Control of chromatin structure is crucial for multicellular development and regulation of cell differentiation. The CHD (chromodomain-helicase-DNA binding) protein family is one of the major ATP-dependent, chromatin remodeling factors that regulate nucleosome positioning and access of transcription factors and RNA polymerase to the eukaryotic genome. There are three mammalian CHD subfamilies and their impaired functions are associated with several human diseases. Here, we identify three CHD orthologs (ChdA, ChdB and ChdC) in Dictyostelium discoideum. These CHDs are expressed throughout development, but with unique patterns. Null mutants lacking each CHD have distinct phenotypes that reflect their expression patterns and suggest functional specificity. Accordingly, using genome-wide (RNA-seq) transcriptome profiling for each null strain, we show that the different CHDs regulate distinct gene sets during both growth and development. ChdC is an apparent ortholog of the mammalian Class III CHD group that is associated with the human CHARGE syndrome, and GO analyses of aberrant gene expression in chdC nulls suggest defects in both cell-autonomous and non-autonomous signaling, which have been confirmed through analyses of chdC nulls developed in pure populations or with low levels of wild-type cells. This study provides novel insight into the broad function of CHDs in the regulation development and disease, through chromatin-mediated changes in directed gene expression.
SNF2; Transcriptome profiling; RNA-seq; Chemotaxis; Growth; Differentiation
Gene targeting by ‘ends-out’ homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR.
Drosophila; Functional genomics; Gene targeting; Homologous recombination
During forebrain morphogenesis, there is extensive reorganisation of the cells destined to form the eyes, telencephalon and diencephalon. Little is known about the molecular mechanisms that regulate region-specific behaviours and that maintain the coherence of cell populations undergoing specific morphogenetic processes. In this study, we show that the activity of the Eph/Ephrin signalling pathway maintains segregation between the prospective eyes and adjacent regions of the anterior neural plate during the early stages of forebrain morphogenesis in zebrafish. Several Ephrins and Ephs are expressed in complementary domains in the prospective forebrain and combinatorial abrogation of their activity results in incomplete segregation of the eyes and telencephalon and in defective evagination of the optic vesicles. Conversely, expression of exogenous Ephs or Ephrins in regions of the prospective forebrain where they are not usually expressed changes the adhesion properties of the cells, resulting in segregation to the wrong domain without changing their regional fate. The failure of eye morphogenesis in rx3 mutants is accompanied by a loss of complementary expression of Ephs and Ephrins, suggesting that this pathway is activated downstream of the regional fate specification machinery to establish boundaries between domains undergoing different programmes of morphogenesis.
Eye field; Neural plate; Morphogenesis; Zebrafish
The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.
Myb; Multiciliogenesis; Centriole amplification
Established transgenesis methods for fish model systems allow efficient genomic integration of transgenes. However, thus far a way of controlling copy number and integration sites has not been available, leading to variable transgene expression caused by position effects. The integration of transgenes at predefined genomic positions enables the direct comparison of different transgenes, thereby improving time and cost efficiency. Here, we report an efficient PhiC31-based site-specific transgenesis system for medaka. This system includes features that allow the pre-selection of successfully targeted integrations early on in the injected generation. Pre-selected embryos transmit the correctly integrated transgene through the germline with high efficiency. The landing site design enables a variety of applications, such as reporter and enhancer switch, in addition to the integration of any insert. Importantly, this allows assaying of enhancer activity in a site-specific manner without requiring germline transmission, thus speeding up large-scale analyses of regulatory elements.
