Julia M. George

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.

Our understanding of bird song, a model system for animal communication and the neurobiology of learning, depends critically on making reliable, validated comparisons between the complex multidimensional syllables that are used in songs. However, most assessments of song similarity are based on human inspection of spectrograms, or computational methods developed from human intuitions. Using a novel automated operant conditioning system, we collected a large corpus of zebra finches’ ( Taeniopygia guttata ) decisions about song syllable similarity. We use this dataset to compare and externally validate similarity algorithms in widely-used publicly available software (Raven, Sound Analysis Pro, Luscinia). Although these methods all perform better than chance, they do not closely emulate the avian assessments. We then introduce a novel deep learning method that can produce perceptual similarity judgements trained on such avian decisions. We find that this new method outperforms the established methods in accuracy and more closely approaches the avian assessments. Inconsistent (hence ambiguous) decisions are a common occurrence in animal behavioural data; we show that a modification of the deep learning training that accommodates these leads to the strongest performance. We argue this approach is the best way to validate methods to compare song similarity, that our dataset can be used to validate novel methods, and that the general approach can easily be extended to other species.

Sound is an essential source of information in many taxa and can notably be used by embryos to programme their phenotypes for postnatal environments. While underlying mechanisms are mostly unknown, there is growing evidence for the involvement of mitochondria—main source of cellular energy (i.e. ATP)—in developmental programming processes. Here, we tested whether prenatal sound programmes mitochondrial metabolism. In the arid-adapted zebra finch, prenatal exposure to ‘heat-calls’—produced by parents incubating at high temperatures—adaptively alters nestling growth in the heat. We measured red blood cell mitochondrial function, in nestlings exposed prenatally to heat- or control-calls, and reared in contrasting thermal environments. Exposure to high temperatures always reduced mitochondrial ATP production efficiency. However, as expected to reduce heat production, prenatal exposure to heat-calls improved mitochondrial efficiency under mild heat conditions. In addition, when exposed to an acute heat-challenge,LEAKrespiration was higher in heat-call nestlings, and mitochondrial efficiency low across temperatures. Consistent with its role in reducing oxidative damage,LEAKunder extreme heat was also higher in fast growing nestlings. Our study therefore provides the first demonstration of mitochondrial acoustic sensitivity, and brings us closer to understanding the underpinning of acoustic developmental programming and avian strategies for heat adaptation.

A species’ success during the invasion of new areas hinges on an interplay between demographic processes and the outcome of localized selection. Invasive European Starlings ( Sturnus vulgaris ) established populations in Australia and North America in the 19 ^th century. Here, we compare whole-genome sequences among native and independently introduced European Starling populations from three continents to determine how demographic processes interact with rapid adaptive evolution to generate similar genetic patterns in these recent and replicated invasions. Our results confirm that a post-bottleneck expansion may in fact support local adaptation. We find that specific genomic regions have differentiated even on this short evolutionary timescale, and suggest that selection best explains differentiation in at least two of these regions. This infamous and highly mobile invader adapted to novel selection (e.g., extrinsic factors), perhaps in part due to the demographic boom intrinsic to many invasions.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species^1–4. To address this issue, the international Genome 10K (G10K) consortium^5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are only available for a few non-microbial species ^1–4 . To address this issue, the international Genome 10K (G10K) consortium ^5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling the most accurate and complete reference genomes to date. Here we summarize these developments, introduce a set of quality standards, and present lessons learned from sequencing and assembling 16 species representing major vertebrate lineages (mammals, birds, reptiles, amphibians, teleost fishes and cartilaginous fishes). We confirm that long-read sequencing technologies are essential for maximizing genome quality and that unresolved complex repeats and haplotype heterozygosity are major sources of error in assemblies. Our new assemblies identify and correct substantial errors in some of the best historical reference genomes. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an effort to generate high-quality, complete reference genomes for all ~70,000 extant vertebrate species and help enable a new era of discovery across the life sciences.

Significance: Our results show that the experience of being alone has immediate consequences on brain gene activity. Clearly this is important for the human context, where loneliness (i.e., perceived social isolation) is a risk factor for psychological and neurodegenerative disorders. As the response occurs in brain areas involved in higher-order cognition and perception, these changes in gene activity may influence the ongoing processing of experience. Prior studies in humans have measured effects of social isolation in circulating leukocytes, but not in brain, and studies in mammalian models have typically focused on effects of much longer isolation periods (weeks or more). The results also bear on interpretation of animal experiments where short-term isolation is used to establish the baseline reference condition.

Songbirds communicate through learned vocalizations, using a forebrain circuit with convergent similarity to vocal-control circuitry in humans. This circuit is incomplete in female zebra finches, hence only males sing. We show that the UTS2B gene, encoding Urotensin-Related Peptide (URP), is uniquely expressed in a key pre-motor vocal nucleus (HVC), and specifically marks the neurons that form a male-specific projection that encodes timing features of learned song. UTS2B-expressing cells appear early in males, prior to projection formation, but are not observed in the female nucleus. We find no expression evidence for canonical receptors within the vocal circuit, suggesting either signalling to other brain regions via diffusion or transduction through other receptor systems. Urotensins have not previously been implicated in vocal control, but we find an annotation in Allen Human Brain Atlas of increased UTS2B expression within portions of human inferior frontal cortex implicated in human speech and singing. Thus UTS2B (URP) is a novel neural marker that may have conserved functions for vocal communication.