A new conditional mouse model for Cre-mediated restoration of Hprt1 expression during brain development

Abstract

A central challenge in genetic neurodevelopmental disorders is determining the developmental windows, brain regions, and cell lineages that are affected by gene dysfunction, and whether the resulting pathology remains reversible. The defined genotype-phenotype relationship in Lesch-Nyhan disease (LND), a monogenic disorder caused by pathogenic variants in HPRT1 and subsequent deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt), provides a well-characterized disease context for such mechanistic investigations. We present a new conditional Hprt1 mouse model carrying a loxP-flanked mCherry and transcriptional stop cassette, permitting Cre-driver-dependent analysis of spatially and temporally controlled restoration of endogenous Hprt1 expression during brain development. Hprt1 reactivation was confirmed at the genomic, transcriptional, and biochemical levels. Using the ubiquitous tamoxifen-inducible CAG-CreER driver, a single maternal tamoxifen dose at embryonic day (E)8.5 restored whole-brain Hprt1 mRNA expression and HGprt enzyme activity in E14.5 embryos to 72.4% and 13.4% of wild-type levels, respectively. Postnatal exposure through lactation during maternal treatment from postnatal day (P)0 to P4 yielded whole-brain HGprt activity equivalent to 28.3% of the WT level at P17. However, ligand-independent CAG-CreER activity and treatment-associated reproductive and developmental complications precluded extended longitudinal rescue studies with this Cre-driver. Developmentally regulated Wnt1-Cre2 activity, from E8 onwards, provided an alternative approach that permitted analysis through P60. Although the Wnt1 lineage constitutes only a fraction of the developing brain, Cre-dependent reactivation produced detectable HGprt activity in the forebrain, midbrain, and hindbrain. RNA fluorescent in situ hybridization (FISH) detected spatially distributed Hprt1 reactivation in the midbrain and prefrontal cortex at E14.5 and across multiple midbrain, cortical, and subcortical regions at P60. These findings establish the conditional Hprt1tm1.1(LoxP-mCherry-Stop-LoxP)Vsr mouse as a model for investigating when and where Hprt1 restoration could rescue abnormal brain development, while demonstrating that Cre-driver properties determine experimental specificity and longitudinal feasibility. Beyond LND, this conditional reactivation strategy provides an approach for defining developmental windows of reversibility and lineage-specific requirements for gene restoration in monogenic neurodevelopmental disorders.