PhiC31 integrase; Fish; Regulatory DNA; In vivo analysis; Position effects
Although progress has been made in resolving the genetic pathways that specify neuronal asymmetries in the brain, little is known about genes that mediate the development of structural asymmetries between neurons on left and right. In this study, we identify daam1a as an asymmetric component of the signalling pathways leading to asymmetric morphogenesis of the habenulae in zebrafish. Daam1a is a member of the Formin family of actin-binding proteins and the extent of Daam1a expression in habenular neuron dendrites mirrors the asymmetric growth of habenular neuropil between left and right. Local loss and gain of Daam1a function affects neither cell number nor subtype organisation but leads to a decrease or increase of neuropil, respectively. Daam1a therefore plays a key role in the asymmetric growth of habenular neuropil downstream of the pathways that specify asymmetric cellular domains in the habenulae. In addition, Daam1a mediates the development of habenular efferent connectivity as local loss and gain of Daam1a function impairs or enhances, respectively, the growth of habenular neuron terminals in the interpeduncular nucleus. Abrogation of Daam1a disrupts the growth of both dendritic and axonal processes and results in disorganised filamentous actin and α-tubulin. Our results indicate that Daam1a plays a key role in asymmetric habenular morphogenesis mediating the growth of dendritic and axonal processes in dorsal habenular neurons.
Asymmetry; Nervous system; Morphogenesis; Daam1; Habenula; Zebrafish
The use of whole-genome pooled shRNA libraries in loss-of-function screening in tissue culture models provides an effective means to identify novel factors acting in pathways of interest. Embryonic stem cells (ESCs) offer a unique opportunity to study processes involved in stem cell pluripotency and differentiation. Here, we report a genome-wide shRNA screen in ESCs to identify novel components involved in repression of the Gata6 locus, using a cell viability-based screen, which offers the benefits of stable shRNA integration and a robust and simple protocol for hit identification. Candidate factors identified were enriched for transcription factors and included known Polycomb proteins and other chromatin-modifying factors. We identified the protein Bcor, which is known to associate in complexes with the Polycomb protein Ring1B, and verified its importance in Gata6 repression in ESCs. Potential further applications of such a screening strategy could allow the identification of factors important for regulation of gene expression and pluripotency.
Embryonic stem cells; Polycomb; shRNA screen; Mouse
The majority of cranial sensory neurons originate in placodes in the surface ectoderm, migrating to form ganglia that connect to the central nervous system (CNS). Interactions between inward-migrating sensory neuroblasts and emigrant cranial neural crest cells (NCCs) play a role in coordinating this process, but how the relationship between these two cell populations is established is not clear. Here, we demonstrate that NCCs generate corridors delineating the path of migratory neuroblasts between the placode and CNS in both chick and mouse. In vitro analysis shows that NCCs are not essential for neuroblast migration, yet act as a superior substrate to mesoderm, suggesting provision of a corridor through a less-permissive mesodermal territory. Early organisation of NCC corridors occurs prior to sensory neurogenesis and can be recapitulated in vitro; however, NCC extension to the placode requires placodal neurogenesis, demonstrating reciprocal interactions. Together, our data indicate that NCC corridors impose physical organisation for precise ganglion formation and connection to the CNS, providing a local environment to enclose migrating neuroblasts and axonal processes as they migrate through a non-neural territory.
Neural crest; Placode; Cranial sensory ganglia
The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis.
C. elegans; KSR scaffolds; Nm23/NDPK; NME; Ras signaling
Syndecan 4 (Sdc4) is a cell-surface heparan sulfate proteoglycan (HSPG) that regulates gastrulation, neural tube closure and directed neural crest migration in Xenopus development. To determine whether Sdc4 participates in Wnt/PCP signaling during mouse development, we evaluated a possible interaction between a null mutation of Sdc4 and the loop-tail allele of Vangl2. Sdc4 is expressed in multiple tissues, but particularly in the non-neural ectoderm, hindgut and otic vesicles. Sdc4;Vangl2Lp compound mutant mice have defective spinal neural tube closure, disrupted orientation of the stereocilia bundles in the cochlea and delayed wound healing, demonstrating a strong genetic interaction. In Xenopus, co-injection of suboptimal amounts of Sdc4 and Vangl2 morpholinos resulted in a significantly greater proportion of embryos with defective neural tube closure than each individual morpholino alone. To probe the mechanism of this interaction, we overexpressed or knocked down Vangl2 function in HEK293 cells. The Sdc4 and Vangl2 proteins colocalize, and Vangl2, particularly the Vangl2Lp mutant form, diminishes Sdc4 protein levels. Conversely, Vangl2 knockdown enhances Sdc4 protein levels. Overall HSPG steady-state levels were regulated by Vangl2, suggesting a molecular mechanism for the genetic interaction in which Vangl2Lp/+ enhances the Sdc4-null phenotype. This could be mediated via heparan sulfate residues, as Vangl2Lp/+ embryos fail to initiate neural tube closure and develop craniorachischisis (usually seen only in Vangl2Lp/Lp) when cultured in the presence of chlorate, a sulfation inhibitor. These results demonstrate that Sdc4 can participate in the Wnt/PCP pathway, unveiling its importance during neural tube closure in mammalian embryos.
