1. Role of midkine in neurogenesis
Midkine (Mdk) and Pleiotrophin (PTN) are two widely expressed heparin-binding growth factors involved in multiple biological processes. In mammalian embryos, Mdk expression is typically found in the developing nervous system during embryogenesis. Although its expression is drastically down-regulated after birth, only persisting in the kidney, the up-regulation of midkine can still be triggered in serval disease conditions. Midkine knockout mice embryos are phenotypically normal at early stage, while striking deficiencies related to neural protection and regeneration are found at their adult stage.
Two Mdk orthologues, mdka and mdkb, exist in the zebrafish genome, which are expressed in mostly non-overlapping patterns in the embryonic nervous system. Their spatial expressions are also highly restricted in the adult brain. Knocking-down of mdka by morpholinos has been previously reported to change the choices of fate between medial floor plate and notochord cells, while the alteration of mdkb expression affects early cell determination in neural crest border.
In our lab, CRISPR/Cas9 genome editing tools are applied to generate zebrafish Mdk and PTN knockout mutants. By analyzing these mutants, we expect the information they provide will assist us in unraveling the conundrum of the functions of these genes. To understand how the concentration gradients of secreted growth factors Mdks are formed and maintained, and how these gradients subsequently influence the developmental process of zebrafish at different stages, we will integrate fluorescent proteins into Mdks gene loci by CRISPR/Cas9 mediated knock-in technology to monitor Mdks proteins in vivo. We aim at an integrated multi-disciplinary approach that combines modern biological techniques with state-of-the-art quantitative bioimaging and computational tools to quantitatively investigate gradient formation within a developing and constantly changing zebrafish embryo. With our collaborators, we will build computational models to help us understand the principles of developmental process controlled by morphogens. These models will also provide testable predictions about developmental outcomes in various mutants. Both the mutants and transgenic genetic tools will eventually facilitate our discovery on the Mdk receptors and downstream signaling effectors.
2. 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.
3. 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.
4. 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.
