Quaking isoforms cooperate to promote the mesenchymal phenotype.

The RNA-binding protein Quaking (QKI) has widespread effects on mRNA regulation including alternative splicing, stability, translation, and localization of target mRNAs. Recently, QKI was found to be induced during epithelial-mesenchymal transition (EMT), where it promotes a mesenchymal alternative splicing signature that contributes to the mesenchymal phenotype. QKI is itself alternatively spliced to produce three major isoforms, QKI-5, QKI-6, and QKI-7. While QKI-5 is primarily localized to the nucleus where it controls mesenchymal splicing during EMT, the functions of the two predominantly cytoplasmic isoforms, QKI-6 and QKI-7, in this context remain uncharacterized. Here we used CRISPR-mediated depletion of QKI in a human mammary epithelial cell model of EMT and studied the effects of expressing the QKI isoforms in isolation and in combination. QKI-5 was required to induce mesenchymal morphology, while combined expression of QKI-5 with either QKI-6 or QKI-7 further enhanced mesenchymal morphology and cell migration. In addition, we found that QKI-6 and QKI-7 can partially localize to the nucleus and contribute to alternative splicing of QKI target genes. These findings indicate that the QKI isoforms function in a dynamic and cooperative manner to promote the mesenchymal phenotype.

The landscape of alternative polyadenylation during EMT and its regulation by the RNA-binding protein Quaking.

Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3' end anchored RNA sequencing, we mapped the alternative polyadenylation (APA) landscape following Transforming Growth Factor (TGF)-ß-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3'UTR lengthening during this cell state transition. Investigation of potential mediators of APA indicated the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, regulates a subset of events including the length of its own transcript. Analysis of QKI crosslinked immunoprecipitation (CLIP)-sequencing data identified the binding of QKI within 3' untranslated regions (UTRs) was enriched near cleavage and polyadenylation sites. Following QKI knockdown, APA of many transcripts is altered to produce predominantly shorter 3'UTRs associated with reduced gene expression. These findings reveal the changes in APA that occur during EMT and identify a potential role for QKI in this process.

Basonuclin-2 regulates extracellular matrix production and degradation.

Epithelial-mesenchymal transition is essential for tissue patterning and organization. It involves both regulation of cell motility and alterations in the composition and organization of the ECM-a complex environment of proteoglycans and fibrous proteins essential for tissue homeostasis, signaling in response to chemical and biomechanical stimuli, and is often dysregulated under conditions such as cancer, fibrosis, and chronic wounds. Here, we demonstrate that basonuclin-2 (BNC2), a mesenchymal-expressed gene, that is, strongly associated with cancer and developmental defects across genome-wide association studies, is a novel regulator of ECM composition and degradation. We find that at endogenous levels, BNC2 controls the expression of specific collagens, matrix metalloproteases, and other matrisomal components in breast cancer cells, and in fibroblasts that are primarily responsible for the production and processing of the ECM within the tumour microenvironment. In so doing, BNC2 modulates the motile and invasive properties of cancers, which likely explains the association of high BNC2 expression with increasing cancer grade and poor patient prognosis.

Neuropilins guide preganglionic sympathetic axons and chromaffin cell precursors to establish the adrenal medulla.

The adrenal medulla is composed of neuroendocrine chromaffin cells that secrete adrenaline into the systemic circulation to maintain physiological homeostasis and enable the autonomic stress response. How chromaffin cell precursors colonise the adrenal medulla and how they become connected to central nervous system-derived preganglionic sympathetic neurons remain largely unknown. By combining lineage tracing, gene expression studies, genetic ablation and the analysis of mouse mutants, we demonstrate that preganglionic axons direct chromaffin cell precursors into the adrenal primordia. We further show that preganglionic axons and chromaffin cell precursors require class 3 semaphorin (SEMA3) signalling through neuropilins (NRP) to target the adrenal medulla. Thus, SEMA3 proteins serve as guidance cues to control formation of the adrenal neuroendocrine system by establishing appropriate connections between preganglionic neurons and adrenal chromaffin cells that regulate the autonomic stress response.

miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing.

Transcriptome profiling reveals expression signatures of cranial neural crest cells arising from different axial levels.

BACKGROUND: Cranial neural crest cells (NCCs) are a unique embryonic cell type which give rise to a diverse array of derivatives extending from neurons and glia through to bone and cartilage. Depending on their point of origin along the antero-posterior axis cranial NCCs are rapidly sorted into distinct migratory streams that give rise to axial specific structures. These migratory streams mirror the underlying segmentation of the brain with NCCs exiting the diencephalon and midbrain following distinct paths compared to those exiting the hindbrain rhombomeres (r). The genetic landscape of cranial NCCs arising at different axial levels remains unknown. RESULTS: Here we have used RNA sequencing to uncover the transcriptional profiles of mouse cranial NCCs arising at different axial levels. Whole transcriptome analysis identified over 120 transcripts differentially expressed between NCCs arising anterior to r3 (referred to as r1-r2 migratory stream for simplicity) and the r4 migratory stream. Eight of the genes differentially expressed between these populations were validated by RT-PCR with 2 being further validated by in situ hybridisation. We also explored the expression of the Neuropilins (Nrp1 and Nrp2) and their co-receptors and show that the A-type Plexins are differentially expressed in different cranial NCC streams. CONCLUSIONS: Our analyses identify a large number of genes differentially regulated between cranial NCCs arising at different axial levels. This data provides a comprehensive description of the genetic landscape driving diversity of distinct cranial NCC streams and provides novel insight into the regulatory networks controlling the formation of specific skeletal elements and the mechanisms promoting migration along different paths.