Neural tube defects; Proteoglycans; Wnt planar cell polarity
Transcription is an essential component of basic cellular and developmental processes. However, early embryonic development occurs in the absence of transcription and instead relies upon maternal mRNAs and proteins deposited in the egg during oocyte maturation. Although the early zebrafish embryo is competent to transcribe exogenous DNA, factors present in the embryo maintain genomic DNA in a state that is incompatible with transcription. The cell cycles of the early embryo titrate out these factors, leading to zygotic transcription initiation, presumably in response to a change in genomic DNA chromatin structure to a state that supports transcription. To understand the molecular mechanisms controlling this maternal to zygotic transition, it is important to distinguish between the maternal and zygotic transcriptomes during this period. Here we use exome sequencing and RNA-seq to achieve such discrimination and in doing so have identified the first zygotic genes to be expressed in the embryo. Our work revealed different profiles of maternal mRNA post-transcriptional regulation prior to zygotic transcription initiation. Finally, we demonstrate that maternal mRNAs are required for different modes of zygotic transcription initiation, which is not simply dependent on the titration of factors that maintain genomic DNA in a transcriptionally incompetent state.
MZT; Maternal; Paternal; Transcriptome; Zebrafish
Notch and Wnt are highly conserved signalling pathways that are used repeatedly throughout animal development to generate a diverse array of cell types. However, they often have opposing effects on cell-fate decisions with each pathway promoting an alternate outcome. Commonly, a cell receiving both signals exhibits only Wnt pathway activity. This suggests that Wnt inhibits Notch activity to promote a Wnt-ON/Notch-OFF output; but what might underpin this Notch regulation is not understood. Here, we show that Wnt acts via Dishevelled to inhibit Notch signalling, and that this crosstalk regulates cell-fate specification in vivo during Xenopus development. Mechanistically, Dishevelled binds and directly inhibits CSL transcription factors downstream of Notch receptors, reducing their activity. Furthermore, our data suggest that this crosstalk mechanism is conserved between vertebrate and invertebrate homologues. Thus, we identify a dual function for Dishevelled as an inhibitor of Notch signalling and an activator of the Wnt pathway that sharpens the distinction between opposing Wnt and Notch responses, allowing for robust cell-fate decisions.
Dishevelled; Notch; Wnt; Signalling crosstalk; Xenopus
The somatosensory and sympathetic innervation of the vertebrate head is derived principally from the neurons of trigeminal and superior cervical ganglia (SCG), respectively. During development, the survival of both populations of neurons and the terminal growth and branching of their axons in the tissues they innervate is regulated by the supply of nerve growth factor (NGF) produced by these tissues. NGF is derived by proteolytic cleavage of a large precursor protein, proNGF, which is recognised to possess distinctive biological functions. Here, we show that proNGF promotes profuse neurite growth and branching from cultured postnatal mouse SCG neurons. In marked contrast, proNGF does not promote the growth of trigeminal neurites. Studies using compartment cultures demonstrated that proNGF acts locally on SCG neurites to promote growth. The neurite growth-promoting effect of proNGF is not observed in SCG neurons cultured from p75NTR-deficient mice, and proNGF does not phosphorylate the NGF receptor tyrosine kinase TrkA. These findings suggest that proNGF selectively promotes the growth of neurites from a subset of NGF-responsive neurons by a p75NTR-dependent mechanism during postnatal development when the axons of these neurons are ramifying within their targets in vivo.
