In the Murine and Bovine Maternal Mammary Gland Signal Transducer and Activator of Transcription 3 is Activated in Clusters of Epithelial Cells around the Day of Birth.

Signal transducers and activators of transcription (STAT) proteins regulate mammary development. Here we investigate the expression of phosphorylated STAT3 (pSTAT3) in the mouse and cow around the day of birth. We present localised colocation analysis, applicable to other mammary studies requiring identification of spatially congregated events. We demonstrate that pSTAT3-positive events are multifocally clustered in a non-random and statistically significant fashion. Arginase-1 expressing cells, consistent with macrophages, exhibit distinct clustering within the periparturient mammary gland. These findings represent a new facet of mammary STAT3 biology, and point to the presence of mammary sub-microenvironments.

Size of supernumerary teats in sheep correlates with complexity of the anatomy and microenvironment.

Supernumerary nipples or teats (polythelia) are congenital accessory structures that may develop at any location along the milk line and have been implicated in the pathogenesis of mastitis. We describe the anatomy and histology of 27 spontaneously occurring supernumerary teats from 16 sheep, delineating two groups of teats - simple and anatomically complex - according to the complexity of the anatomy and microenvironment. Anatomically complex supernumerary teats exhibited significantly increased length and barrel diameter compared with simple supernumerary teats. A teat canal and/or teat cistern was present in anatomically complex teats, with smooth muscle fibres forming a variably well-organised encircling teat sphincter. Complex supernumerary teats also exhibited immune cell infiltrates similar to those of normal teats, including lymphoid follicle-like structures at the folds of the teat cistern-teat canal junction, and macrophages that infiltrated the peri-cisternal glandular tissue. One complex supernumerary teat exhibited teat end hyperkeratosis. These anatomical and histological features allow inference that supernumerary teats may be susceptible to bacterial ingress through the teat canal and we hypothesise that this may be more likely in those teats with less well-organised encircling smooth muscle. The teat cistern of anatomically complex teats may also constitute a focus of milk accumulation and thus a possible nidus for bacterial infection, potentially predisposing to mastitis. We suggest that size of the supernumerary teat, and relationship to the main teats, particularly in the case of 'cluster teats', should be considerations if surgical removal is contemplated.

N-terminal phosphorylation of xHes1 controls inhibition of primary neurogenesis in Xenopus.

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.

Multi-site phosphorylation controls the neurogenic and myogenic activity of E47.

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.

Cell cycle-dependent phosphorylation and regulation of cellular differentiation.

Embryogenesis requires an exquisite regulation of cell proliferation, cell cycle withdrawal and differentiation into a massively diverse range of cells at the correct time and place. Stem cells also remain to varying extents in different adult tissues, acting in tissue homeostasis and repair. Therefore, regulated proliferation and subsequent differentiation of stem and progenitor cells remains pivotal throughout life. Recent advances have characterised the cell cycle dynamics, epigenetics, transcriptome and proteome accompanying the transition from proliferation to differentiation, revealing multiple bidirectional interactions between the cell cycle machinery and factors driving differentiation. Here, we focus on a direct mechanistic link involving phosphorylation of differentiation-associated transcription factors by cell cycle-associated Cyclin-dependent kinases. We discuss examples from the three embryonic germ layers to illustrate this regulatory mechanism that co-ordinates the balance between cell proliferation and differentiation.

Nervous decision-making: to divide or differentiate.

The intricate balance between proliferation and differentiation is of fundamental importance in the development of the central nervous system (CNS). The division versus differentiation decision influences both the number and identity of daughter cells produced, thus critically shaping the overall microstructure and function of the CNS. During the past decade, significant advances have been made to characterise the changes in the cell cycle during differentiation, and to uncover the multiple bidirectional links that coordinate these two processes. Here, we explore the nature and mechanistic basis of these links in the context of the developing CNS, highlighting new insights into transcriptional, post-translational, and epigenetic levels of interaction.

An oncologist׳s friend: How Xenopus contributes to cancer research.

