Methodological Challenges for Ablation of BMPR1A in Hypothalamic Tanycytes-A Cautionary Tale

The hypothalamus coordinates energy-balance regulation and is also a neurogenic/plastic region in the adult brain. Tanycytes, a specialized population of radial glial-like cells lining the third ventricle, reside at the critical interface between the blood, cerebrospinal fluid, and hypothalamic parenchyma. This unique positioning enables them to sense metabolic and nutrient-derived signals, and to shuttle molecules between periphery and brain. Tanycytes can respond to glucose and lipids, as demonstrated by a calcium transient down their long processes that extend into the hypothalamic nuclei. Tanycytes are also capable of self-renewal and differentiation after brain injury, supporting their classification as putative neural stem cells in the adult hypothalamus. Bone morphogenetic protein (BMP) signaling regulates neuroplasticity and contributes to metabolic regulation, including appetite and sympathetic drive to adipose. We previously demonstrated that central administration of BMP7 suppresses appetite, and BMP receptor 1A (BMPR1A) in anorectic hypothalamic POMC neurons impacts appetite regulation. BMPR1A is also tightly and highly co-expressed in hypothalamic tanycytes. Here, we attempted to genetically inactivate BMPR1A in adult tanycytes to explore its functional roles. Using the Rax-CreER^T2xBMPR1Aflox mouse line, we tested multiple routes of tamoxifen administration, as well as its metabolite, without success. Cre recombinase activity was successfully induced via dietary tamoxifen (shown by recombination of the BMPR1A locus and fluorescent reporter induction), but efficient BMPR1A knockout in adult tanycytes was not achieved. Similarly, adeno-associated viral (AAV)-mediated BMPR1A knockdown via the Dio2 promoter and intracerebroventricular delivery yielded limited efficiency, despite confirmed Cre activity indicated by reporter expression. We also observed a compensatory increase in BMPR1A in cells not targeted by these knock-out/knockdown systems, as we observed previously with POMC-Cre knockout of BMPR1A, indicating a responsiveness of the hypothalamic niche to manipulation of BMPR1A levels. Together, our findings support that Cre-driven reporter activity doesn't guarantee gene depletion, and demonstrate that current strategies for loss of function of BMPR1A in adult hypothalamic tanycytes remain technically challenging and require careful validation before interpretation of phenotypes. More efficient and reliable methods are required to elucidate the molecular signaling and functional roles of molecules expressed in adult tanycytes.