Proneurotrophins; p75NTR; Neurite growth; Sympathetic neurons; Mouse
The thymus is the central site of T-cell development and thus is of fundamental importance to the immune system, but little information exists regarding molecular regulation of thymus development in humans. Here we demonstrate, via spatial and temporal expression analyses, that the genetic mechanisms known to regulate mouse thymus organogenesis are conserved in humans. In addition, we provide molecular evidence that the human thymic epithelium derives solely from the third pharyngeal pouch, as in the mouse, in contrast to previous suggestions. Finally, we define the timing of onset of hematopoietic cell colonization and epithelial cell differentiation in the human thymic primordium, showing, unexpectedly, that the first colonizing hematopoietic cells are CD45+CD34int/-. Collectively, our data provide essential information for translation of principles established in the mouse to the human, and are of particular relevance to development of improved strategies for enhancing immune reconstitution in patients.
Thymus development; Human; Hematopoietic; Epithelium; Mesenchyme; Colonization
Signals that promote germ cell self-renewal by preventing premature meiotic entry are well understood. However, signals that control mitotic proliferation to promote meiotic differentiation have not been well characterized. In Caenorhabditis elegans, GLP-1 Notch signalling promotes the proliferative fate by preventing premature meiotic entry. The germline niche cell, which is the source of the ligand for GLP-1, spatially restricts GLP-1 signalling and thus enables the germ cells that have moved away from the niche to enter meiosis. Here, we show that the suppression of RAS/MAP kinase signalling in the mitotic and meiotic-entry regions is essential for the regulation of the mitosis-meiosis switch by niche signalling. We provide evidence that the conserved PUF family RNA-binding protein PUF-8 and the RAS GAP protein GAP-3 function redundantly to suppress the LET-60 RAS in the mitotic and meiotic entry regions. Germ cells missing both PUF-8 and GAP-3 proliferate in an uncontrolled fashion and fail to undergo meiotic development. MPK-1, the MAP kinase downstream of the LET-60 RAS, is prematurely activated in these cells; downregulation of MPK-1 activation eliminates tumours and restores differentiation. Our results further reveal that PUF-8 negatively regulates LET-60 expression at a post-transcriptional step. LET-60 is misexpressed in the puf-8(-) mutant germlines and PUF-8 physically interacts with the let-60 3′ UTR. Furthermore, PUF-8 suppresses let-60 3′ UTR-mediated expression in the germ cells that are transitioning from the mitotic to meiotic fate. These results reveal that PUF-8-mediated inhibition of the RAS/MAPK pathway is essential for mitotic-to-meiotic fate transition.
PUF-8; GAP-3; RAS; MAPK; C. elegans; Germ cells; Mitosis-meiosis decision
The asymmetric localisation of core planar polarity proteins at apicolateral junctions is required to specify cell polarity in the plane of epithelia. This asymmetric distribution of the core proteins is proposed to require amplification of an initial asymmetry by feedback loops. In addition, generation of asymmetry appears to require the regulation of core protein levels, but the importance of such regulation and the underlying mechanisms is unknown. Here we show that ubiquitylation acts through more than one mechanism to control core protein levels in Drosophila, and that without this regulation cellular asymmetry is compromised. Levels of Dishevelled at junctions are regulated by a Cullin-3-Diablo/Kelch ubiquitin ligase complex, the activity of which is most likely controlled by neddylation. Furthermore, activity of the deubiquitylating enzyme Fat facets is required to maintain Flamingo levels at junctions. Notably, ubiquitylation does not alter the total cellular levels of Dishevelled or Flamingo, but only that of the junctional population. When junctional core protein levels are either increased or decreased by disruption of the ubiquitylation machinery, their asymmetric localisation is reduced and this leads to disruption of planar polarity at the tissue level. Loss of asymmetry by altered core protein levels can be explained by reference to feedback models for amplification of asymmetry.
Planar polarity; PCP; Ubiquitination; Neddylation; Dishevelled; Drosophila
Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment.
Adhesion; CD46; Delta; Endocytosis
The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.
