Grainyhead-like 2 interacts with noggin to regulate tissue fusion in mouse.

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.

The Role of PLAG1 in Mouse Brain Development and Neurogenesis.

The pleomorphic adenoma gene 1 (Plag1) is a transcription factor involved in the regulation of growth and cellular proliferation. Here, we report the spatial distribution and functional implications of PLAG1 expression in the adult mouse brain. We identified Plag1 promoter-dependent ß-galactosidase expression in various brain structures, including the hippocampus, cortex, choroid plexus, subcommisural organ, ependymal cells lining the third ventricle, medial and lateral habenulae and amygdala. We noted striking spatial-restriction of PLAG1 within the cornu ammonis (CA1) region of the hippocampus and layer-specific cortical expression, with abundant expression noted in all layers except layer 5. Furthermore, our study delved into the role of PLAG1 in neurodevelopment, focusing on its impact on neural stem/progenitor cell proliferation. Loss of Plag1 resulted in reduced proliferation and decreased production of neocortical progenitors in vivo, although ex vivo neurosphere experiments revealed no cell-intrinsic defects in the proliferative or neurogenic capacity of Plag1-deficient neural progenitors. Lastly, we explored potential target genes of PLAG1 in the cortex, identifying that Neurogenin 2 (Ngn2) was significantly downregulated in Plag1-deficient mice. In summary, our study provides novel insights into the spatial distribution of PLAG1 expression in the adult mouse brain and its potential role in neurodevelopment. These findings expand our understanding of the functional significance of PLAG1 within the brain, with potential implications for neurodevelopmental disorders and therapeutic interventions.

In Situ Hybridization to Characterize Neurulation and Midbrain-Hindbrain Boundary Formation in Zebrafish.

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.

Neural Stem/Progenitor Cell (NSPC) Extraction and Culture.

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.

Cryosectioning and Immunohistochemistry Using Frozen Adult Murine Brain Neural Tissue.

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.

Organophosphate Insecticide Toxicity in Neural Development, Cognition, Behaviour and Degeneration: Insights from Zebrafish.

Organophosphate (OP) insecticides are used to eliminate agricultural threats posed by insects, through inhibition of the neurotransmitter acetylcholinesterase (AChE). These potent neurotoxins are extremely efficacious in insect elimination, and as such, are the preferred agricultural insecticides worldwide. Despite their efficacy, however, estimates indicate that only 0.1% of organophosphates reach their desired target. Moreover, multiple studies have shown that OP exposure in both humans and animals can lead to aberrations in embryonic development, defects in childhood neurocognition, and substantial contribution to neurodegenerative diseases such as Alzheimer's and Motor Neurone Disease. Here, we review the current state of knowledge pertaining to organophosphate exposure on both embryonic development and/or subsequent neurological consequences on behaviour, paying particular attention to data gleaned using an excellent animal model, the zebrafish (Danio rerio).

Crucial Convolution: Genetic and Molecular Mechanisms of Coiling during Epididymis Formation and Development in Embryogenesis.

As embryonic development proceeds, numerous organs need to coil, bend or fold in order to establish their final shape. Generally, this occurs so as to maximise the surface area for absorption or secretory functions (e.g., in the small and large intestines, kidney or epididymis); however, mechanisms of bending and shaping also occur in other structures, notably the midbrain-hindbrain boundary in some teleost fish models such as zebrafish. In this review, we will examine known genetic and molecular factors that operate to pattern complex, coiled structures, with a primary focus on the epididymis as an excellent model organ to examine coiling. We will also discuss genetic mechanisms involving coiling in the seminiferous tubules and intestine to establish the final form and function of these coiled structures in the mature organism.

Transcription Factors in Cancer.

This Special Issue comprises three original studies and five review articles [...].

Grainyhead-like (Grhl) Target Genes in Development and Cancer.

Grainyhead-like (GRHL) factors are essential, highly conserved transcription factors (TFs) that regulate processes common to both natural cellular behaviours during embryogenesis, and de-regulation of growth and survival pathways in cancer. Serving to drive the transcription, and therefore activation of multiple co-ordinating pathways, the three GRHL family members (GRHL1-3) are a critical conduit for modulating the molecular landscape that guides cellular decision-making processes during proliferation, epithelial-mesenchymal transition (EMT) and migration. Animal models and in vitro approaches harbouring GRHL loss or gain-of-function are key research tools to understanding gene function, which gives confidence that resultant phenotypes and cellular behaviours may be translatable to humans. Critically, identifying and characterising the target genes to which these factors bind is also essential, as they allow us to discover and understand novel genetic pathways that could ultimately be used as targets for disease diagnosis, drug discovery and therapeutic strategies. GRHL1-3 and their transcriptional targets have been shown to drive comparable cellular processes in Drosophila, C. elegans, zebrafish and mice, and have recently also been implicated in the aetiology and/or progression of a number of human congenital disorders and cancers of epithelial origin. In this review, we will summarise the state of knowledge pertaining to the role of the GRHL family target genes in both development and cancer, primarily through understanding the genetic pathways transcriptionally regulated by these factors across disparate disease contexts.

