Hes1 marks peri-condensation mesenchymal cells that generate both chondrocytes and perichondrial cells in early bone development.

Bone development starts with condensations of undifferentiated mesenchymal cells that set a framework for future bones within the primordium. In the endochondral pathway, mesenchymal cells inside the condensation differentiate into chondrocytes and perichondrial cells in a SOX9-dependent mechanism. However, the identity of mesenchymal cells outside the condensation and how they participate in developing bones remain undefined. Here we show that mesenchymal cells surrounding the condensation contribute to both cartilage and perichondrium, robustly generating chondrocytes, osteoblasts, and marrow stromal cells in developing bones. Single-cell RNA-seq analysis of Prrx1-cre-marked limb bud mesenchymal cells at E11.5 reveals that Notch effector Hes1 is expressed in a mutually exclusive manner with Sox9 that is expressed in pre-cartilaginous condensations. Analysis of a Notch signaling reporter CBF1:H2B-Venus reveals that peri-condensation mesenchymal cells are active for Notch signaling. In vivo lineage-tracing analysis using Hes1-creER identifies that Hes1+ early mesenchymal cells surrounding the SOX9+ condensation at E10.5 contribute to both cartilage and perichondrium at E13.5, subsequently becoming growth plate chondrocytes, osteoblasts of trabecular and cortical bones, and marrow stromal cells in postnatal bones. In contrast, Hes1+ cells in the perichondrium at E12.5 or E14.5 do not generate chondrocytes within cartilage, contributing to osteoblasts and marrow stromal cells only through the perichondrial route. Therefore, Hes1+ peri-condensation mesenchymal cells give rise to cells of the skeletal lineage through cartilage-dependent and independent pathways, supporting the theory that early mesenchymal cells outside the condensation also play important roles in early bone development.

Role of Dach1 revealed using a novel inner ear-specific Dach1-knockdown mouse model.

The Dach1 gene is expressed in the inner ear of normal mouse embryos in the area that differentiates into the cochlear stria vascularis (SV). We hypothesised that Dach1 downregulation in the inner ear would lead to SV dysplasia. However, because Dach1 knockout is embryonic lethal in mice, the role of Dach1 in the inner ear is unclear. Here, we established inner ear-specific Dach1-knockdown mice and showed that Dach1 downregulation resulted in hearing loss, reduced endocochlear potential and secondary outer hair cell loss. There were no abnormalities in marginal cells and basal cells in the SV or spiral ligament in inner ear-specific Dach1-knockdown mature mice. However, intermediate cell dysplasia and thinning of the SV were observed. Moreover, dynamic changes in the expression of key genes related to the epithelial-mesenchymal transition were observed in the lateral wall of the cochlear epithelium, which differentiated into the SV in inner ear-specific Dach1-knockdown mice at embryonic stages. In summary, suppression of Dach1 expression in the inner ear caused the epithelial-mesenchymal transition in the lateral wall of cochlear epithelium, resulting in loss of intermediate cells in the SV and SV dysplasia.This article has an associated First Person interview with the first author of the paper.

Genome-Wide Association Analysis Identifies Dcc as an Essential Factor in the Innervation of the Peripheral Vestibular System in Inbred Mice.

This study aimed to investigate the genetic causes of vestibular dysfunction. We used vestibular sensory-evoked potentials (VsEPs) to characterize the vestibular function of 35 inbred mouse strains selected from the Hybrid Mouse Diversity Panel and demonstrated strain-dependent phenotypic variation in vestibular function. Using these phenotypic data, we performed the first genome-wide association study controlling for population structure that has revealed two highly suggestive loci, one of which lies within a haplotype block containing five genes (Stard6, 4930503L19Rik, Poli, Mbd2, Dcc) on Chr. 18 (peak SNP rs29632020), one gene, deleted in colorectal carcinoma (Dcc) has a well-established role in nervous system development. An in-depth analysis of Dcc-deficient mice demonstrated elevation in mean VsEP threshold for Dcc (+/-) mice (-11.86 dB) compared to wild-type (-9.68 dB) littermates. Synaptic ribbon studies revealed Dcc (-/-) (P0) and Dcc (+/-) (6-week-old) mice showed lower density of the presynaptic marker (CtBP2) as compared to wild-type controls. Vestibular ganglion cell counts of Dcc (-/-) (P0) was lower than controls. Whole-mount preparations showed abnormal innervation of the utricle, saccule, and crista ampullaris at E14.5, E16.5, and E18.5. Postnatal studies were limited by the perinatal lethality in Dcc (-/-) mice. Expression analyses using in situ hybridization and immunohistochemistry showed Dcc expression in the mouse vestibular ganglion (E15.5), and utricle and crista ampullaris (6-week-old), respectively. In summary, we report the first GWAS for vestibular functional variation in inbred mice and provide evidence for the role of Dcc in the normal innervation of the peripheral vestibular system.

