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Wellcome Open Research - Research Note - Raw data files.

Wellcome Open Research - Research Note - raw data files.

Wellcome Open Research - Research Note - Raw data files

The superfamily of basic-Helix-Loop-Helix (bHLH) transcription factors influence cell fate in all three embryonic germ layers, and the tissue-specific class II factors have received prominent attention for their potent ability to direct differentiation during development and in cellular reprogramming. The activity of many class II bHLH proteins driving differentiation, and the inhibitory class VI bHLH factor Hes1, is controlled by phosphorylation on multiple sites by Cyclin-dependent kinases (Cdks). As class II proteins are generally thought to be active through hetero-dimerisation with the ubiquitously expressed class I E proteins, regulation of class I transcription factors such as E47 may influence the activity of multiple tissue-specific bHLH proteins. Using differentiation of nerve and muscle in Xenopus frog embryos as a model system, we set out to explore whether with the ubiquitously expressed class I E protein E47 that hetero-dimerises with Class II bHLHs to control their activity, is also regulated by multi-site phosphorylation. We demonstrate that E47 can be readily phosphorylated by Cdks on multiple sites in vitro, while ectopically-expressed E47 exists in multiple phosphorylated forms in Xenopus embryos. Preventing multi-site phosphorylation using a phospho-mutant version of E47 enhances the neurogenic and myogenic activity of three different class II bHLH reprogramming factors, and also when E47 acts in hetero-dimerisation with endogenous proteins. Mechanistically, unlike phospho-regulation of class II bHLH factors, we find that preventing phosphorylation of E47 increases the amount of chromatin-bound E47 protein but without affecting its overall protein stability. Thus, multi-site phosphorylation is a conserved regulatory mechanism across the bHLH superfamily that can be manipulated to enhance cellular differentiation.

Xenopus embryos provide a rapid and accessible in vivo model, expressing a plethora of endogenous kinase and phosphatase enzymes that control protein phosphorylation, often affecting physiological function. Traditionally, detection of protein phosphorylation has been achieved by radioisotope phosphate labelling of proteins, sometimes with in vitro assays using recombinant proteins, or by site-specific phospho-antibodies. However, target phospho-sites and kinases responsible are often unknown, and use of radioactive isotopes is not always desirable. Therefore, as a first step to determining functional significance of potential phosphorylation, it is useful to show that a protein can be phosphorylated in vivo in Xenopus eggs and/or embryos. This protocol describes a non-radioactive method to visualise protein phosphorylation by exposing the protein to the egg/embryo kinase environment and observing a difference in protein migration on SDS-PAGE and western blot with and without treatment with lamda phosphatase enzyme. Subsequent investigation of the ability of site-specific phospho-mutant proteins to recapitulate the effect of phosphatase treatment can be used to explore the identity of the phosphorylated sites. Moreover, detection of multiple bands of the protein of interest even after phosphatase treatment points to the presence of other types of post-translational modifications., Manuscript amended to include: L.H. was funded by a Medical Research Council Studentship. Research in AP’s laboratory is supported by Medical Research Council grants MR/K018329/1, MR/L021129/1, and core support from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. LH is supported by a Peterhouse Fellowship.

Xenopus embryos have long been used to demonstrate phenotypic effects following overexpression of proteins of interest such as transcription factors, while post-translational modification of these proteins can dramatically alter the extent of the observed phenotype by inhibiting or enhancing protein activity. In order to determine the mechanisms controlling transcription factor activity, it is useful to compare relative levels of chromatin-bound protein, as this can reveal altered chromatin association in addition to changes in overall protein accumulation seen in the cytoplasm. Assaying protein binding to the bulk DNA described here compliments alternative assays such as EMSA and ChIP that measure sitespecific DNA binding. This protocol describes a method to prepare and analyse chromatin and cytoplasmic extracts from embryos over-expressing the proteins of interest, and uses a robust fractionation procedure that results in clear separation of cytoplasmic tubulin from histone-H3 enriched chromatin. This assay for relative chromatin-bound protein is most suitable for comparing modified forms of a single protein, for example to investigate the effects of point mutations on chromatin association. Optimisation is required for the specific protein of interest but guide ranges are provided.