Wholemount In-Situ Hybridization (WISH) in Zebrafish Embryos to Analyze Craniofacial Development.

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.

Methodology for Free-Floating Organ Culture of Mid-gestation Maxillary Primordial Tissue.

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.

Grhl3 promotes retention of epidermal cells under endocytic stress to maintain epidermal architecture in zebrafish.

Epithelia such as epidermis cover large surfaces and are crucial for survival. Maintenance of tissue homeostasis by balancing cell proliferation, cell size, and cell extrusion ensures epidermal integrity. Although the mechanisms of cell extrusion are better understood, how epithelial cells that round up under developmental or perturbed genetic conditions are reintegrated in the epithelium to maintain homeostasis remains unclear. Here, we performed live imaging in zebrafish embryos to show that epidermal cells that round up due to membrane homeostasis defects in the absence of goosepimples/myosinVb (myoVb) function, are reintegrated into the epithelium. Transcriptome analysis and genetic interaction studies suggest that the transcription factor Grainyhead-like 3 (Grhl3) induces the retention of rounded cells by regulating E-cadherin levels. Moreover, Grhl3 facilitates the survival of MyoVb deficient embryos by regulating cell adhesion, cell retention, and epidermal architecture. Our analyses have unraveled a mechanism of retention of rounded cells and its importance in epithelial homeostasis.

Delineating the roles of Grhl2 in craniofacial development through tissue-specific conditional deletion and epistasis approaches in mouse.

BACKGROUND: The highly conserved Grainyhead-like (Grhl) family of transcription factors play critical roles in the development of the neural tube and craniofacial skeleton. In particular, deletion of family member Grainyhead-like 2 (Grhl2) leads to mid-gestational embryonic lethality, maxillary clefting, abdominoschisis, and both cranial and caudal neural tube closure defects. These highly pleiotropic and systemic defects suggest that Grhl2 plays numerous critical developmental roles to ensure correct morphogenesis and patterning. RESULTS: Here, using four separate Cre-lox conditional deletion models, as well as one genetic epistasis approach (Grhl2+/- ;Edn1+/- double heterozygous mice) we have investigated tissue-specific roles of Grhl2 in embryonic development, with a particular focus on the craniofacial skeleton. We find that loss of Grhl2 in the pharyngeal epithelium (using the ShhCre driver) leads to low-penetrance micrognathia, whereas deletion of Grhl2 within the ectoderm of the pharynx (NestinCre ) leads to small, albeit significant, differences in the proximal-distal elongation of both the maxilla and mandible. Loss of Grhl2 in endoderm (Sox17-2aiCre ) resulted in noticeable lung defects and a single instance of secondary palatal clefting, although formation of other endoderm-derived organs such as the stomach, bladder and intestines was not affected. Lastly, deletion of Grhl2 in cells of the neural crest (Wnt1Cre ) did not lead to any discernible defects in craniofacial development, and similarly, our epistasis approach did not detect any phenotypic consequences of loss of a single allele of both Grhl2 and Edn1. CONCLUSION: Taken together, our study identifies a pharyngeal-epithelium intrinsic, non-cell-autonomous role for Grhl2 in the patterning and formation of the craniofacial skeleton, as well as an endoderm-specific role for Grhl2 in the formation and establishment of the mammalian lung.

Effect of Pleomorphic Adenoma Gene 1 Deficiency on Selected Behaviours in Adult Mice.

The proto-oncogene pleomorphic adenoma gene 1 (Plag1) encodes a zinc finger transcription factor. PLAG1 is part of the high motility group AT hook-2 (HGMA2)-PLAG1-insulin-like growth factor 2 (IGF2) pathway that, when disrupted, leads to Silver-Russell syndrome, a severe form of intrauterine growth restriction. With little known about PLAG1's role in normal physiology, this study is the first to characterise the behavioural phenotype of PLAG1-deficient mice. Mice were tested for differences in circadian locomotor activity and body temperature, sleep-like behaviour, anxiety-like behaviour, cognition, social behaviour, and sensorimotor gating. Overall, the behavioural phenotype of the Plag1 knock-out (KO) mice was mild: no significant differences were seen in circadian activity levels, locomotion, object recognition, spatial memory or sociability compared to wild-type mice. However, the cued test of fear conditioning, prepulse inhibition of the startle response and Preyer's reflex test suggest that Plag1 KO mice may have a hearing impairment. This implies that PLAG1 plays an important role in proper functioning and/or development of the neural circuitry behind the auditory processes or interacts with genes involved in those processes.

