Maternal high-fat diet exposure is associated with altered hypothalamic microglial development and reduced early postnatal TGFβ1 signaling in male offspring

The hypothalamus plays a central role in maintaining energy homeostasis by integrating peripheral metabolic signals. Maternal high-fat diet (HFD) exposure has been shown to disrupt hypothalamic development and increase offspring susceptibility to metabolic disorders. Microglia, as key regulators of neuroimmune interactions and synaptic remodeling, are increasingly recognized as critical mediators of metabolic programming. During early postnatal development, microglia undergo a transition toward a homeostatic phenotype characterized by markers such as TMEM119, a process that is critically dependent on canonical TGFβ1/SMAD3 signaling. However, whether maternal HFD exposure interferes with the acquisition of homeostatic-like microglial features during this sensitive developmental window remains unclear.

We characterized the postnatal trajectory of hypothalamic microglia-associated developmental features using single-cell RNA sequencing and temporally resolved immunohistochemical analyses in male offspring of HFD-fed dams. Microglial abundance, morphology, and expression of homeostatic markers were assessed across developmental stages in the mediobasal hypothalamus (MBH). Canonical TGFβ1 signaling activity was evaluated by pSMAD3 immunoreactivity, and Parkin-associated signals were quantified within hypothalamic tissue and microglia-defined regions of interest. To assess whether restoration of local TGFβ1 signaling influences microglia-associated developmental features, neonatal MBH supplementation with recombinant TGFβ1 was performed.

Single-cell transcriptomic analysis revealed that hypothalamic microglia progressively acquired homeostatic-associated transcriptional features during early postnatal development, with increasing enrichment of homeostatic marker-associated signatures by the juvenile stage. Maternal HFD exposure attenuated the developmental increase of TMEM119⁺ cells in the MBH and was associated with altered microglial abundance and morphology across postnatal stages. During a critical early postnatal window, male offspring born to HFD-fed dams exhibited reduced pSMAD3 immunoreactivity in IBA1⁺ microglia and decreased Parkin-associated signal in both hypothalamic tissue and microglial regions. Neonatal TGFβ1 supplementation was associated with increased pSMAD3 immunoreactivity in IBA1-positive cells, increased Parkin-associated signal within standardized IBA1-centered regions of interest, and increased TMEM119-positive cell abundance at later juvenile stages.

These findings support a developmental association between maternal HFD exposure, reduced early postnatal TGFβ1/SMAD3 signaling within the MBH, and altered acquisition of homeostatic-like microglial features in the hypothalamus. These developmental neuroimmune alterations may contribute to increased susceptibility to later metabolic dysfunction in male offspring.