The processes of cell proliferation and differentiation are intimately linked during embryogenesis, and the superfamily of (basic) Helix-Loop-Helix (bHLH) transcription factors play critical roles in these events. For example, neuronal differentiation is promoted by class II bHLH proneural proteins such as Ngn2 and Ascl1, while class VI Hes proteins act to restrain differentiation and promote progenitor maintenance. We have previously described multi-site phosphorylation as a key regulator of tissue specific class II bHLH proteins in all three embryonic germ layers, and this enables coordination of differentiation with the cell cycle. Hes1 homologues also show analogous conserved proline directed kinase sites. Here we have used formation of Xenopus primary neurons to investigate the effects of xHes1 multi-site phosphorylation on both endogenous and ectopic proneural protein-induced neurogenesis. We find that xHes1 is phosphorylated in vivo, and preventing phosphorylation on three conserved SP/TP sites in the N terminus of the protein enhances xHes1 protein stability and repressor activity. Mechanistically, compared to wild-type xHes1, phospho-mutant xHes1 exhibits greater repression of Ngn2 transcription as well as producing a greater reduction in Ngn2 protein stability and chromatin binding. We propose that cell cycle dependent phosphorylation of class VI Hes proteins may act alongside similar regulation of class II bHLH proneural proteins to co-ordinate their activity.

Proneural basic-helix-loop-helix (bHLH) proteins, such as Neurogenin2 (Ngn2) and Ascl1, are critical regulators at the onset of neuronal differentiation. Endogenously they have largely complementary expression patterns, and have conserved roles in the specification of distinct neuronal subtypes. In Xenopus embryos, xNgn2 is the master regulator of primary neurogenesis forming sensory, inter- and motor neurons within the neural plate, while xAscl1 is the master regulator of autonomic neurogenesis, forming noradrenergic neurons in the antero-ventral region of the embryo. Here we characterise neuronal subtype identity of neurons induced by xNgn2 in the ectoderm of whole Xenopus embryos in comparison with xAscl1, and in ectodermal “animal cap” explants. We find that the transcriptional cascades mediating primary and autonomic neuron formation are distinct, and while xNgn2 and xAscl1 can upregulate genes associated with a non-endogenous cascade, this expression is spatially restricted within the embryo. xNgn2 is more potent than xAscl1 at inducing primary neurogenesis as assayed by neural-β-tubulin. In ectoderm of the intact embryo, these induced primary neurons have sensory characteristics with no upregulation of motor neuron markers. In contrast, xNgn2 is able to up-regulate both sensory and motor neuron markers in naïve ectoderm of animal cap explants, suggesting a non-permissive environment for motor identity in the patterned ectoderm of the whole embryo., This work is supported by the Wellcome Trust [203151] (AP). LH has been supported by an MRC Doctoral Training Award and Peterhouse Research Fellowship.

The use of the Xenopus model system has provided diverse contributions to cancer research, not least because of the striking parallels between tumour pathogenesis and early embryo development. Cell cycle regulation, signalling pathways, and cell behaviours such as migration are frequently perturbed in cancers; all have been investigated using Xenopus, and these developmental events can additionally act as an assay for drug development studies. In this mini-review, we focus our discussion primarily on whole embryo Xenopus models informing cancer biology; the contributions to date and future potential. Insights into tumour immunity, oncogene function, and visualisation of vascular responses during tumour formation have all been achieved with naturally occurring tumours and induced-tumour-like-structures in Xenopus. Finally, as we are now entering the era of genetically modified Xenopus models, we can harness genome editing techniques to recapitulate human disease through creating embryos with analogous genetic abnormalities. With the speed, versatility and accessibility that epitomise the Xenopus system, this new range of pre-clinical Xenopus models has great potential to advance our mechanistic understanding of oncogenesis and provide an early in vivo model for chemotherapeutic development.