One of the most striking features of the Xenopus system is the versatility in providing a unique range of both in vitro and in vivo models that are rapid, accessible and easily manipulated. Here we present an overview of the diverse contribution that Xenopus has made to advance our understanding of tumour biology and behaviour; a contribution that goes beyond the traditional view of Xenopus as a developmental model organism. From the utility of the egg and oocyte extract system to the use of whole embryos as developmental or induced tumour models, the Xenopus system has been fundamental to investigation of cell cycle mechanisms, cell metabolism, cell signalling and cell behaviour, and has allowed an increasing appreciation of the parallels between early development and the pathogenesis of tumour progression and metastasis. Although not the prototypical oncological model system, we propose that Xenopus is an adaptable and multifunctional tool in the oncologist׳s arsenal.

MyoD phosphorylation on multiple C terminal sites regulates myogenic conversion activity.

MyoD is a master regulator of myogenesis with a potent ability to redirect the cell fate of even terminally differentiated cells. Hence, enhancing the activity of MyoD is an important step to maximising its potential utility for in vitro disease modelling and cell replacement therapies. We have previously shown that the reprogramming activity of several neurogenic bHLH proteins can be substantially enhanced by inhibiting their multi-site phosphorylation by proline-directed kinases. Here we have used Xenopus embryos as an in vivo developmental and reprogramming system to investigate the multi-site phospho-regulation of MyoD during muscle differentiation. We show that, in addition to modification of a previously well-characterised site, Serine 200, MyoD is phosphorylated on multiple additional serine/threonine sites during primary myogenesis. Through mutational analysis, we derive an optimally active phospho-mutant form of MyoD that has a dramatically enhanced ability to drive myogenic reprogramming in vivo. Mechanistically, this is achieved through increased protein stability and enhanced chromatin association. Therefore, multi-site phospho-regulation of class II bHLH proteins is conserved across cell lineages and germ layers, and manipulation of phosphorylation of these key regulators may have further potential for enhancing mammalian cell reprogramming.

Cell cycle regulation of proliferation versus differentiation in the central nervous system.

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.

Complex domain interactions regulate stability and activity of closely related proneural transcription factors.

Characterising post-translational regulation of key transcriptional activators is crucial for understanding how cell division and differentiation are coordinated in developing organisms and cycling cells. One important mode of protein post-translational control is by regulation of half-life via ubiquitin-mediated proteolysis. Two key basic Helix-Loop-Helix transcription factors, Neurogenin 2 (Ngn2) and NeuroD, play central roles in development of the central nervous system but despite their homology, Ngn2 is a highly unstable protein whilst NeuroD is, by comparison, very stable. The basis for and the consequences of the difference in stability of these two structurally and functionally related proteins has not been explored. Here we see that ubiquitylation alone does not determine Ngn2 or NeuroD stability. By making chimeric proteins, we see that the N-terminus of NeuroD in particular has a stabilising effect, whilst despite their high levels of homology, the most conserved bHLH domains of these proneural proteins alone can confer significant changes in protein stability. Despite widely differing stabilities of Ngn2, NeuroD and the chimeric proteins composed of domains of both, there is little correlation between protein half-life and ability to drive neuronal differentiation. Therefore, we conclude that despite significant homology between Ngn2 and NeuroD, the regulation of their stability differs markedly and moreover, stability/instability of the proteins is not a direct correlate of their activity.

Breed-related expression patterns of Ki67, γH2AX, and p21 during ageing in the canine liver.

Cellular senescence is a molecular hallmark of ageing that is associated with multiple pathologies, and DNA damage marker γH2AX, together with cell cycle inhibitor p21, have been used as senescence markers in multiple species including dogs. Idiopathic canine chronic hepatitis has recognised breed-related differences in predisposition and prognosis, but reasons behind this are poorly understood. This retrospective study using archived post mortem tissue aimed to provide insight into liver ageing in 51 microscopically normal canine livers across seven breed categories, including those with and without increased risk of chronic hepatitis. Immunohistochemistry was conducted for γH2AX, p21, and cell proliferation marker Ki67, and the mean number of positive hepatocytes per high power field was determined. All three markers were strongly correlated to each other, but no age-dependent expression was seen in the combined study population. Overall expression levels were low in most dogs, with median values representing less than 1.5% of hepatocytes, but this increased to 20-30% in individual dogs at the upper end of the range. Individual breed differences were noted in two breeds that have increased risk of chronic hepatitis, with English Springer Spaniels having lower expression of Ki67 than other dogs, and Labradors having higher expression of Ki67 and γH2AX than other dogs. These results warrant further investigation in these breeds and highlight a need to validate reliable markers of cellular senescence in dogs.