Oct4; Wnt signaling; Mouse embryonic stem cells; Pluripotency; β-Catenin
PUF family proteins are well-conserved regulators of cell proliferation in different developmental processes. They regulate target mRNAs by promoting degradation or by influencing translation through interaction with the translation initiation machinery. Here we show that Caenorhabditis elegans PUF-8 functions redundantly with the nuclear protein TCER-1 in the post-transcriptional maintenance of at least six germline mRNAs. The levels of spliced mRNAs in the puf-8(-) tcer-1(-) double mutant are only 10-30% of the wild type, whereas the unspliced forms increase by ∼2- to 3-fold compared with the wild type. These two proteins colocalise at the inner nuclear periphery, and their absence leads to reduced germ cell proliferation and to sterility. A yeast two-hybrid screen of 31 components of the nuclear pore complex and mRNA processing machineries identified seven proteins involved in mRNA export as potential partners of PUF-8. One of these, the nuclear cap-binding protein NCBP-2, colocalises with PUF-8 in the nucleus. A 50 amino acid N-terminal domain of PUF-8 is essential for interaction with NCBP-2 and for PUF-8 to function redundantly with TCER-1. These results reveal two important unexpected aspects of PUF proteins: that, in addition to the C-terminal PUF domain, the N-terminal domain is crucial for PUF function, and that PUF proteins have a novel role in mRNA maintenance. We propose that PUF proteins, in addition to their known cytoplasmic roles, participate in nuclear processing and/or export of mRNAs.
PUF-8; TCER-1; C. elegans; Germ cells; mRNA processing; mRNA export
Polarised tissue elongation during morphogenesis involves cells within epithelial sheets or tubes making and breaking intercellular contacts in an oriented manner. Growing evidence suggests that cell adhesion can be modulated by endocytic trafficking of E-cadherin (E-cad), but how this process can be polarised within individual cells is poorly understood. The Frizzled (Fz)-dependent core planar polarity pathway is a major regulator of polarised cell rearrangements in processes such as gastrulation, and has also been implicated in regulation of cell adhesion through trafficking of E-cad; however, it is not known how these functions are integrated. We report a novel role for the core planar polarity pathway in promoting cell intercalation during tracheal tube morphogenesis in Drosophila embryogenesis, and present evidence that this is due to regulation of turnover and levels of junctional E-cad by the guanine exchange factor RhoGEF2. Furthermore, we show that core pathway activity leads to planar-polarised recruitment of RhoGEF2 and E-cad turnover in the epidermis of both the embryonic germband and the pupal wing. We thus reveal a general mechanism by which the core planar polarity pathway can promote polarised cell rearrangements.
Planar polarity; E-cadherin; Morphogenesis
Nuclear receptor interacting protein (Nrip1), also known as RIP140, is a co-regulator for nuclear receptors that plays an essential role in ovulation by regulating the expression of the epidermal growth factor-like family of growth factors. Although several studies indicate a role for RIP140 in breast cancer, its role in the development of the mammary gland is unclear. By using RIP140-null and RIP140 transgenic mice, we demonstrate that RIP140 is an essential factor for normal mammary gland development and that it functions by mediating oestrogen signalling. RIP140-null mice exhibit minimal ductal elongation with no side-branching, whereas RIP140-overexpressing mice show increased cell proliferation and ductal branching with age. Tissue recombination experiments demonstrate that RIP140 expression is required in both the mammary epithelial and stromal compartments for ductal elongation during puberty and that loss of RIP140 leads to a catastrophic loss of the mammary epithelium, whereas RIP140 overexpression augments the mammary basal cell population and shifts the progenitor/differentiated cell balance within the luminal cell compartment towards the progenitors. For the first time, we present a genome-wide global view of oestrogen receptor-α (ERα) binding events in the developing mammary gland, which unravels 881 ERα binding sites. Unbiased evaluation of several ERα binding sites for RIP140 co-occupancy reveals selectivity and demonstrates that RIP140 acts as a co-regulator with ERα to regulate directly the expression of amphiregulin (Areg), the progesterone receptor (Pgr) and signal transducer and activator of transcription 5a (Stat5a), factors that influence key mitogenic pathways that regulate normal mammary gland development.
