Foxi3GFP and Foxi3CreER mice allow identification and lineage labeling of pharyngeal arch ectoderm and endoderm, and tooth and hair placodes.

BACKGROUND: FOXI3 is a forkhead family transcription factor that is expressed in the progenitors of craniofacial placodes, epidermal placodes, and the ectoderm and endoderm of the pharyngeal arch region. Loss of Foxi3 in mice and pathogenic Foxi3 variants in dogs and humans cause a variety of craniofacial defects including absence of the inner ear, severe truncations of the jaw, loss or reduction in external and middle ear structures, and defects in teeth and hair. RESULTS: To allow for the identification, isolation, and lineage tracing of Foxi3-expressing cells in developing mice, we targeted the Foxi3 locus to create Foxi3GFP and Foxi3CreER mice. We show that Foxi3GFP mice faithfully recapitulate the expression pattern of Foxi3 mRNA at all ages examined, and Foxi3CreER mice can trace the derivatives of pharyngeal arch ectoderm and endoderm, the pharyngeal pouches and clefts that separate each arch, and the derivatives of hair and tooth placodes. CONCLUSIONS: Foxi3GFP and Foxi3CreER mice are new tools that will be of use in identifying and manipulating pharyngeal arch ectoderm and endoderm and hair and tooth placodes.

The mouse Foxi3 transcription factor is necessary for the development of posterior placodes.

The inner ear develops from the otic placode, one of the cranial placodes that arise from a region of ectoderm adjacent to the anterior neural plate called the pre-placodal domain. We have identified a Forkhead family transcription factor, Foxi3, that is expressed in the pre-placodal domain and down-regulated when the otic placode is induced. We now show that Foxi3 mutant mice do not form otic placodes as evidenced by expression changes in early molecular markers and the lack of thickened placodal ectoderm, an otic cup or otocyst. Some preplacodal genes downstream of Foxi3-Gata3, Six1 and Eya1-are not expressed in the ectoderm of Foxi3 mutant mice, and the ectoderm exhibits signs of increased apoptosis. We also show that Fgf signals from the hindbrain and cranial mesoderm, which are necessary for otic placode induction, are received by pre-placodal ectoderm in Foxi3 mutants, but do not initiate otic induction. Finally, we show that the epibranchial placodes that develop in close proximity to the otic placode and the mandibular division of the trigeminal ganglion are missing in Foxi3 mutants. Our data suggest that Foxi3 is necessary to prime pre-placodal ectoderm for the correct interpretation of inductive signals for the otic and epibranchial placodes.

Transcriptional dynamics of delaminating neuroblasts in the mouse otic vesicle.

An abundance of research has recently highlighted the susceptibility of cochleovestibular ganglion (CVG) neurons to noise damage and aging in the adult cochlea, resulting in hearing deficits. Furthering our understanding of the transcriptional cascades that contribute to CVG development may provide insight into how these cells can be regenerated to treat inner ear dysfunction. Here we perform a high-depth single-cell RNA sequencing analysis of the E10.5 otic vesicle and its surrounding tissues, including CVG precursor neuroblasts and emerging CVG neurons. Clustering and trajectory analysis of otic-lineage cells reveals otic markers and the changes in gene expression that occur from neuroblast delamination toward the development of the CVG. This dataset provides a valuable resource for further identifying the mechanisms associated with CVG development from neurosensory competent cells within the otic vesicle.

Cellular reprogramming with ATOH1, GFI1, and POU4F3 implicate epigenetic changes and cell-cell signaling as obstacles to hair cell regeneration in mature mammals.

Reprogramming of the cochlea with hair-cell-specific transcription factors such as ATOH1 has been proposed as a potential therapeutic strategy for hearing loss. ATOH1 expression in the developing cochlea can efficiently induce hair cell regeneration but the efficiency of hair cell reprogramming declines rapidly as the cochlea matures. We developed Cre-inducible mice to compare hair cell reprogramming with ATOH1 alone or in combination with two other hair cell transcription factors, GFI1 and POU4F3. In newborn mice, all transcription factor combinations tested produced large numbers of cells with the morphology of hair cells and rudimentary mechanotransduction properties. However, 1 week later, only a combination of ATOH1, GFI1 and POU4F3 could reprogram non-sensory cells of the cochlea to a hair cell fate, and these new cells were less mature than cells generated by reprogramming 1 week earlier. We used scRNA-seq and combined scRNA-seq and ATAC-seq to suggest at least two impediments to hair cell reprogramming in older animals. First, hair cell gene loci become less epigenetically accessible in non-sensory cells of the cochlea with increasing age. Second, signaling from hair cells to supporting cells, including Notch signaling, can prevent reprogramming of many supporting cells to hair cells, even with three hair cell transcription factors. Our results shed light on the molecular barriers that must be overcome to promote hair cell regeneration in the adult cochlea.

The Pipeline CURE: An Iterative Approach to Introduce All Students to Research Throughout a Biology Curriculum.

Participation in research provides personal and professional benefits for undergraduates. However, some students face institutional barriers that prevent their entry into research, particularly those from underrepresented groups who may stand to gain the most from research experiences. Course-based undergraduate research experiences (CUREs) effectively scale research availability, but many only last for a single semester, which is rarely enough time for a novice to develop proficiency. To address these challenges, we present the Pipeline CURE, a framework that integrates a single research question throughout a biology curriculum. Students are introduced to the research system - in this implementation, C. elegans epigenetics research - with their first course in the major. After revisiting the research system in several subsequent courses, students can choose to participate in an upper-level research experience. In the Pipeline, students build resilience via repeated exposure to the same research system. Its iterative, curriculum-embedded approach is flexible enough to be implemented at a range of institutions using a variety of research questions. By uniting evidence-based teaching methods with ongoing scientific research, the Pipeline CURE provides a new model for overcoming barriers to participation in undergraduate research.

A Resource for the Allele-Specific Analysis of DNA Methylation at Multiple Genomically Imprinted Loci in Mice.

Genomically imprinted loci are expressed mono-allelically, dependent upon the parent of origin. Their regulation not only illuminates how chromatin regulates gene expression but also how chromatin can be reprogrammed every generation. Because of their distinct parent-of-origin regulation, analysis of imprinted loci can be difficult. Single nucleotide polymorphisms (SNPs) are required to accurately assess these elements allele specifically. However, publicly available SNP databases lack robust verification, making analysis of imprinting difficult. In addition, the allele-specific imprinting assays that have been developed employ different mouse strains, making it difficult to systemically analyze these loci. Here, we have generated a resource that will allow the allele-specific analysis of many significant imprinted loci in a single hybrid strain of Mus musculus This resource includes verification of SNPs present within 10 of the most widely used imprinting control regions and allele-specific DNA methylation assays for each gene in a C57BL/6J and CAST/EiJ hybrid strain background.

The role of foxi family transcription factors in the development of the ear and jaw.

The mammalian outer, middle, and inner ears have different embryonic origins and evolved at different times in the vertebrate lineage. The outer ear is derived from first and second branchial arch ectoderm and mesoderm, the middle ear ossicles are derived from neural crest mesenchymal cells that invade the first and second branchial arches, whereas the inner ear and its associated vestibule-acoustic (VIIIth) ganglion are derived from the otic placode. In this chapter, we discuss recent findings in the development of these structures and describe the contributions of members of a Forkhead transcription factor family, the Foxi family to their formation. Foxi transcription factors are critical for formation of the otic placode, survival of the branchial arch neural crest, and developmental remodeling of the branchial arch ectoderm.