Developing an Isoform-Specific Knock Out Mouse to Investigate the Roles of Non-Canonical Signaling by ErbB4 In Vivo

Abstract

ErbB4 is a receptor tyrosine kinase (RTK) of the epidermal growth factor receptor (EGFR) family that regulates numerous biological processes, from development to physiological function and degeneration. ErbB4 is important in many aspects of neurodevelopment including astrogenesis, neural migration, dendrite formation and synapse maturation. ErbB4 is also necessary for normal development of the heart, mammary glands, immune cells, and lungs. Furthermore, ErbB4 plays important roles in brain function and synaptic transmission in structures such as the hippocampus, amygdala, nucleus accumbens and substantia nigra. ErbB4, and its cognate ligand neuregulin 1 (NRG1) are both schizophrenia susceptibility genes. Mice lacking ErbB4 expression during development or in adulthood display ‘schizophrenia-like behavior,’ indicating that ErbB4 plays important roles in both development and maintenance of normal brain function. ErbB4, like all RTKs, signals through canonical signaling via phosphorylation-mediated signaling cascades. ErbB4 can also signal through a non-canonical mechanism whereby ErbB4 is cleaved and releases an intracellular domain (E4ICD) that shuttles to the nucleus and regulates gene transcription. Non-canonical signaling by ErbB4 requires an isoform-specific inclusion of a cleavage site in the extracellular juxtamembrane domain (EJD). Alternative splicing of the ERBB4 can generate two different ErbB4 isoforms: ErbB4-JMb, which is uncleavable, and ErbB4-JMa, which includes a cleavage site required to signal through the non-canonical mechanism. Non-canonical signaling by ErbB4-JMa has been implicated in the regulation of cortical, cardiac, mammary gland, pulmonary and immune cell development. While ErbB4-JMa was the first RTK identified to be capable of non-canonical signaling, it is now known that most RTKs can be cleaved and utilize direct ICD signaling. However, most studies of this non-canonical signaling by RTKs, including ErbB4, have been performed in vitro. Loss-of-function mouse models, in which non-canonical RTK signaling is abrogated, have not yet been developed to study non-canonical RTK signaling in vivo. The central aim of this dissertation is to create a novel mutant mouse in which non-canonical signaling by ErbB4-JMa is abolished and to phenotypically assess the roles of ErbB4 signaling in the intact organism. To accomplish this, I used germline CRISPR/Cas9 gene editing of mouse ERBB4 exon 16a to target ErbB4-JMa and create an isoform-specific knock out mouse, designated ErbB4-JMa-/-. In this mutant mouse expression of ErbB4-JMa, and hence non-canonical E4ICD signaling, is eliminated, while ErbB4-JMb canonical signaling is intact. I first confirmed that formation of E4ICD was abolished due to the absence of ErbB4-JMa transcription. I then used the ErbB4-JMa-/- mouse to showed that non-canonical signaling by ErbB4-JMa regulates the expression of GFAP during cortical development, consistent with previous results. Next, I used an unbiased gene-discovery approach to identify genes that are regulated by ErbB4 during cortical development. I found that the regulation of two genes, CRYM and DBi, is dependent on ErbB4-JMa. Finally, I used the ErbB4-JMa-/- mouse to assess whether non-canonical ErbB4 signaling plays a role in ErbB4-related developmental phenotypes. I found that ErbB4-JMa signaling is not required for the development of the heart, mammary glands, sensory ganglia or cortical interneurons, but may regulate the development of immune cells. Together these findings show that the ErbB4-JMa-/- mouse is a robust tool to study the role of non-canonical ErbB4-JMa signaling in vivo.