Formation of the central nervous system requires a period of extensive progenitor cell proliferation, accompanied or closely followed by differentiation; the balance between these two processes in various regions of the central nervous system gives rise to differential growth and cellular diversity. The correlation between cell cycle lengthening and differentiation has been reported across several types of cell lineage and from diverse model organisms, both in vivo and in vitro. Furthermore, different cell fates might be determined during different phases of the preceding cell cycle, indicating direct cell cycle influences on both early lineage commitment and terminal cell fate decisions. Significant advances have been made in the last decade and have revealed multi-directional interactions between the molecular machinery regulating the processes of cell proliferation and neuronal differentiation. Here, we first introduce the modes of proliferation in neural progenitor cells and summarise evidence linking cell cycle length and neuronal differentiation. Second, we describe the manner in which components of the cell cycle machinery can have additional and, sometimes, cell-cycle-independent roles in directly regulating neurogenesis. Finally, we discuss the way that differentiation factors, such as proneural bHLH proteins, can promote either progenitor maintenance or differentiation according to the cellular environment. These intricate connections contribute to precise coordination and the ultimate division versus differentiation decision.

The superfamily of basic-Helix-Loop-Helix (bHLH) transcription factors influence cell fate in all three embryonic germ layers, and the tissue-specific class II factors have received prominent attention for their potent ability to direct differentiation during development and in cellular reprogramming. The activity of many class II bHLH proteins driving differentiation, and the inhibitory class VI bHLH factor Hes1, is controlled by phosphorylation on multiple sites by Cyclin-dependent kinases (Cdks). As class II proteins are generally thought to be active through hetero-dimerisation with the ubiquitously expressed class I E proteins, regulation of class I transcription factors such as E47 may influence the activity of multiple tissue-specific bHLH proteins. Using differentiation of nerve and muscle in Xenopus frog embryos as a model system, we set out to explore whether with the ubiquitously expressed class I E protein E47 that hetero-dimerises with Class II bHLHs to control their activity, is also regulated by multi-site phosphorylation. We demonstrate that E47 can be readily phosphorylated by Cdks on multiple sites in vitro, while ectopically-expressed E47 exists in multiple phosphorylated forms in Xenopus embryos. Preventing multi-site phosphorylation using a phospho-mutant version of E47 enhances the neurogenic and myogenic activity of three different class II bHLH reprogramming factors, and also when E47 acts in hetero-dimerisation with endogenous proteins. Mechanistically, unlike phospho-regulation of class II bHLH factors, we find that preventing phosphorylation of E47 increases the amount of chromatin-bound E47 protein but without affecting its overall protein stability. Thus, multi-site phosphorylation is a conserved regulatory mechanism across the bHLH superfamily that can be manipulated to enhance cellular differentiation.

The proneural basic-helix-loop-helix (bHLH) transcription factor Ascl1 is a master regulator of neurogenesis in both central and peripheral nervous systemsin vivo,and is a central driver of neuronal reprogrammingin vitro. Over the last three decades, assaying primary neuron formation inXenopusembryos in response to transcription factor overexpression has contributed to our understanding of the roles and regulation of proneural proteins like Ascl1, with homologues from different species usually exhibiting similar functional effects. Here we demonstrate that the mouse Ascl1 protein is twice as active as theXenopusprotein in inducing neural-β-tubulin expression inXenopusembryos, despite there being little difference in protein accumulation or ability to undergo phosphorylation, two properties known to influence Ascl1 function. This superior activity of the mouse compared to theXenopusprotein is dependent on the presence of the non-conserved N terminal region of the protein, and indicates species-specific regulation that may necessitate care when interpreting results in cross-species experiments.