Hepatocyte Growth Factor-c-MET Signaling Mediates the Development of Nonsensory Structures of the Mammalian Cochlea and Hearing.

UNLABELLED: The stria vascularis is a nonsensory structure that is essential for auditory hair cell function by maintaining potassium concentration of the scala media. During mouse embryonic development, a subpopulation of neural crest cell-derived melanocytes migrates and incorporates into a subregion of the cochlear epithelium, forming the intermediate cell layer of the stria vascularis. The relation of this developmental process to stria vascularis function is currently unknown. In characterizing the molecular differentiation of developing peripheral auditory structures, we discovered that hepatocyte growth factor (Hgf) is expressed in the future stria vascularis of the cochlear epithelium. Its receptor tyrosine kinase, c-Met, is expressed in the cochlear epithelium and melanocyte-derived intermediate cells in the stria vascularis. Genetic dissection of HGF signaling via c-MET reveals that the incorporation of the melanocytes into the future stria vascularis of the cochlear duct requires c-MET signaling. In addition, inactivation of either the ligand or receptor developmentally resulted in a profound hearing loss at young adult stages. These results suggest a novel connection between HGF signaling and deafness via melanocyte deficiencies. SIGNIFICANCE STATEMENT: We found the roles of hepatocyte growth factor (HGF) signaling in stria vascularis development for the first time and that lack of HGF signaling in the inner ear leads to profound hearing loss in the mouse. Our findings reveal a novel mechanism that may underlie human deafness DFNB39 and DFNB97. Our findings reveal an additional example of context-dependent c-MET signaling diversity, required here for proper cellular invasion developmentally that is essential for specific aspects of auditory-related organogenesis.

Foxi3 Deficiency Compromises Hair Follicle Stem Cell Specification and Activation.

The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ (HG). Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here, we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary HG marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary HG activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. Stem Cells 2016;34:1896-1908.

SOX9 controls epithelial branching by activating RET effector genes during kidney development.

Congenital abnormalities of the kidney and urinary tract are some of the most common defects detected in the unborn child. Kidney growth is controlled by the GDNF/RET signalling pathway, but the molecular events required for the activation of RET downstream targets are still poorly understood. Here we show that SOX9, a gene involved in campomelic dysplasia (CD) in humans, together with its close homologue SOX8, plays an essential role in RET signalling. Expression of SOX9 can be found from the earliest stages of renal development within the ureteric tip, the ureter mesenchyme and in a segment-specific manner during nephrogenesis. Using a tissue-specific knockout approach, we show that, in the ureteric tip, SOX8 and SOX9 are required for ureter branching, and double-knockout mutants exhibit severe kidney defects ranging from hypoplastic kidneys to renal agenesis. Further genetic analysis shows that SOX8/9 are required downstream of GDNF signalling for the activation of RET effector genes such as Sprouty1 and Etv5. At later stages of development, SOX9 is required to maintain ureteric tip identity and SOX9 ablation induces ectopic nephron formation. Taken together, our study shows that SOX9 acts at multiple steps during kidney organogenesis and identifies SOX8 and SOX9 as key factors within the RET signalling pathway. Our results also explain the aetiology of kidney hypoplasia found in a proportion of CD patients.

Hey2 regulation by FGF provides a Notch-independent mechanism for maintaining pillar cell fate in the organ of Corti.

The organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Here, we show that Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and that-unlike other Hes/Hey factors-Hey2 expression is Notch independent. Hey2 is activated by FGF and blocks hair cell differentiation, whereas mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. We speculate that co-option of FGF signaling to render Hey2 Notch independent also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear.