Hydrocephalus in Nfix-/- Mice Is Underpinned by Changes in Ependymal Cell Physiology.

Nuclear factor one X (NFIX) is a transcription factor required for normal ependymal development. Constitutive loss of Nfix in mice (Nfix-/-) is associated with hydrocephalus and sloughing of the dorsal ependyma within the lateral ventricles. Previous studies have implicated NFIX in the transcriptional regulation of genes encoding for factors essential to ependymal development. However, the cellular and molecular mechanisms underpinning hydrocephalus in Nfix-/- mice are unknown. To investigate the role of NFIX in hydrocephalus, we examined ependymal cells in brains from postnatal Nfix-/- and control (Nfix+/+) mice using a combination of confocal and electron microscopy. This revealed that the ependymal cells in Nfix-/- mice exhibited abnormal cilia structure and disrupted localisation of adhesion proteins. Furthermore, we modelled ependymal cell adhesion using epithelial cell culture and revealed changes in extracellular matrix and adherens junction gene expression following knockdown of NFIX. Finally, the ablation of Nfix from ependymal cells in the adult brain using a conditional approach culminated in enlarged ventricles, sloughing of ependymal cells from the lateral ventricles and abnormal localisation of adhesion proteins, which are phenotypes observed during development. Collectively, these data demonstrate a pivotal role for NFIX in the regulation of cell adhesion within ependymal cells of the lateral ventricles.

Deletion of NFIX results in defective progression through meiosis within the mouse testis†.

Members of the nuclear factor I (NFI) family are key regulators of stem cell biology during development, with well-documented roles for NFIA, NFIB, and NFIX in a variety of developing tissues, including brain, muscle, and lung. Given the central role these factors play in stem cell biology, we posited that they may be pivotal for spermatogonial stem cells or further developing spermatogonia during testicular development. Surprisingly, in stark contrast to other developing organ systems where NFI members are co-expressed, these NFI family members show discrete patterns of expression within the seminiferous tubules. Sertoli cells (spermatogenic supporting cells) express NFIA, spermatocytes express NFIX, round spermatids express NFIB, and peritubular myoid cells express each of these three family members. Further analysis of NFIX expression during the cycle of the seminiferous epithelium revealed expression not in spermatogonia, as we anticipated, but in spermatocytes. These data suggested a potential role for NFIX in spermatogenesis. To investigate, we analyzed mice with constitutive deletion of Nfix (Nfix-null). Assessment of germ cells in the postnatal day 20 (P20) testes of Nfix-null mice revealed that spermatocytes initiate meiosis, but zygotene stage spermatocytes display structural defects in the synaptonemal complex, and increased instances of unrepaired DNA double-strand breaks. Many developing spermatocytes in the Nfix-null testis exhibited multinucleation. As a result of these defects, spermatogenesis is blocked at early diplotene and very few round spermatids are produced. Collectively, these novel data establish the global requirement for NFIX in correct meiotic progression during the first wave of spermatogenesis.

Genome-wide transcriptomic analysis of the forebrain of postnatal Slc13a4+/- mice.

OBJECTIVE: Sulfation is an essential physiological process that regulates the function of a wide array of molecules involved in brain development. We have previously shown expression levels for the sulfate transporter Slc13a4 to be elevated during postnatal development, and that sulfate accumulation in the brains of Slc13a4+/- mice is reduced, suggesting a role for this transporter during this critical window of brain development. In order to understand the pathways regulated by cellular sulfation within the brain, we performed a bulk RNA-sequencing analysis of the forebrain of postnatal day 20 (P20) Slc13a4 heterozygous mice and wild-type litter mate controls. DATA DESCRIPTION: We performed an RNA transcriptomic based sequencing screen on the whole forebrain from Slc13a4+/- and Slc13a4+/+mice at P20. Differential expression analysis revealed 90 differentially regulated genes in the forebrain of Slc13a4+/- mice (a p-value of 0.1 was considered as significant). Of these, 55 were upregulated, and 35 were downregulated in the forebrain of heterozygous mice. Moreover, when we stratified further with a ± 1.2 fold-change, we observed 38 upregulated, and 16 downregulated genes in the forebrain of heterozygous mice. This resource provides a useful tool to interrogate which pathways may require elevated sulfate levels to drive normal postnatal development of the brain.

Common Regulatory Targets of NFIA, NFIX and NFIB during Postnatal Cerebellar Development.

Transcriptional regulation plays a central role in controlling neural stem and progenitor cell proliferation and differentiation during neurogenesis. For instance, transcription factors from the nuclear factor I (NFI) family have been shown to co-ordinate neural stem and progenitor cell differentiation within multiple regions of the embryonic nervous system, including the neocortex, hippocampus, spinal cord and cerebellum. Knockout of individual Nfi genes culminates in similar phenotypes, suggestive of common target genes for these transcription factors. However, whether or not the NFI family regulates common suites of genes remains poorly defined. Here, we use granule neuron precursors (GNPs) of the postnatal murine cerebellum as a model system to analyse regulatory targets of three members of the NFI family: NFIA, NFIB and NFIX. By integrating transcriptomic profiling (RNA-seq) of Nfia- and Nfix-deficient GNPs with epigenomic profiling (ChIP-seq against NFIA, NFIB and NFIX, and DNase I hypersensitivity assays), we reveal that these transcription factors share a large set of potential transcriptional targets, suggestive of complementary roles for these NFI family members in promoting neural development.

A simple, web-based repository for the management, access and analysis of micrographic images.

Microscopy is advancing at a rapid pace, enabling high-speed, high-resolution analyses to be conducted in a wide range of cellular contexts. For example, the capacity to quickly capture high-resolution images from multiple optical sections over multiple channels with confocal microscopy has allowed researchers to gain deeper understanding of tissue morphology via techniques such as three-dimensional rendering, as have more recent advances such as lattice light sheet microscopy and superresolution structured illumination microscopy. With this, though, comes the challenge of storing, curating, analysing and sharing data. While there are ways in which this has been attempted previously, few approaches have provided a central repository in which all of these different aspects of microscopy can be seamlessly integrated. Here, we describe a web-based storage and analysis platform called Microndata, that enables relatively straightforward storage, annotation, tracking, analysis and multi-user access to micrographs. This easy to use tool will simplify and harmonise laboratory work flows, and, importantly, will provide a central storage repository that is readily accessed, even after the researcher responsible for capturing the images has left the laboratory. Microndata is open-source software, available at http://www.microndata.net/.

Granule neuron precursor cell proliferation is regulated by NFIX and intersectin 1 during postnatal cerebellar development.

Cerebellar granule neurons are the most numerous neuronal subtype in the central nervous system. Within the developing cerebellum, these neurons are derived from a population of progenitor cells found within the external granule layer of the cerebellar anlage, namely the cerebellar granule neuron precursors (GNPs). The timely proliferation and differentiation of these precursor cells, which, in rodents occurs predominantly in the postnatal period, is tightly controlled to ensure the normal morphogenesis of the cerebellum. Despite this, our understanding of the factors mediating how GNP differentiation is controlled remains limited. Here, we reveal that the transcription factor nuclear factor I X (NFIX) plays an important role in this process. Mice lacking Nfix exhibit reduced numbers of GNPs during early postnatal development, but elevated numbers of these cells at postnatal day 15. Moreover, Nfix-/- GNPs exhibit increased proliferation when cultured in vitro, suggestive of a role for NFIX in promoting GNP differentiation. At a mechanistic level, profiling analyses using both ChIP-seq and RNA-seq identified the actin-associated factor intersectin 1 as a downstream target of NFIX during cerebellar development. In support of this, mice lacking intersectin 1 also displayed delayed GNP differentiation. Collectively, these findings highlight a key role for NFIX and intersectin 1 in the regulation of cerebellar development.

Transcriptional regulation of ependymal cell maturation within the postnatal brain.

BACKGROUND: Radial glial stem cells within the developing nervous system generate a variety of post-mitotic cells, including neurons and glial cells, as well as the specialised multi-ciliated cells that line the walls of the ventricular system, the ependymal cells. Ependymal cells separate the brain parenchyma from the cerebrospinal fluid and mediate osmotic regulation, the flow of cerebrospinal fluid, and the subsequent dispersion of signalling molecules via the co-ordinated beating of their cilia. Deficits to ependymal cell development and function have been implicated in the formation of hydrocephalus, but the transcriptional mechanisms underpinning ependymal development remain poorly characterised. FINDINGS: Here, we demonstrate that the transcription factor nuclear factor IX (NFIX) plays a central role in the development of the ependymal cell layer of the lateral ventricles. Expression of ependymal cell-specific markers is delayed in the absence of Nfix. Moreover, Nfix-deficient mice exhibit aberrant ependymal cell morphology at postnatal day 15, culminating in abnormal thickening and intermittent loss of this cell layer. Finally, we reveal Foxj1, a key factor promoting ependymal cell maturation, as a target for NFIX-mediated transcriptional activation. CONCLUSIONS: Collectively, our data indicate that ependymal cell development is reliant, at least in part, on NFIX expression, further implicating this transcription factor as a mediator of multiple aspects of radial glial biology during corticogenesis.