Sympathoadrenal neural crest cells: the known, unknown and forgotten?

Neural crest cells (NCCs) are highly migratory progenitor cells that give rise to a vast array of differentiated cell types. One of their key derivatives is the autonomic nervous system (ANS) that is comprised in part from chromaffin cells of the adrenal medulla and organ of Zuckerkandl, the sympathetic chain and additional prevertebral ganglia such as the celiac ganglia, suprarenal ganglia and mesenteric ganglia. In this review we discuss recent advances toward our understanding of how the NCC precursors of the ANS migrate to their target regions, how they are instructed to differentiate into the correct cell types, and the morphogenetic signals controlling their development. Many of these processes remain enigmatic to developmental biologists worldwide. Taking advantage of lineage tracing mouse models one of our own aims is to address the morphogenetic events underpinning the formation of the ANS and to identify the molecular mechanisms that help to segregate a mixed population of NCCs into pathways specific for the sympathetic ganglia, sensory ganglia or adrenal medulla.

Neuropilins define distinct populations of neural crest cells.

BACKGROUND: Neural crest cells (NCCs) are a transient embryonic cell type that give rise to a wide spectrum of derivatives, including neurons and glia of the sensory and autonomic nervous system, melanocytes and connective tissues in the head. Lineage-tracing and functional studies have shown that trunk NCCs migrate along two distinct paths that correlate with different developmental fates. Thus, NCCs migrating ventrally through the anterior somite form sympathetic and sensory ganglia, whereas NCCs migrating dorsolaterally form melanocytes. Although the mechanisms promoting migration along the dorsolateral path are well defined, the molecules providing positional identity to sympathetic and sensory-fated NCCs that migrate along the same ventral path are ill defined. Neuropilins (Nrp1 and Nrp2) are transmembrane glycoproteins that are essential for NCC migration. Nrp1 and Nrp2 knockout mice have disparate phenotypes, suggesting that these receptors may play a role in sorting NCCs biased towards sensory and sympathetic fates to appropriate locations. RESULTS: Here we have combined in situ hybridisation, immunohistochemistry and lineage-tracing analyses to demonstrate that neuropilins are expressed in a non-overlapping pattern within NCCs. Whereas Nrp1 is expressed in NCCs emigrating from hindbrain rhombomere 4 (r4) and within trunk NCCs giving rise to sympathetic and sensory ganglia, Nrp2 is preferentially expressed in NCCs emigrating from r2 and in trunk NCCs giving rise to sensory ganglia. By generating a tamoxifen-inducible lineage-tracing system, we further demonstrate that Nrp2-expressing NCCs specifically populate sensory ganglia including the trigeminal ganglia (V) in the head and the dorsal root ganglia in the trunk. CONCLUSIONS: Taken together, our results demonstrate that Nrp1 and Nrp2 are expressed in different populations of NCCs, and that Nrp2-expressing NCCs are strongly biased towards a sensory fate. In the trunk, Nrp2-expressing NCCs specifically give rise to sensory ganglia, whereas Nrp1-expressing NCCs likely give rise to both sensory and sympathetic ganglia. Our findings therefore suggest that neuropilins play an essential role in coordinating NCC migration with fate specification.

The ubiquitin ligase Nedd4 regulates craniofacial development by promoting cranial neural crest cell survival and stem-cell like properties.

The integration of multiple morphogenic signalling pathways and transcription factor networks is essential to mediate neural crest (NC) cell induction, delamination, survival, stem-cell properties, fate choice and differentiation. Although the transcriptional control of NC development is well documented in mammals, the role of post-transcriptional modifications, and in particular ubiquitination, has not been explored. Here we report an essential role for the ubiquitin ligase Nedd4 in cranial NC cell development. Our analysis of Nedd4(-/-) embryos identified profound deficiency of cranial NC cells in the absence of structural defects in the neural tube. Nedd4 is expressed in migrating cranial NC cells and was found to positively regulate expression of the NC transcription factors Sox9, Sox10 and FoxD3. We found that in the absence of these factors, a subset of cranial NC cells undergo apoptosis. In accordance with a lack of cranial NC cells, Nedd4(-/-) embryos have deficiency of the trigeminal ganglia, NC derived bone and malformation of the craniofacial skeleton. Our analyses therefore uncover an essential role for Nedd4 in a subset of cranial NC cells and highlight E3 ubiquitin ligases as a likely point of convergence for multiple NC signalling pathways and transcription factor networks.

Li-gazing at the crest: modulation of the neural crest by the ubiquitin pathway.

Neural crest cells are a transient population of stem cells that give rise to a diverse range of cell types during embryonic development. Through gain-of-function and loss-of-function studies in several model organisms many key signalling pathways and cell-type specific transcription factors essential for neural crest cell development have been identified. However, the role of post-translational regulation remains largely unexplored. Here we review this cell type with a foray into the known and potential roles of the ubiquitination pathway in key signalling events during neural crest cell development.