Amphiregulin; Mammary gland; Mammary progenitors; Oestrogen receptor; Progesterone receptor; RIP140; Mouse
The physiological activities of organs are underpinned by an interplay between the distinct cell types they contain. However, little is known about the genetic control of patterned cell differentiation during organ development. We show that the conserved Teashirt transcription factors are decisive for the differentiation of a subset of secretory cells, stellate cells, in Drosophila melanogaster renal tubules. Teashirt controls the expression of the water channel Drip, the chloride conductance channel CLC-a and the Leukokinin receptor (LKR), all of which characterise differentiated stellate cells and are required for primary urine production and responsiveness to diuretic stimuli. Teashirt also controls a dramatic transformation in cell morphology, from cuboidal to the eponymous stellate shape, during metamorphosis. teashirt interacts with cut, which encodes a transcription factor that underlies the differentiation of the primary, principal secretory cells, establishing a reciprocal negative-feedback loop that ensures the full differentiation of both cell types. Loss of teashirt leads to ineffective urine production, failure of homeostasis and premature lethality. Stellate cell-specific expression of the teashirt paralogue tiptop, which is not normally expressed in larval or adult stellate cells, almost completely rescues teashirt loss of expression from stellate cells. We demonstrate conservation in the expression of the family of tiptop/teashirt genes in lower insects and establish conservation in the targets of Teashirt transcription factors in mouse embryonic kidney.
Cell differentiation; Drosophila; Kidney; Malpighian tubule; Organogenesis; Tiptop/Teashirt
The neurohypophysis is a crucial component of the hypothalamo-pituitary axis, serving as the site of release of hypothalamic neurohormones into a plexus of hypophyseal capillaries. The growth of hypothalamic axons and capillaries to the forming neurohypophysis in embryogenesis is therefore crucial to future adult homeostasis. Using ex vivo analyses in chick and in vivo analyses in mutant and transgenic zebrafish, we show that Fgf10 and Fgf3 secreted from the forming neurohypophysis exert direct guidance effects on hypothalamic neurosecretory axons. Simultaneously, they promote hypophyseal vascularisation, exerting early direct effects on endothelial cells that are subsequently complemented by indirect effects. Together, our studies suggest a model for the integrated neurohemal wiring of the hypothalamo-neurohypophyseal axis.
FGF; Guidance; Hypothalamus; Zebrafish; Chick
In the developing lung, it is thought that the terminal buds of elongating airways contain a population of multipotent epithelial progenitors. As the bronchial tree extends, descendants of these cells give rise to lineage-restricted progenitors in the conducting airways via Notch signaling, which is involved in the establishment of epithelial Clara, ciliated and pulmonary neuroendocrine (NE) cell populations. However, the precise molecular details of this selection process are still emerging. Our stepwise removal of the three Notch receptors from the developing lung epithelium reveals that, whereas Notch2 mediates the Clara/ciliated cell fate decision with negligible contributions from Notch1 and Notch3, all three Notch receptors contribute in an additive manner to regulate the abundance of NE cells and the size of the presumptive pulmonary neuroepithelial body (pNEB) as a result of mutual interactions between NE cells and the Notch-dependent, SSEA-1+, CC10− cell population surrounding the pNEB (SPNC cells). Ectopic expression of the Notch1 or Notch2 intracellular domain was sufficient to induce SSEA-1+ cells and to suppress pNEB formation without expending Clara cells. We provide evidence that the additive functions of Notch receptors, together with other signaling pathways, maintains the expression of Hes1, a key regulator of NE cell fate, and that maintenance of Hes1 expression in epithelial cells is key to the regulation of pNEB size. These results suggest that two different assemblies of Notch receptors coordinate the numbers and distribution of the major epithelial cell types in the conducting airway during lung organogenesis.
Lung; Notch signaling; Neuroendocrine; Mouse
During murine embryogenesis, the Ets factor Erg is highly expressed in endothelial cells of the developing vasculature and in articular chondrocytes of developing bone. We identified seven isoforms for the mouse Erg gene. Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes. The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells. Homozygous ErgΔEx3/ΔEx3 knockout mice are viable, fertile and do not display any overt phenotype. By contrast, homozygous ErgΔEx4/ΔEx4 knockout mice are embryonic lethal, which is associated with a marked reduction in endocardial-mesenchymal transformation (EnMT) during cardiac valve morphogenesis. We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.
Ets factor; Erg isoforms; Valve morphogenesis; EMT; EnMT; Snail1 (Snai1); Snail2 (Snai2)