a zebrafish model for Retinitis pigmentosa
Retinitis pigmentosa (RP) is an inherited eye disease characterized by progressive photoreceptor degeneration. 12% of patients suffering from RP carry mutations in the RNA splicing factors PRPF3, PRPF8 and PRPF31. How mutations in these ubiquitous splicing factors selectively cause degeneration of photoreceptors remains unknown. We have established a zebrafish model to address this problem. This model allows a unique combination of powerful genetics and dynamic bioimaging in vivo.
Using advanced confocal microscopy, we were the first to visualize the effects of distinct PRPF31 mutations on photoreceptor degeneration in an in vivo setting. Furthermore, we were the first to identify aberrantly spliced mRNA targets in this model, yielding important new insight into the mechanism of splice factor induced neuron degeneration.
In vivo imaging of osteoblast-osteoclast interaction in a medaka model for osteoporosis
We are interested in the cellular mechanisms that control bone homeostasis. Fish, such as medaka, have bone cells very similar to humans. Also, the genetic networks regulating formation of bone-forming osteoblasts and bone-resorbing osteoclasts are highly conserved.
We have established several transgenic medaka lines that express fluorescent reporters in bone cells at distinct stages of differentiation, or express RANKL, an osteoclast-inducing factor, under control of a heatshock promoter. Upon heatshock, RANKL induces the formation and activation of ectopic osteoclasts. This results in degradation of bone matrix in a manner very similar to the situation in human osteoporosis patients. This unique in vivo model allows visualization of osteoblast/osteoclast interaction in an intact living animal during bone degradation as well as regeneration.
Neurogenesis and neural differentiation in the embryonic spinal cord
Our nervous system consists of billions of neurons that interconnect in a very precise manner to allow proper function of the nervous system. To achieve this extraordinary complexity, neurons need to be born at exactly defined time points and positions in the developing embryo and make specific interconnections. Using the zebrafish model, we analyze how growth factors (Midkines), their receptors (Alk, RPTPs) and a class of transcription factors (the Dmrt family) control timing and position of neuron birth and differentiation in brain and spinal cord.
FRET-based Ca2+ sensors to visualize neuron-glia interaction during synapse establishment in a zebrafish model for Spinal Muscular Atrophy
We have generated transgenic zebrafish lines that express a FRET-based ratiometric Ca2+ sensor in motoneurons and surrounding glia cells. These lines allow imaging of Ca2+ influx and thus activaty of neurons and glia during synapse establishment and maintenance in intact embryos. Ca2+ influx is analyzed during normal development and in our zebrafish model for Spinal Muscular Atrophy (SMA), a common neurodegenerative disorder characterized by progressive motoneuron degeneration with unclear etiology.
Witten, P.E., Harris, M.P., Huysseune, A., Winkler C. (2016). Small teleost fish provide new insights into human skeletal diseases. Methods Cell Biol. In Press.
Yu, T., and Winkler, C. (2016). Drug treatment and in vivo imaging of osteoblast-osteoclast interactions in a medaka fish osteoporosis model. Journal of Visualized Experiments. In Press.
Spiró, Z., Koh, A., Tay, S., See, K., Winkler, C. (2016). Transcriptional enhancement of Smn levels in motoneurons is crucial for proper axon morphology in zebrafish. Scientific Reports 6:27470.
Yu, T., Buettner, A., To, T.T., Witten, P.E., Huysseune, A., Winkler, C. (2016). Live imaging of osteoclast inhibition by bisphosphonates in a medaka osteoporosis model. Disease Models & Mechanisms 9(2), 155-163.
To, T.T., Witten, P.E., Huysseune, A., Winkler, C. (2015). An adult osteopetrosis model in medaka reveals importance of osteoclast function for bone remodeling in teleost fish. Comp Biochem Physiol C Toxicol Pharmacol 178:68-75.
Willems, B., Tao, S., Yu, T., Huysseune, A., Witten, P.E., Winkler, C. (2015). The Wnt co-receptor Lrp5 is required for cranial neural crest cell migration in zebrafish. PLoS ONE 10(6):e0131768.
Graf, M., Teo Qi-Wen, E.R., Sarusie, M.V., Rajaei, F., Winkler, C. (2015). Dmrt5 controls corticotrope and gonadotrope differentiation in the zebrafish pituitary. Mol Endocrinol 29, 187-99.
See, K., Yadav, P., Giegerich, M., Cheong, P.S., Graf, M., Vyas, H., Lee, S.G.P, Mathavan, S., Fischer, U., Sendtner, M., Winkler, C. (2014). SMN-deficiency alters Nrxn2 expression and splicing in zebrafish and mouse models of spinal muscular atrophy. Human Molecular Genetics 23, 1754-1770.
Winkler, C. and Yao, S. (2014). The midkine family of growth factors: Diverse roles in nervous system formation and maintenance. British Journal of Pharmacology 171, 905-912.
Renn, J., Buettner, A., To, T.T., Chan, S.J.H., Winkler, C. (2013). A novel col10a1:nlGFP transgenic reporter line displays putative osteoblast precursors at the medaka notochordal sheath prior to mineralization. Developmental Biology 381, 134-143.
Yao, S., Cheng, M.G., Zhang, Q., Wasik, M., Kelsh, R., Winkler, C. (2013). Anaplastic lymphoma kinase is required for neurogenesis in the developing central nervous system of zebrafish. PLoS ONE 8(5): e63757.
Lim, J.W., Yao, S., Graf, M., Winkler, C., Yang, D.W. (2013). Structure-function analysis of full-length midkine reveals novel residues important for heparin-binding and zebrafish embryogenesis. Biochemical Journal 451, 407-415.
To, T.T., Witten, P.E., Renn, J., Bhattacharya, D., Huysseune, A., Winkler, C. (2012). RANKL induced osteoclastogenesis leads to loss of mineralization in a Medaka osteoporosis model. Development 139, 141-150.
Willems, B., Buettner, A., Huysseune, A., Renn, J., Witten, P.E., Winkler, C. (2012). Conditional ablation of osteoblasts in Medaka. Developmental Biology 364, 128-137.
Tran, L.D., Hino, H., Quach, H., Lim, S., Shindo, A., Mimori-Kiyosue, Y., Mione, M., Ueno, N., Winkler, C., Hibi, M., Sampath, K. (2012). Dynamic Microtubules at the Vegetal Cortex Predict the Embryonic Axis in Zebrafish. Development 139, 3644-3652.
Yin, J., Brocher, J., Fischer, U., Winkler, C. (2011). Mutant Prpf31 causes pre-mRNA splicing defects and rod photoreceptor cell degeneration in a zebrafish model for Retinitis pigmentosa. Molecular Neurodegeneration 6:56.
Linder, B., Dill, H., Hirmer, A., Brocher, J., Lee G.K., Mathavan, S., Bolz, H.J., Winkler, C., Laggerbauer, B., Fischer, U. (2011). Systemic splice factor deficiency causes tissue-specific defects: A zebrafish model for Retinitis pigmentosa. Human Molecular Genetics 20, 368-377.