 Good morning. My name is Rajesh Sipal. I am an assistant professor of stem cells and regenerative medicine in Manipal University, India. So, together with my graduate student Paul Ami Banerjee, we have done some interesting work about the effect of metformin on neural crest development. This work is described in our paper published in stem cells earlier this month. But we would like to tell you the highlights of this work in this short video clip. Well, the neural crest is a unique multi-portal stem cell population that gives rise to diverse cell types of the peripheral nervous system, skin, bone and cartilage. The development of neural crest appears to take place in three distinct stages. But more importantly, the neural crest undergoes a complete and gradual segregation from the neural epithelium through epithelial to sm-chymal transition. This is minutely orchestrated by sequential interplay of several genes and proteins via multiple signaling pathways. Therefore, this has emerged as a relevant model to understand EMT in a non-malignant environment. Recent epidemiological studies indicate the involvement of various drugs and food supplements to congenital abnormalities and developmental defects with impaired neural crest formation. In fact, according to an earlier report, one-third of birth defects are associated with neural crest development. Metformin is one such popular IT diabetic drug often prescribed during pregnancy. But recently it is shown that metformin can have an inhibitory role during EMT. Even patients with prolonged treatment with metformin suffered from problems related to peripheral neuropathy. Now, one of the current focus of my lab has been about the mechanism of metformin action in type 3 EMT as well. Based on this background, our current study not only deals with the development of a suitable platform to evaluate developmental neurotoxicity, but also intended to understand the mode of metformin action on neural crest development using both in vitro and in vivo models. On a whole, we envisage that this study would not only aid in modeling developmental toxicity, but also offer significant therapeutic insights by revealing the associated side effects of metformin. In order to understand the molecular network underlying metformin action during neural crest formation, we first differentiated the murine embryonic stem cells into neural crest cells and demonstrated the spatial temporal regulation of key markers like SOX1, SOX9, HNQ1 and P75. We further authenticated the multilinear differentiation potential of these naïve neural crest cells by guiding them to form Schwann cells, smooth muscle cells, onrocytes and endipocytes. In the next step, when we initiated treatment with metformin along the entire course of differentiation, we witnessed a delay in delamination and migration of these neural crest cells. We observed a decreased expression of key markers like SNEL, SOX9, HNQ1, P75, CTC42, MP2 and 9 with a contrasting increment in the expression of a typical non-neuroepithelial gene, ecotherm. Our data also revealed that metformin impits wind access, a major signaling pathway acted during neural crest formation via DVL3 inhibition, which in turn allows a potential downregulation of ecotherm repressors like SNEL, SLUG and CEP, thus supporting the persistent expression of ecotherm. Moreover, incomplete rescue of NCC determinants using exogenous recombinant wind 3A helped us to reason that perhaps a parallel mechanism besides the wind cascade is active in driving this phenomenon. Thereafter, we took advantage of bioinformatics tools like Ingenuity Pathome Analysis, TargetScan and Miranda for screening NCC-specific target genes and identified the putative role of a candidate set of MIRMS. Further studies involving loss of gain of functions confirmed that NCC-specifiers like SOXMON and SOX9 are direct targets of MIR-200 and MIR-145 respectively and that they are essentially modulated by metform. Additionally, in the last decade, zebrafish models have emerged as an excellent tool for phenotype-based small molecule screen. For example, zebrafish screens have been successfully used to identify the effects of nicotine on spinal motor neuron development. Therefore, employing zebrafish as an environmental model, we confirmed that metformin treatment indeed inhibits induction, migration and differentiation of NCC cells during early embryonic development and leading to an impairment in melanocyte development at a later stage. Taken together for the first time, we demonstrate that metformin can induce a delayed onset of developmental EMT during neural press formation by interfering with canonical win signaling and misregulation of MIR-145 and MIR-200.