Prakrit Subba

Mitral valve prolapse (MVP) is a common congenital valve disorder that has been linked to defects in primary cilia during cardiac development, yet the mechanisms connecting ciliary regulation to valvulogenesis remain incompletely understood. As DZIP1 is required for ciliogenesis and has been shown to be involved in MVP pathogenesis, we sought to identify interacting partners that may regulate its localization and function. Using an unbiased yeast two-hybrid screen of a human heart cDNA library, we identified BRD10, a previously uncharacterized bromodomain-containing protein, as a DZIP1-interacting partner. Co-immunoprecipitation confirmed the DZIP1–BRD10 interaction and mapped the binding region to a conserved domain containing a putative nuclear localization sequence. RNAscope and immunofluorescence demonstrated that BRD10 and DZIP1 are co-expressed in developing murine valve cells, with BRD10 localizing near the basal body and transition zone of the primary cilium. BRD10 also interacted with acetylated α-tubulin, supporting a relationship with ciliary microtubule structures. Nuclear–cytoplasmic fractionation further showed that BRD10 promotes cytoplasmic enrichment of DZIP1. Together, these findings identify BRD10 as a novel regulator of DZIP1 subcellular localization and suggest that bromodomain-mediated recruitment at the primary cilium may contribute to normal valvulogenesis and developmental mechanisms relevant to MVP.

Climate change is driving more frequent and severe heat events, yet the developmental mechanisms by which organisms anticipate and adapt to thermal stress remain poorly understood. In zebra finches ( Taeniopygia guttata ), parents exposed to extreme heat emit distinctive “heat call” vocalizations during late incubation that trigger adaptive phenotypic responses in offspring, including altered growth, thermoregulation, and reproductive success. Recent transcriptomic analysis revealed that prenatal heat call exposure induces hypothalamic gene expression changes enriched in non-neuronal populations, but cellular heterogeneity has obscured which cell types are primary targets and the regulatory mechanisms underlying acoustic programming. Here, we performed single-nucleus multiome sequencing to generate a cell-type-resolved atlas of gene expression and chromatin accessibility in the late-stage embryonic zebra finch hypothalamus. We find that heat call exposure selectively remodels astrocyte regulatory landscapes, with most playback-responsive chromatin changes localized to glial populations like astrocytes, challenging neuron-centric models of developmental programming. Motif enrichment and gene regulatory network analysis showed Nuclear Factor I-C, AP-1, and Notch pathway components - canonical drivers of the gliogenic switch - coordinate this response. Chromatin co-accessibility analysis revealed extensive astrocyte-specific enhancer-promoter rewiring at developmental signaling hubs, and pseudotemporal analysis identified condition-associated shifts in developmental trajectories and astrocyte gene-expression dynamics consistent with altered gliogenic programs. In parallel, we identified sex-dimorphic regulation of transthyretin in glutamatergic neurons of the paraventricular nucleus, linking acoustic programming to thyroid-dependent neuroendocrine pathways. These findings suggest that prenatal acoustic experience engages astrocyte-selective epigenetic priming and neuroendocrine transcriptional responses that may reshape hypothalamic developmental programs under predicted thermal environments.

Developmental plasticity allows organisms to adjust their phenotypes to match environmental conditions, but how sensory cues program specific physiological systems remains poorly understood. In Australian zebra finches, incubating parents emit heat calls during extreme temperatures, and embryos exposed to these acoustic signals develop enhanced thermal tolerance and altered growth trajectories as adults, a striking example of anticipatory programming. We hypothesized that heat call exposure alters embryonic hypothalamic gene expression, given this brain region's central role in integrating environmental signals and regulating metabolism, thermoregulation and growth. We exposed zebra finch embryos to playback of parental heat calls or control calls during late incubation and used RNA-sequencing of hypothalamic tissue to identify transcriptional responses. Contrary to predictions of widespread neuroendocrine reprogramming, heat call exposure produced targeted changes: robust downregulation of genes regulating vascular smooth muscle contraction and cytoskeletal dynamics, with coordinated isoform switching. Cell-type analyses revealed these molecular changes localized to vascular endothelial cells, smooth muscle cells and ependymal cells, the cellular components that control cerebral blood flow and regulate the brain's vascular barrier. Gene expression patterns suggest increased vascular plasticity that may protect against heat-induced cellular damage. Remarkably, these adaptive modifications occurred in response to an acoustic signal alone, without thermal exposure. Our results provide transcriptional evidence that prenatal acoustic cues may program cerebrovascular function through cell type-specific gene regulation, providing a novel mechanism for sensory-mediated developmental plasticity. This targeted vascular programming may represent a conserved strategy for anticipatory adaptation to predictable thermal challenges across endothermic vertebrates.

: Organisms have evolved mechanisms to adjust to rapid environmental change. A dramatic example is the Australian zebra finch, where incubating parents produce an acoustic signal ("heat call") during extreme heat, triggering adaptive phenotypic plasticity in their offspring growth, thermoregulation, and reproductive success. To elucidate for the first time the molecular mechanisms underlying heat call-induced programming, we hypothesized a prenatal shift in hypothalamic gene expression, given the hypothalamus’s central role in neuroendocrine signaling controlling metabolism and growth. In addition, we tested whether prenatal heat-call exposure induces local changes in the brain, to protect this highly heat-sensitive organ from upcoming heat challenges. We exposed zebra finch embryos to chronic playback of parental heat calls or control calls, then isolated the hypothalamus for RNA sequencing to identify differentially expressed genes and gene regulatory networks. Heat-call exposure elicited modest neuroendocrine gene expression changes, but robust downregulation of genes tied to muscle contraction and cytoskeletal dynamics, with evidence of isoform usage shifts. These changes were prominently localized to hypothalamic neurovascular endothelial, mural, and ependymal cell populations, forming the blood–brain barrier (BBB). Because embryos experienced heat-associated sound, but not heat itself, and changes matched the loosening of the BBB to avoid breakages, these transcriptomic shifts likely represent an anticipatory response to enhance subsequent brain resilience to heat. Our study provides the first genome-wide characterization of embryonic hypothalamic gene expression in a songbird and reveals that prenatal acoustic cues can developmentally program neurovascular systems, expanding current understanding of developmental plasticity under climate change.

Studies of early development in birds typically rely on PCR analysis of genomic DNA to identify embryonic or neonatal sex. In zebra finches and other birds, males are the homogametic sex (ZZ) while females are heterogametic (ZW), and females are distinguished by the presence of specific sequences on the female-specific W chromosome. However, when only a single W locus is analyzed, lack of a PCR product in a sample could potentially arise from genetic variation or technical failure of the amplification, leading to false identification of female samples as males. To mitigate this concern, we developed an approach that targets two different W loci, using SYBR-based quantitative PCR to analyze amplification curves. We applied this method to determine sex of 30 zebra finch embryos (embryonic day 13) and subsequently confirmed genetic sex by brain transcriptome sequencing. We also identified and tested primer sets that are effective for sex determination in chickens.