Deciphering BMP signaling in human lateral plate mesoderm

Embryonic development is controlled by signals that coordinate specification and morphogenesis of tissue. The posterior lateral plate mesoderm (PLPM) is a special type of tissue that generates both limb and body wall structures. Mutations that influence its development can lead to severe human disabilities. Currently, we dont have human models that replicate morphogenesis (e.g. formation of body cavity) and specification of PLPM into subtypes (splanchnic, somatic). This limitation is hindering research on the molecular causes of genetic disorders. During early embryology, the BMP (bone morphogenetic protein) pathway is crucial in the context of PLPM development. However, it is still unclear how BMP signaling specifically acts on the different PLPM subtypes due to its highly context- dependent nature and timing. We created a platform using human pluripotent stem cells to differentiate PLPM in 2D and 3D formats, which paved the way for studying the role of BMP during PLPM development. Using this model our objective is to dissect the role of BMP signaling at a molecular level through the following hypotheses and questions. A) How does BMP signaling impact the earliest stages of PLPM development? Our hypothesis is that the levels of BMP signaling at an early stage (induction) governs the separation into splanchnic/somatic PLPM versus intermediate mesoderm (a different subtype of mesoderm, giving rise to kidney and reproductive system). Only after that (patterning stage) splanchnic and somatic subtypes are specified. B) What is the role of BMP in regulating morphogenetic processes that eventually determine lumen formation in PLPM? We hypothesize that BMP differentially regulates the extracellular matrix, cell polarity and contractile machinery to create splanchnic and somatic PLPM cavities. Our goal is to identify BMP signaling targets during the PLPM induction and patterning stages using SLAMseq. The specific role of selected targets will then be validated by functional genetics and RNA sequencing. The role of transcription factors will be confirmed through ChIP-seq and data integration to show how it affects the specification of somatic and splanchnic PLPM. In addition to this unbiased approach, we will study the role of candidate pathways using immunostaining. Complementary, we will investigate the BMP- regulated phosphorylation of the morphogenetic machinery during PLPM cavitation using global phospho-proteomics. Finally, we will genetically and pharmacologically confirm the BMP-dependent candidate regulators responsible for forming the PLPM cavity. With this project ,we aim to develop the first extensive human PLPM organoid model to study the role of BMP in PLPM subtype specification (part A) and morphogenesis (part B). This will pave the way to create human models for studying PLPM congenital disorders.