Analysis of Chromatin Binding of Ectopically Expressed Proteins in Early Xenopus Embryos.

Xenopus embryos have long been used to show phenotypic effects following overexpression of proteins of interest such as transcription factors. Posttranslational modification of these proteins can dramatically alter the extent of the observed phenotype by inhibiting or enhancing protein activity. 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 electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) that measure site-specific DNA binding. This protocol describes a method to prepare and analyze chromatin and cytoplasmic extracts from embryos overexpressing proteins of interest, and it 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 (e.g., to investigate the effects of point mutations on chromatin association). Optimization is required for the specific protein of interest but guide ranges are provided.

Analysis of Phosphorylation Status of Ectopically Expressed Proteins in Early Xenopus Embryos.

Xenopus embryos provide a rapid and accessible in vivo model, expressing a plethora of endogenous kinase and phosphatase enzymes that control protein phosphorylation and, in turn, affect physiological function. Traditionally, the detection of protein phosphorylation has been achieved by radioisotope phosphate labeling of proteins, sometimes with in vitro assays using recombinant proteins or with site-specific phospho-antibodies. However, the target phospho-sites and kinases responsible are often unknown, and the use of radioactive isotopes is not always desirable. Therefore, as a first step in determining the 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 nonradioactive method to visualize protein phosphorylation by exposing the protein to the egg/embryo kinase environment and then observing a difference in protein migration (as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] and western blot analysis) with and without treatment with the lambda 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, the detection of multiple bands of the protein of interest even after phosphatase treatment points to the presence of other types of posttranslational modifications.

The N terminus of Ascl1 underlies differing proneural activity of mouse and Xenopus Ascl1 proteins.

The proneural basic-helix-loop-helix (bHLH) transcription factor Ascl1 is a master regulator of neurogenesis in both central and peripheral nervous systems in vivo, and is a central driver of neuronal reprogramming in vitro. Over the last three decades, assaying primary neuron formation in Xenopus embryos 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 the Xenopus protein in inducing neural-ß-tubulin expression in Xenopus embryos, 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 the Xenopus protein 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.

Interaction between opposing modes of phospho-regulation of the proneural proteins Ascl1 and Ngn2.

From the relatively simple nervous system of Drosophila to the elaborate mammalian cortex, neurogenesis requires exceptional spatial and temporal precision to co-ordinate progenitor cell proliferation and subsequent differentiation to a diverse range of neurons and glia. A limited number of transiently expressed proneural basic-helix-loop-helix (bHLH) transcription factors, for example achaete-scute-complex (as-c) and atonal (ato) in Drosophila and the vertebrate homologues Ascl1 and Neurogenin2 (Ngn2), are able to orchestrate the onset of neuronal determination, context-dependent subtype selection and even influence later aspects of neuronal migration and maturation. Within the last decade, two models have emerged to explain how the temporal activity of proneural determination factors is regulated by phosphorylation at distinct sites. One model describes how cell-cycle associated phosphorylation on multiple sites in the N and C termini of vertebrate proneural proteins limits neuronal differentiation in cycling progenitor cells. A second model describes phosphorylation on a single site in the bHLH domain of Drosophila atonal that acts as a binary switch, where phosphorylation terminates proneural activity. Here we combine activating mutations of phosphorylation sites in the N- and C- termini with an inhibitory phospho-mimetic mutation in the bHLH domain of Ascl1 and Ngn2 proteins, and test their functions in vivo using Xenopus embryos to determine which mode of phospho-regulation dominates. Enhancing activity by preventing N- and C terminal phosphorylation cannot overcome the inhibitory effect of mimicking phosphorylation of the bHLH domain. Thus we have established a hierarchy between these two modes of proneural protein control and suggest a model of temporal regulation for proneural protein activity.

Xenopus Models of Cancer: Expanding the Oncologist's Toolbox.

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.

Multi-site phosphorylation regulates NeuroD4 activity during primary neurogenesis: a conserved mechanism amongst proneural proteins.

