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Drosophila R8 photoreceptor cell subtype specification requires Notch and hibris.

Cell differentiation and cell fate determination in sensory systems are essential for stimulus discrimination and coding of environmental stimuli. Color vision is based on the differential color sensitivity of retinal photoreceptors, however the developmental programs that control photoreceptor cell differentiation and specify color sensitivity are poorly understood. In Drosophila melanogaster, there is evidence that the color sensitivity of different photoreceptors in the compound eye is regulated by inductive signals between cells, but the exact nature of these signals and how they are propagated remains unknown. We conducted a genetic screen to identify additional regulators of this process and identified a novel mutation in the hibris gene. hibris encodes an irre cell recognition module protein (IRM). These immunoglobulin super family cell adhesion molecules include human neph and nephrin (NPHS1). hibris is expressed dynamically in the developing Drosophila melanogaster eye and loss-of-function mutations give rise to a diverse range of mutant phenotypes including disruption of the specification of R8 photoreceptors cell diversity. The specification of blue or green sensitivity in R8 cells is also dependent upon Notch signaling. We demonstrate that hibris is required within the retina, non-cell autonomously for these effects, suggesting an additional layer of complexity in the signaling process that produces paired expression of opsin genes in adjacent R7 and R8 photoreceptor cells.\n\nAuthor SummaryAs humans, our ability to distinguish different colors is dependent upon the presence of three different types of cone cell neurons in the retina of the eye. The cone cells express blue, green or red absorbing visual pigments that detect and discriminate between these colors. The principle of color discrimination by neurons \"tuned\" to different colors is an evolutionarily conserved specialization that occurs in many different animals. This specialization requires 1) visual pigments that detect different colors and 2) a developmental program that regulates the expression of these pigments in different types of cells. In this study we discovered that the fruit fly (Drosophila melanogaster) gene hibris is required for the developmental program that produces blue sensitive neurons in the fly retina. When we over-expressed hibris throughout the developing retina, extra blue sensitive cells were produced. These results demonstrate that if there is not enough hibris, too few blue sensitive cells form, but if there is too much hibris, too many blue sensitive cells form. Finally, we discovered that the hibris gene does not act in color sensitive neurons of the retina themselves. This surprising discovery suggests that hibris may influence development of the retina in a completely new and different way.

developmental biology

The transmembrane protein Crb2a regulates cardiomyocyte apicobasal polarity and adhesion in zebrafish

Tissue morphogenesis requires changes in cell-cell adhesion as well as in cell shape and polarity. Cardiac trabeculation is a morphogenetic process essential to form a functional ventricular wall. Here we show that zebrafish hearts lacking Crb2a, a component of the Crumbs polarity complex, display compact wall integrity defects and fail to form trabeculae. Crb2a localization is very dynamic, at a time when other cardiomyocyte junctional proteins also relocalize. Before the initiation of cardiomyocyte delamination to form the trabecular layer, Crb2a is expressed in all ventricular cardiomyocytes colocalizing with the junctional protein ZO-1. Subsequently, Crb2a becomes localized all along the apical membrane of compact layer cardiomyocytes and is downregulated by those delaminating. We show that blood flow and Nrg/ErbB2 signaling regulate these Crb2a localization changes. crb2a mutants display a multilayered wall with polarized cardiomyocytes, a unique phenotype. Our data further indicate that Crb2a regulates cardiac trabeculation by controlling the localization of tight and adherens junctions in cardiomyocytes. Importantly, transplantation data show that Crb2a controls trabeculation in a CM-autonomous manner. Altogether, our study reveals a critical role for Crb2a during cardiac development.\n\nSummary statementInvestigation of the Crumbs polarity protein Crb2a in zebrafish reveals a novel role in cardiac development via regulation of cell-cell adhesion and apicobasal polarity.

developmental biology

Regulation of tensile stress in response to external forces coordinates epithelial cell shape transitions with organ growth and elongation

