Embryonic development is driven by a series of molecular instructions encoded by the transcription factors (TFs) that drive the formation of the three primary germ layers: the ectoderm, mesoderm and endoderm. Analysis of mouse genetic models has demonstrated that key TFs such as LIM homeobox 1 (Lhx1) and Otx2 are indispensable for head and face development, with severe head truncation phenotype seen in the knock-out mice. The aim of this project is to identify and functionally characterise the genomic targets of LHX1 to refine the gene regulatory network for embryonic head formation. We have conducted RNA-sequencing and ATAC-sequencing on gastrulating mouse embryos to identify genomic regions that are either directly regulated by LHX1 or Lhx1 downstream targets. In addition, we mapped by DamID technique the DNA binding sites of LHX1 through a mutant Dam methylase fusion enzyme of improved sensitivity and resolution. LHX1 proteins have been shown to act together with LDB1 and SSDP1 to regulate transcription. To discover other interacting proteins that may be present in this TF complex, we engineered embryonic stem (ES) cell lines to express in the ES cells a BioID2 moiety fused to Lhx1 by doxycycline activation. The BioID2-Lhx1 fusion protein can biotinylate any proximal proteins, which could then be separated by streptavidin pulldown. Identifying protein interactors of LHX1 in both ES cells and differentiated embryoid bodies will help elucidate the composition of LHX1 TF complex. Finally, the functional role of LHX1 downstream genes, such as Foxd4, Zfhx4 and Zkscan2, and the interacting proteins will be characterised. Chimera mouse embryos were generated using CRISPR-Cas9 edited ES cells to examine the requirement of the candidate factors and downstream genes for head formation.