BACKGROUND: Basic Helix Loop Helix (bHLH) proneural transcription factors are master regulators of neurogenesis that act at multiple stages in this process. We have previously demonstrated that multi-site phosphorylation of two members of the proneural protein family, Ngn2 and Ascl1, limits their ability to drive neuronal differentiation when cyclin-dependent kinase levels are high, as would be found in rapidly cycling cells. Here we investigate potential phospho-regulation of proneural protein NeuroD4 (also known as Xath3), the Xenopus homologue of Math3/NeuroM, that functions downstream of Ngn2 in the neurogenic cascade. RESULTS: Using the developing Xenopus embryo system, we show that NeuroD4 is expressed and phosphorylated during primary neurogenesis, and this phosphorylation limits its ability to drive neuronal differentiation. Phosphorylation of up to six serine/threonine-proline sites contributes additively to regulation of NeuroD4 proneural activity without altering neuronal subtype specification, and number rather than location of available phospho-sites is the key for limiting NeuroD4 activity. Mechanistically, a phospho-mutant NeuroD4 displays increased protein stability and enhanced chromatin binding relative to wild-type NeuroD4, resulting in transcriptional up-regulation of a range of target genes that further promote neuronal differentiation. CONCLUSIONS: Multi-site phosphorylation on serine/threonine-proline pairs is a widely conserved mechanism of limiting proneural protein activity, where it is the number of phosphorylated sites, rather than their location that determines protein activity. Hence, multi-site phosphorylation is very well suited to allow co-ordination of proneural protein activity with the cellular proline-directed kinase environment.

Emergence of neuronal diversity from patterning of telencephalic progenitors.

During central nervous system (CNS) development, hundreds of distinct neuronal subtypes are generated from a single layer of multipotent neuroepithelial progenitor cells. Within the rostral CNS, initial regionalization of the telencephalon marks the territories where the cerebral cortex and the basal ganglia originate. Subsequent refinement of the primary structures determines the formation of domains of differential gene expression, where distinct fate-restricted progenitors are located. To understand how diversification of neural progenitors and neurons is achieved in the telencephalon, it is important to address early and late patterning events in this context. In particular, important questions include: How does the telencephalon become specified and regionalized along the major spatial axes? Within each region, are the differences in neuronal subtypes established at the progenitor level or at the postmitotic stage? If distinct progenitors exist that are committed to subtype-specific neuronal lineages, how does the diversification emerge? What is the contribution of positional and temporal cues and how is this information integrated into the intrinsic programs of cell identity? WIREs For further resources related to this article, please visit the WIREs website.

Ascl1 phospho-status regulates neuronal differentiation in a Xenopus developmental model of neuroblastoma.

Neuroblastoma (NB), although rare, accounts for 15% of all paediatric cancer mortality. Unusual among cancers, NBs lack a consistent set of gene mutations and, excluding large-scale chromosomal rearrangements, the genome seems to be largely intact. Indeed, many interesting features of NB suggest that it has little in common with adult solid tumours but instead has characteristics of a developmental disorder. NB arises overwhelmingly in infants under 2 years of age during a specific window of development and, histologically, NB bears striking similarity to undifferentiated neuroblasts of the sympathetic nervous system, its likely cells of origin. Hence, NB could be considered a disease of development arising when neuroblasts of the sympathetic nervous system fail to undergo proper differentiation, but instead are maintained precociously as progenitors with the potential for acquiring further mutations eventually resulting in tumour formation. To explore this possibility, we require a robust and flexible developmental model to investigate the differentiation of NB's presumptive cell of origin. Here, we use Xenopus frog embryos to characterise the differentiation of anteroventral noradrenergic (AVNA) cells, cells derived from the neural crest. We find that these cells share many characteristics with their mammalian developmental counterparts, and also with NB cells. We find that the transcriptional regulator Ascl1 is expressed transiently in normal AVNA cell differentiation but its expression is aberrantly maintained in NB cells, where it is largely phosphorylated on multiple sites. We show that Ascl1's ability to induce differentiation of AVNA cells is inhibited by its multi-site phosphorylation at serine-proline motifs, whereas overexpression of cyclin-dependent kinases (CDKs) and MYCN inhibit wild-type Ascl1-driven AVNA differentiation, but not differentiation driven by a phospho-mutant form of Ascl1. This suggests that the maintenance of ASCL1 in its multiply phosphorylated state might prevent terminal differentiation in NB, which could offer new approaches for differentiation therapy in NB.