The role of actomyosin contractility at epithelial adherens junctions has been extensively studied. However, little is known about how external forces are integrated to establish epithelial cell and organ shape in vivo. We use the Drosophila follicle epithelium to investigate how tension at adherens junctions is regulated to integrate external forces arising from changes in germline size and shape. We find that overall tension in the epithelium decreases despite pronounced growth of enclosed germline cells, suggesting that the epithelium relaxes to accommodate growth. However, we find local differences in adherens junction tension correlate with apposition to germline nurse cells or the oocyte. We demonstrate that medial Myosin II coupled to corrugating adherens junctions resists nurse cell-derived forces and thus maintains apical surface areas and cuboidal cell shapes. Furthermore, medial reinforcement of the apical surface ensures cuboidal-to-columnar cell shape transitions and imposes circumferential constraints on nurse cells guiding organ elongation. Our study provides insight into how tension within an adherens junction network integrates growth of a neighbouring tissue, mediates cell shape transitions and channels growth into organ elongation.

developmental biology

Epigenetic factors coordinate intestinal development

Intestinal epithelium development depends on epigenetic modifications, but whether that is also the case for other intestinal tract cell types remains unclear. We found that functional loss of a DNA methylation machinery component, ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1), leads to reduced enteric neuron number, changes in neuronal morphology, and severe intestinal smooth muscle disruption. Genetic chimeras revealed that Uhrf1 functions both cell-autonomously in enteric neuron progenitors and cell-non-autonomously in surrounding intestinal cells. Uhrf1 recruits the DNA methyltransferase Dnmt1 to unmethylated DNA during replication. Dnmt1 is also expressed in enteric neuron and smooth muscle progenitors. dnmt1 mutants show a strong reduction in enteric neuron number and disrupted intestinal smooth muscle. Because dnmt1;uhrf1 double mutants have a similar phenotype to dnmt1 and uhrf1 single mutants, Dnmt1 and Uhrf1 must function together during enteric neuron and intestinal muscle development. This work shows that genes controlling epigenetic modifications are important in coordinating intestinal tract development, provides the first demonstration that these genes are important in ENS development, and advances uhrf1 and dnmt1 as potential new Hirschsprung disease candidates.\n\nSummaryThis work provides evidence that DNA methylation factors are important in all cell types that contribute to development of a functional intestine.

developmental biology

SWI/SNF chromatin remodeling controls Notch-responsive enhancer accessibility

Notch signaling plays a key role in many cell fate decisions during development by directing different gene expression programs via the transcription factor CSL, known as Su(H) in Drosophila. Which target genes are responsive to Notch signaling is influenced by the chromatin state of enhancers, yet how this is regulated is not fully known. Detecting an increase in the histone variant H3.3 in response to Notch signaling, we tested which chromatin remodelers or histone chaperones were required for the changes in enhancer accessibility to Su(H) binding. This revealed a crucial role for the Brahma SWI/SNF chromatin remodeling complex in conferring enhancer accessibility and enabling the transcriptional response. The Notch-responsive regions had high levels of nucleosome turnover which were dependent on the Brahma complex, increased with Notch signaling and primarily involved histone H3.3. Together these results highlight the importance of SWI/SNF-mediated nucleosome turnover in rendering enhancers responsive to Notch.

developmental biology

EGFR SIGNALING COORDINATES PATTERNING WITH CELL SURVIVAL DURING DROSOPHILA EPIDERMAL DEVELOPMENT

Extensive apoptosis is often seen in patterning mutants, suggesting that tissues can detect and eliminate potentially harmful mis-specified cells. Here we show that the pattern of apoptosis in the embryonic epidermis of Drosophila is not a response to fate mis-specification but can instead be explained by the limiting availability of pro-survival signalling molecules released from locations determined by patterning information. In wild type embryos, the segmentation cascade elicits the segmental production of several EGFR ligands, including the TGF-alpha, Spitz and the Neuregulin, Vein. This leads to an undulating pattern of signalling activity, which prevents expression of the pro-apoptotic gene hid throughout the epidermis. In segmentation mutants, where specific peaks of EGFR ligands fail to form, gaps in signalling activity appear, leading to coincident hid upregulation and subsequent cell death. These data provide a mechanistic understanding of how cell survival, and thus appropriate tissue size, is made contingent on correct patterning.

developmental biology

Basolateral localization of MMP14 drives apicobasal polarity change during EMT independently of its catalytic activity