Recent Discoveries on the Involvement of Krüppel-Like Factor 4 in the Most Common Cancer Types.

Krüppel-like factor 4 (KLF4) is a transcription factor highly conserved in evolution. It is particularly well known for its role in inducing pluripotent stem cells. In addition, KLF4 plays many roles in cancer. The results of most studies suggest that KLF4 is a tumor suppressor. However, the functioning of KLF4 is regulated at many levels. These include regulation of transcription, alternative splicing, miRNA, post-translational modifications, subcellular localization, protein stability and interactions with other molecules. Simple experiments aimed at assaying transcript levels or protein levels fail to address this complexity and thus may deliver misleading results. Tumor subtypes are also important; for example, in prostate cancer KLF4 is highly expressed in indolent tumors where it impedes tumor progression, while it is absent from aggressive prostate tumors. KLF4 is important in regulating response to many known drugs, and it also plays a role in tumor microenvironment. More and more information is available about upstream regulators, downstream targets and signaling pathways associated with the involvement of KLF4 in cancer. Furthermore, KLF4 performs critical function in the overall regulation of tissue homeostasis, cellular integrity, and progression towards malignancy. Here we summarize and analyze the latest findings concerning this fascinating transcription factor.

Transcriptome analysis of the epididymis from Plag1 deficient mice suggests dysregulation of sperm maturation and extracellular matrix genes.

BACKGROUND: The transcription factor pleomorphic adenoma gene 1 (PLAG1) is required for male fertility. Mice deficient in PLAG1 exhibit decreased sperm motility and abnormal epididymal tubule elongation and coiling, indicating impaired sperm maturation during epididymal transit. However, the downstream transcriptomic profile of the Plag1 knockout (KO; Plag1-/- ) murine epididymis is currently unknown. RESULTS: In this study, the PLAG1-dependent epididymal transcriptome was characterised using RNA sequencing. Several genes important for the control of sperm maturation, motility, capacitation and the acrosome reaction were dysregulated in Plag1-/- mice. Surprisingly, several cell proliferation genes were upregulated, and Ki67 analysis indicated that cell proliferation is aberrantly upregulated in the cauda epididymis stroma of Plag1-/- mice. Gene ontology analysis showed an overall upregulation of genes encoding extracellular matrix components, and an overall downregulation of genes encoding metalloendopeptidases in the epididymides from Plag1-/- mice. CONCLUSION: Together, these results suggest a defect in the epididymal extracellular matrix in Plag1-/- mice. These results imply that in addition to maintaining epididymal integrity directly, PLAG1 may also regulate several genes involved in the regulation of sperm maturation and capacitation. Moreover, PLAG1 may also be involved in regulating tissue homeostasis and ensuring proper structure and maintenance of the extracellular matrix in the epididymis.

A rat model of valproate teratogenicity from chronic oral treatment during pregnancy.

OBJECTIVE: Sodium valproate (VPA), the most effective antiepileptic drug for patients with genetic generalized epilepsy (GGE), is a potent human teratogen that increases the risk of a range of congenital malformations, including spina bifida. The mechanisms underlying this teratogenicity are not known, but may involve genetic risk factors. This study aimed to develop an animal model of VPA-induced birth defects. METHODS: We used three different rat strains: inbred Genetic Absence Epilepsy Rats From Strasbourg (GAERS), a model of GGE with absence seizures; inbred Non-Epileptic Controls (NEC); and outbred nonepileptic Wistars. Female rats were fed standard chow or VPA (20 g/kg food) mixed in standard chow for 2 weeks prior to conception, and then mated with same-strain males. Treatment continued throughout pregnancy. Fetuses were extracted via C-section on gestational day 21 and examined for birth defects, including external assessment and spinal measurements. RESULTS: VPA-exposed pups showed significant reductions in weight, length, and whole-body development compared with controls of all three strains (P < .0001). Gestational VPA treatment altered intravertebral distances, and resulted in underdeveloped vertebral arches between thoracic region T11 and caudal region C2 in most pups (GAERS, 100%; NEC, 95%; Wistar, 80%), more frequently than in controls (9%, 13%, 19%). SIGNIFICANCE: Gestational VPA treatment results in similar developmental and morphological abnormalities in three rat strains, including one with GGE, indicating that the genetic underpinnings of epilepsy do not contribute markedly to VPA-induced birth defects. This model may be used in future studies to investigate mechanisms involved in the pathogenesis of antiepileptic drug-induced birth defects.

Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure.

Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (Grhl1-3) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2-null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2-null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1-/- mice is evident, with rescue of both palatal and facial fusion seen in Grhl2-/-;Zeb1-/- embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.