Scott A. Juntti - US grants

Throughout the animal kindgdom, territorial and reproductive behaviors are often innate, indicating that their underlying neural substrates are genetically hardwired. Among vertebrates, many of these behaviors are controlled by the steroid hormone testosterone and its metabolites. Exposure of developing and adult brains to testosterone results in the masculinization of behavior, and in males of most species testosterone (or a metabolite) is necessary for the elicitation of mating and aggressive behavior as adults. The production of gonadal steroids such as testosterone is controlled by neurons of the hypothalamus expressing the neuropeptide gonadotropin releasing hormone (GnRH1). Some of these neurons project to the pituitary, where they control the release of the gonadotropins luteinizing hormone and follicle stimulating hormone into the bloodstream. These hormones bind receptors in the testes, stimulating the production of testosterone. Another subset of GnRH1 neurons project elsewhere in the brain, but the functional relevance of these neurons has not been tested. GnRH1 neurons exhibit dynamic cellular, electrophysiological, and gene transcription correlates of reproductive and territorial behaviors, making them candidate neurons to regulate behavioral outputs. To manipulate GnRH1 neurons, I will transgenically modify a cichlid fish, Astatotilapia burtoni, to express nitroreductase specifically in these cells, enabling their ablation in a temporally controlled manner. Experiments utilizing these fish in this proposal will directly test the function of GnRH1 neurons for behavior in transgenic adult A. burtoni. Further, comparison of the behavioral effects of hormone manipulation to GnRH1 neuron ablation will reveal the relative contributions of GnRH1 neurons to reproductive and territorial behavior via hormone synthesis versus direct connections to the neural circuits that generate these behaviors. This transgenic animal model in which GnRH1 neurons can be conditionally ablated in adult fish will provide a system to understand how these neurons contribute to behavior, and may provide insight into their regulation of fertility, puberty, and menopause.

How does the genome encode the information for proper development and function of the body? This important question is technically and ethically difficult to address in humans, and there is a need for additional animal models that can support gaining this foundational understanding. The cichlid fish are an excellent system to use because these species have wide variation in anatomy, physiology, and behavior. Because their genomes have been sequenced recently, correlations between animal features and gene sequences can be determined. However, the causal links between gene sequence and function must be tested directly to gain meaningful understanding. Causal testing of the links requires the development and deployment of new technologies. The work supported by this grant develops the tools to obtain cichlid embryos and modify gene sequences, enabling researchers to understand how specific genes control the form and function of the body. Given that cichlids and humans share most of the same genes, this work should allow new insights into how genes function in health and disease. The work also allows many opportunities for undergraduate researches from diverse backgrounds to participate directly in the research enterprise, and it includes workshops for investigators from other institutions to enable them to adopt the new protocols and tools in their own research programs.

The >2000 species of cichlid fish exhibit a stunning variety of phenotypes in morphology, physiology, and behavior. Genome-wide scans have mapped these traits to candidate genetic loci. However, functional genetic manipulations are required for direct tests of gene function, and technology for such experiments has lagged behind. CRISPR/Cas has emerged as a potent gene editing tool, and proof-of-principle experiments have been performed in two cichlid species. The work of this grant involves development of experimental pipelines that enable gene editing experiments in a wider variety of cichlid species. Specifically, hormonal treatment regimens that support the reproductive cycle of cichlids and derivation of fertilized eggs are identified, and protocols that permit the modification of genes in a variety of species, through creation of loss-of-function mutations and the insertion of species-specific sequence variants into other species, are developed. Insights and knowledge obtained through these efforts are shared with the broader community through workshops, websites, and publications. Together, the newly developed approaches will support the study of genetic variation and its effects on form and function.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

This award is supported by the Major Research Instrumentation and the Chemistry Research Instrumentation programs. Professor Peter Nemes from the University of Maryland College Park and colleagues Daniel Falvey, Lyle Isaacs, Lisa Taneyhill and Scott Juntti are acquiring a high-resolution, high-pressure liquid chromatograph mass spectrometer with electrospray ionization capabilities (HR-HPLC-ESI-MS). In general, mass spectrometry (MS) is one of the key analytical methods used to identify and characterize small quantities of chemical species embedded in complex samples. In a typical experiment, the components flow into a mass spectrometer where they are ionized into ions and the ions' masses are measured. This highly sensitive technique allows the structure of molecules in complex mixtures to be studied. An instrument with a liquid chromatograph can separate mixtures of compounds before they reach the mass spectrometer. In the electrospray technique a high voltage is applied to a liquid to create an aerosol. This voltage is useful to produce ions from large molecules by avoiding the propensity of macromolecules to fragment when ionized. The acquisition strengthens the research infrastructure at the University and regional area. The instrument broadens participation by involving diverse groups of students in research and research training using this modern analytical technique. The acquisition also provides training opportunities to many undergraduate and graduate students as well as postdoctoral fellows at this institution. The new capability to measure both small biological and organic molecules in a shared Mass Spectrometry Facility has a broad impact on scientists and students in the District of Columbia-Maryland-Virginia region through workshops as well as curriculum modernization and collaborations with Bowie State University.


The award of the mass spectrometer is aimed at enhancing research and education at all levels. It especially impacts studies of metabolic effectors of embryonic development and hormone metabolite activity in nervous system functions. The instrumentation is also used for research on mitochondrial metabolism in the liver as well as molecular mechanisms in neural crest and dental placode cells. In addition, the MS provides information useful for the search of the next generation enzymatic kinetic isotope effects and for studying binders for the valosine-containing protein (VCP) that segregate protein molecules from large cellular structures. The mass spectrometer is also used to inform the design of efficient syntheses for molecular guest compounds. It is also utilized in investigations of electrophilic nitrogen containing species and their reactivity with proteins and nucleic acids.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Juntti | Project Summary The overarching goal of my research program is to understand how hormones shape the neural pathways that control social behavior. Gonadal hormones are key regulators of development and adult neural function, and their signaling leads to profound differences in vertebrate animal reproductive and aggressive behaviors across sexes. Differences in hormone signaling across men and women could contribute to discrepancies in incidence of neuropsychiatric conditions, but the neuronal and transcriptional basis for their actions are not clear. These hormones may determine the connectivity of cells, their activity patterns, or both. Reproductive behaviors in cichlid fish are an ideal model system to understand how hormones impact gene transcription and neural circuit function. Cichlid social behaviors have been studied for nearly a century, and are complex yet stereotyped and quantifiable. Dozens of cichlid species have sequenced genomes, and we have pioneered CRISPR gene editing and transgenesis in these species. We have identified a population of neurons sensitive to the hormones progestin and prostaglandin F2? (PGF) that is essential for spawning behavior. Progestin receptor (PR) signaling regulates gene expression in these cells, while PGF and its receptor, Ptgfr, are sufficient and necessary to initiate spawning. We seek to determine the contribution of Ptgfr+ cells to a variety of social behaviors through genetically directed manipulation of these cells. The implementation of the QF-QUAS system for the first time in cichlids will permit studies of these and other cells. Like other steroid hormone receptors, PR acts as a ligand-activated transcription factor. However, its genomic targets are not known in brain, and so it is not clear how it regulates behavior. We will identify how PR regulates cichlid behavior routines and determine the genomic targets of PR that underlie its actions. The production of progestin (and other steroids) is regulated by the hypothalamic-pituitary-gonadal axis. Sensory inputs regulate the production of progestin in cichlids: male odorants promote its synthesis. We will identify cell groups that mediate this signaling from olfaction to the endocrine system. These approaches will build upon our previous findings and leverage technologies that we have developed, together yielding insights into how the vertebrate brain regulates and responds to sex hormones. These experiments will lay a foundation for understanding the gene networks coordinated by hormones and delineating the neurons that regulate social behaviors.

The genomic revolution has ushered in a new era of biological research. Genomes have been sequenced for not only humans and traditional laboratory animal models such as mice and fruit flies, but thousands of diverse organisms. However, to turn this data into a deep understanding of how genes function, scientists need the ability to manipulate these genes in the newly sequenced species. Recent years have seen dramatic technological improvements to genome editing in several of these species, enabling direct tests. An NSF-funded symposium at the Society for Integrative Biology (SICB) in Austin, TX in January 2023 will shine a spotlight on research in diverse species to reveal new principles of biological function. Work presented aims to accelerate development of these tools in additional species by gathering gene editing experts to teach their methods to others. Using tools like CRISPR/Cas9 gene editing, speakers have modified the genomes of species from jellyfish to mammals and many species in between. Studying many of the unique model organisms represented at SICB may have broad impacts including transformative knowledge advances, an understanding of ecologically important species, or medical insights. This symposium will help democratize gene editing tools to many species and advance knowledge of fundamental biological principles. <br/><br/>The advent of genetic manipulations has enabled incredible insights into genetic mechanisms that control biological processes. However, it has also promoted the consolidation of research into a limited number of species (eg, fruit fly, mouse, zebrafish, C. elegans). As a result, the genetic regulation of a tremendous variety of evolutionary adaptations across species have been understudied at a mechanistic level. The development of CRISPR/Cas genome editing, in particular, enables an expansion of the number of species in which genetic experiments can be performed. Some progress has been made in using gene-edited species to understand organismal biology in ways inaccessible with traditional model organisms. The goal of this symposium is to catalyze the transfer of knowledge and skills from researchers who have applied these technologies in new model organisms to scientists who would like to bring these approaches to their own research programs. A symposium at the 2023 Society for Integrative and Comparative Biology (SICB) conference, which will feature talks, discussions, and a workshop. The speakers (who span institution types, locations, genders, along a spectrum of career stages) have modified species from jellyfish to mammals and many species in between. There will also be an educational event in which speakers from the symposium provide small-group consultations plus a panel discussion. To disseminate this work, all speakers have agreed to publish data presented at SICB in Integrative and Comparative Biology.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.