The transmembrane Matrix Metalloproteinase MMP14/MT1-MMP is known to promote cell migration by cleavage of the extracellular matrix. To initiate migration, epithelial cells need to gain mesenchymal attributes. They reduce cell-cell junctions and apicobasal polarity and gain migratory capabilities. This process is named epithelial-mesenchymal transition (EMT). MMP14s implication in EMT is still ill-defined. We used chick neural crest (NC) cells as a model to explore the function of MMP14 in physiological EMT. Our results show that MMP14 is expressed by chick NC cells. However, it is its subcellular localization, rather than its expression, that correlates with EMT. MMP14 is first apical and switches to basolateral domains during EMT. Loss of function and rescue experiments show that MMP14 is involved in EMT independently of its catalytic activity. It lies downstream of pro-EMT genes and upstream of cell polarity. We found that basolateral localization of MMP14 is required and sufficient to induce polarity change in NC cells and neuroepithelial cells, respectively. These effects on polarity occur without impact on cell-cell adhesion or the extracellular matrix. Overall, our data points to a new function of MMP14 in EMT that will need to be further explored in other systems such as cancer cells.

developmental biology

Over-expression of the photoperiod response regulator ZmCCT10 modifies plant architecture, flowering time and inflorescence morphology in maize

Maize originated as a tropical plant that required short days to transition from vegetative to reproductive development. ZmCCT10 [CO, CONSTANS, CO-LIKE and TIMING OF CAB1 (CCT) transcription factor family] is a photoperiod regulator and was identified as a major QTL controlling photoperiod sensitivity in maize. We modulated expression of ZmCCT10 in transgenic maize using two constitutive promoters which cause differing expression levels. Transgenic plants over expressing ZmCCT10 with either promoter was delayed in their transition from vegetative to reproductive development but were not affected in their change from juvenile-to-adult vegetative growth. Strikingly, transgenic plants containing the stronger expressing construct had a very prolonged period of vegetative growth accompanied with dramatic modifications to plant architecture that impacted both vegetative and reproductive traits. These plants did not produced ears, but tassels were heavily branched, and more than half of the transgenic plants showed conversion of shoot apices into \"bushy tops\", which were composed of vegetative reversion plantlets. Analysis of expression modules controlling the floral transition and meristem identity linked these networks to photoperiod dependent regulation, whereas phase change modules appeared to be photoperiod independent. Results from this study clarified the influence of the photoperiod pathway on vegetative and reproductive development and allowed to fine-tune the flowering time model for maize.

developmental biology

Wnt/Fgf crosstalk is required for the specification of tracheal basal progenitor cells

Basal progenitor cells are critical for the establishment and maintenance of the tracheal epithelium. However, it remains unclear how these progenitor cells are specified during foregut development. Here, we found that ablation of the Wnt chaperon protein Gpr177 (also known as Wntless) in the epithelium causes significant reduction in the numbers of basal progenitor cells accompanied by cartilage loss in Shh-Cre;Gpr177loxp/loxp mutants. Consistent with the association between cartilage and basal cell development, Nkx2.1+p63+ basal cells are co-present with cartilage nodules in Shh-Cre;Ctnnb1DM/loxp mutants which keep partial cell-cell adhesion but not the transcription regulation function of {beta}-catenin. More importantly, deletion of Ctnnb1 in the mesenchyme leads to the loss of basal cells and cartilage concomitant with the reduced transcript levels of Fgf10 in Dermo1-Cre;Ctnnb1loxp/loxp mutants. Furthermore, deletion of Fgf receptor 2 (Fgfr2) in the epithelium also leads to significantly reduced numbers of basal cells, supporting the importance of the Wnt/Fgf crosstalk in early tracheal development.

developmental biology

Sensory neurons control heritable adaptation to stress through germline reprogramming

