Central nervous system (CNS) development is regulated by regionally expressed transcription factors that impart initial cell identity, connectivity, and function to neural circuits through complex molecular genetic cascades. genomic screen homeobox 1 and 2 (gsx1 and gsx2) encode homeobox transcription factors expressed in the developing CNS in multiple vertebrates examined to date. However, we have limited knowledge of the expression of these transcription factors and the gene networks that they regulate across developing brain regions in zebrafish. The objective of this study was to comprehensively examine gsx1 and gsx2 expression throughout neurodevelopment and characterize gsx1 and gsx2 mutants to study the essential roles of these closely related transcription factors. Using RT-PCR, whole-mount in situ hybridization (WISH), and fluorescence in situ hybridization, we examine gsx1 and gsx2 expression from early embryonic to late larval stages. gsx1 is expressed initially in the hindbrain and diencephalon and later in the optic tectum, pretectum, and cerebellar plate. Comparatively, gsx2 is expressed in the early telencephalon and later in the pallium and olfactory bulb. gsx1 and gsx2 are regionally co-expressed in the hypothalamus, preoptic area, and hindbrain, however rarely co-localize in the same cells. To identify forebrain target genes, we utilize mutants made with Transcription activator-like effector nucleases (TALEN). gsx1 mutant zebrafish exhibit stunted growth, however, they survive through adulthood and are fertile. gsx2 mutant zebrafish experience swim bladder inflation failure that prevents survival past larval stage. Using WISH and RT-qPCR we demonstrate altered expression of genes including, distal-less homeobox genes and forkhead box gene foxp2. This work provides novel tools with which other target genes and functions of Gsx1 and Gsx2 can be characterized across the CNS to better understand the unique and overlapping roles of these highly conserved transcription factors.

Background Homeobox transcription factor encoding genes, genomic screen homeobox 1 and 2 (gsx1 and gsx2), are expressed during neurodevelopment in multiple vertebrates. However, we have limited knowledge of the dynamic expression of these genes through developmental time and the gene networks that they regulate in zebrafish. Results We confirmed that gsx1 is expressed initially in the hindbrain and diencephalon and later in the optic tectum, pretectum, and cerebellar plate. gsx2 is expressed in the early telencephalon and later in the pallium and olfactory bulb. gsx1 and gsx2 are co‐expressed in the hypothalamus, preoptic area, and hindbrain, however, rarely co‐localize in the same cells. gsx1 and gsx2 mutant zebrafish were made with TALENs. gsx1 mutants exhibit stunted growth, however, they survive to adulthood and are fertile. gsx2 mutants experience swim bladder inflation failure that prevents survival. We also observed significantly reduced expression of multiple forebrain patterning distal‐less homeobox genes in mutants, and expression of foxp2 was not significantly affected. Conclusions This work provides novel tools with which other target genes and functions of Gsx1 and Gsx2 can be characterized across the central nervous system to better understand the unique and overlapping roles of these highly conserved transcription factors.

Early research experiences positively affect students' interest in STEM careers, and develop practical science and critical thinking skills. However, outreach opportunities are not equally accessible for all students. In states like West Virginia, where many students live in rural Appalachian communities, opportunities for engaging in STEM experiences are limited. In addition, rural teachers may not be equipped to provide authentic research experiences for students due to lack of resources or support. For many students in West Virginia, the Health Sciences and Technology Academy (HSTA) is a major opportunity for STEM engagement. Since its inception in 1998, HSTA has spread to 26 of 55 counties in West Virginia. The program recruits first-generation, low-socioeconomic status, rurally living, and African American high school students who are under-represented in STEM fields. Our research laboratory partnered with HSTA to implement an innovative, hands-on research camp using zebrafish for students participating in their annual junior-level biomedical sciences summer camp. Our camp was held in-person and adapted to an online format during the Covid-19 pandemic. We used pre–post surveys in both camps to assess impacts on science confidence and to collect information about general perceptions of zebrafish, research, and STEM fields. We found that students participating in the in-person and online camps experienced similar overall gains in science confidence. We also identified strong interest in zebrafish, research, and STEM degrees among online students. Online students did not prefer virtual learning experiences; however, they still enjoyed our camp. We also surveyed high school teachers volunteering for HSTA to identify factors that would encourage use of zebrafish in classrooms. The most prominent needs include classroom supplies, experience, and funding. Our successful science-education partnership demonstrates that zebrafish research experiences foster positive outcomes for under-represented students, and can inform future outreach efforts and collaborations with teachers.