Dissecting Hhip1 Function <i>In Vivo</i> Using a Conditional Knockout Mouse Model

The Hedgehog (Hh) signaling pathway is essential for organ development and tissue homeostasis; its dysregulation is associated with various congenital anomalies and human diseases. Hedgehog-interacting protein 1 (Hhip1) functions as a membrane-bound negative regulator of Hh signaling by directly binding to Hh ligands and limiting their activity in target tissues. While global knockout models have demonstrated the importance of <i>Hhip1</i> during embryonic development, perinatal lethality has precluded investigations into its postnatal functions. To overcome this limitation, a novel conditional allele of <i>Hhip1</i> (hereafter referred to as the <i>Hhip1^flox</i> or floxed allele, in which exon 2 is flanked by loxP sites) was generated using CRISPR/Cas9-based gene targeting. Cre-mediated excision of exon 2 produced a conditional null allele (<i>Hhip1^ex2Δ</i> ) with a frameshift mutation that annulled the Hedgehog ligand-binding domain. Germline recombination through CMV-Cre recapitulated the known pulmonary defects of global <i>Hhip1</i> knockout mice, validating the model's functionality. Furthermore, using Prx1-Cre to generate limb-specific <i>Hhip1</i> conditional knockout (cKO) mice, a previously unrecognized role for <i>Hhip1</i> in maintaining postnatal growth plate architecture was detected. cKO mice exhibited progressive growth plate expansion and long bone overgrowth, together with sustained upregulation of <i>Gli1</i> expression. These findings establish the <i>Hhip1^flox</i> model as a powerful tool for tissue- and stage-specific functional studies of <i>Hhip1</i> and provide new insights into the spatiotemporal regulation of Hedgehog signaling in development and disease. Importantly, this model offers broad utility for dissecting Hedgehog signaling mechanisms across diverse biological contexts and disease models.