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Vitamin D plays an important pleiotropic role in maintaining global homeostasis of the human body. Its functions go far beyond skeletal health, playing a crucial role in a plethora of cellular functions, as well as in extraskeletal health, ensuring the proper functioning of multiple human organs, including the skin. Genes from the Grainyhead-like (GRHL) family code for transcription factors necessary for the development and maintenance of various epithelia. Even though they are involved in many processes regulated by vitamin D, a direct link between vitamin D-mediated cellular pathways and GRHL genes has never been described. We employed various bioinformatic methods, quantitative real-time PCR, chromatin immunoprecipitation, reporter gene assays, and calcitriol treatments to investigate this issue. We report that the vitamin D receptor (VDR) binds to a regulatory region of the Grainyhead-like 1 (GRHL1) gene and regulates its expression. Ectopic expression of VDR and treatment with calcitriol alters the expression of the GRHL1 gene. The evidence presented here indicates a role of VDR in the regulation of expression of GRHL1 and correspondingly a role of GRHL1 in mediating the actions of vitamin D. [ABSTRACT FROM AUTHOR]

Defective tissue fusion during mammalian embryogenesis results in congenital anomalies, such as exencephaly, spina bifida and cleft lip and/or palate. The highly conserved transcription factor grainyhead-like 2 (Grhl2) is a crucial regulator of tissue fusion, with mouse models lacking GRHL2 function presenting with a fully penetrant open cranial neural tube, facial and abdominal clefting (abdominoschisis), and an open posterior neuropore. Here, we show that GRHL2 interacts with the soluble morphogen protein and bone morphogenetic protein (BMP) inhibitor noggin (NOG) to impact tissue fusion during development. The maxillary prominence epithelium in embryos lacking Grhl2 shows substantial morphological abnormalities and significant upregulation of NOG expression, together with aberrantly distributed pSMAD5-positive cells within the neural crest cell-derived maxillary prominence mesenchyme, indicative of disrupted BMP signalling. Reducing this elevated NOG expression (by generating Grhl2−/−;Nog+/− embryos) results in delayed embryonic lethality, partial tissue fusion rescue, and restoration of tissue form within the craniofacial epithelia. These data suggest that aberrant epithelial maintenance, partially regulated by noggin-mediated regulation of BMP-SMAD pathways, may underpin tissue fusion defects in Grhl2−/− mice. [ABSTRACT FROM AUTHOR]

Neural stem-progenitor cells (NSPCs) are multipotent, self-renewing cells that generate radial glial cells (RGC). RGCs then give rise to neurons and glia during neural development. Here, we describe the process of NSPC isolation and culturing to form clonal aggregates termed neurospheres. There are multiple assays outlined in this chapter that allow us to quantify differences in proliferation, self-renewal potential, and differentiation of these cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Whole-mount in situ hybridization is cable to harness the inherent advantages of zebrafish as a model organism for developmental biology, particularly when visualizing the formation of the neural tube, specifically at the level of the midbrain-hindbrain boundary. The size and transparency of developing zebrafish embryos allow for the visualization of neural markers in vivo along the length of the developing zebrafish central nervous system. In practice, this technique is useful for examining defects in neurulation and midbrain-hindbrain boundary formation that may arise following gene manipulation, for example, CRISPR mutagenesis. This method describes the process of embryo collection and preparation, RNA probe transcription, probe hybridization in vivo, as well as the process of probe detection and visualization., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cryopreservation and immunohistochemistry offer a comprehensive, robust, and simple methodology to investigate neural patterning and cellular function. Rapid freezing of the whole brain allows excellent preservation of neural ultrastructure and tissue architecture without destroying sensitive protein epitopes that are often compromised following standard paraffin embedding histological techniques. Here, we present a rapid and simple protocol for employing cryosectioning and subsequent immunohistochemistry in the study of adult murine brain neural tissue., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Craniofacial defects, such as cleft palate, are prevalent congenital malformations that present an interesting research challenge due to the complex and multifactorial nature of their etiology. In vitro modeling of craniofacial morphogenesis provides valuable insight into the developmental processes critical to the presentation of these conditions. One such technique, termed a submerged or free-floating organ culture, allows culturing and observation of isolated craniofacial tissue without the need for specialized supporting equipment. Outlined here is a detailed protocol for isolating and culturing maxillary and palatal tissue as a midfacial tissue section. This protocol has been modified from a previously established technique to accommodate culturing tissue from developmental time-points as early as embryonic day 10.5. This allows for greater control over genotypic variance within litters and provides a simplified, accessible methodology., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Wholemount in-situ hybridization in zebrafish is a powerful technique for visualizing spatiotemporal gene expression during development. Here we describe a technique to detect endogenous mRNA expression in zebrafish that can be adapted to use on embryos from the single-cell stage until 5 days postfertilization., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)