Embryonic development is driven by a series of molecular instructions encoded by the transcription factors (TFs) that induces the formation of the three primary germ layers; the ectoderm, mesoderm and endoderm. Analysis of mouse knock-out models has demonstrated that key TFs such as LIM homeobox 1 (Lhx1) and Otx2 are indispensable for development, with severe head truncation abnormalities seen in the knock-out mice. While Lhx1 function is essential for development, the molecular targets and interacting proteins of LHX1 have not been fully elucidated. The aim of this project is to identify and functionally characterise the targets of LHX1 in embryonic head development. RNA-sequencing on gastrulating mouse embryos identified genes that are either directly regulated by or downstream targets of Lhx1. For validation, RNA from chimera mouse embryos over-expressing Lhx1 for 24h were collected a E7.75, at the stage of early head-fold formation. qPCR validated the upregulation of four genes for further functional analysis; Kctd1, Zfhx4, Zkscan2 and Colec12. To discover novel protein-protein interactions of LHX1, a method to screen for proximate and interacting proteins called BioID will be used. We have engineered embryonic stem (ES) cell lines to express the BioID moiety fused to Lhx1 following doxycycline treatment. The BioID-Lhx1 fusion protein can biotinylate any proximal proteins when expressed in the ES cells, which enables them to be separated from all other proteins using streptavidin pulldown. Mass-spectrometry is used to identify these interacting proteins in the LHX1 TF complex. Finally, to characterise the functional role of these candidate genes and interacting proteins in-vivo, chimera embryos will be generated using CRISPR-Cas9 edited ES cells to examine whether the function of the candidate factors are required for head formation in mice. The outcome of this project will lead to building a gene regulatory network for embryonic head formation.