Maternal neuronal signaling has been reported to program adaptive changes in offspring physiology in diverse organisms [1, 2]. However, the mechanisms for the inheritance of adaptive maternal effects through the germline are largely unknown. In the nematode Auanema freiburgensis, stress-resistance and sex of the offspring depend on environmental cues experienced by the mother. Maternal sensing of high population densities results in the production of stress-resistant larvae (dauers) that develop into hermaphrodites. Ablation of the maternal ASH chemosensory neurons results only in non-dauer offspring that develop into males or females. High population densities correlate with changes in the methylation status of H3K4 and H3K9 in the maternal germline. Inhibition of JMJD histone demethylases prevents mothers from producing dauers and hermaphrodite offspring in high-density conditions. Our results demonstrate a case of soma-to-germline transmission of environmental information that influences the phenotype of the following generation through changes in histone modifications of the maternal germline.\n\nHighlightsO_LIHigh population density leads to the production of hermaphrodite offspring.\nC_LIO_LIThe ASH neuron in the hermaphrodite mother senses population density.\nC_LIO_LIHistone modifications in the maternal germline correlate with the sex of offspring.\nC_LIO_LIInhibition of histone demethylases results in female offspring in all conditions.\nC_LI

developmental biology

A screen for targets of the Drosophila pseudokinase Tribbles identifies Neuralized and Mindbomb, ubiquitin ligases that mediate Notch signaling

Drosophila Tribbles (Trbl) is the founding member of a family of pseudokinases with conserved roles in antagonizing cell division, tissue growth and cell differentiation. In humans, three Tribbles isoforms serve as adaptor proteins, binding targets such as Cdc25 phosphatase, Akt kinase or the transcription factor C/EBP to block their activity or direct their proteosomal degradation. Mutations in Tribbles family members are associated with susceptibility to diabetes and cancer, notably Notch-induced tumor growth. Trbl misexpression in the fly wing disk leads to a block in mitosis associated with decreased levels of String/Cdc25 and increased levels of Cyclin B leading to reduced overall wing size and reduced trichome density. We show these Trbl growth-restricting phenotypes can be suppressed by manipulating levels of known Trbl targets, and use this sensitized wing system to screen a collection of growth regulating open reading frames (ORFs) to search for enhancers and suppressors affecting cell and tissue size. By precisely measuring morphometric changes in wing phenotypes using a computer-based tool, we detected synthetic interactions with several E3 ubiquitin ligases, and focused our analysis on the Notch pathway components Neuralized (Neur) and Mindbomb1 (Mib1). In the wing, notum and egg chamber epithelia, Trbl misexpression suppressed Neur and Mib1 activities and stabilized the accumulation of both proteins. To understand these interactions, we used yeast two-hybrid assays to show Trbl physically bound to both Neur and Mib1. Our data are consistent with published reports that mammalian Tribbles3 modulates Notch responses by binding and stabilizing Mindbomb and indicate that a wing misexpression approach is useful to identify novel components in a conserved Tribbles signaling pathway.\n\nAUTHOR SUMMARYTribbles pseudokinases are adaptor molecules, binding diverse targets regulating cell differentiation, growth and proliferation and directing their proteasomal degradation. To search for novel targets of Drosophila Tribbles, we adopted a wing co-misexpression scheme and measured changes in cell/tissue size to identify enhancers and suppressors of the Tribbles phenotype. We show the Notch pathway components Neuralized and Mindbomb1 E3 ligases act as Tribbles suppressors and demonstrate that Tribbles modulates their levels and activites. Recent demonstration that mammalian Tribbles 3 binds the E3 ligase Mindbomb to promote ligand-mediated Notch activation implies a conserved role for Tribbles family members in Notch activation.

developmental biology

WNT ligands stimulate transient signaling in human pluripotent cells and synergize with TGF-β ligands to stimulate sustained signaling during differentiation

WNT/{beta}-catenin signaling is crucial to all stages of life. It controls early morphogenetic events in embryos, maintains stem-cell niches in adults, and is disregulated in many types of cancer. Despite its ubiquity, little is known about the dynamics of signal transduction or whether it varies across contexts. Here we probe the dynamics of signaling by monitoring nuclear accumulation of {beta}-catenin, the primary transducer of canonical WNT signals, using quantitative live-cell imaging. We show that {beta}-catenin signaling responds adaptively to constant WNT signaling in pluripotent stem cells, and that these dynamics become sustained upon differentiation. Varying dynamics were also observed in the response to WNT in commonly used mammalian cell-lines. Signal attenuation in pluripotent cells is controlled by both intra- and extra-cellular negative regulation of WNT signaling. TGF{beta}-superfamily ligands Activin and BMP, which coordinate with WNT signaling to pattern the gastrula, increase the {beta}-catenin response in a manner independent of their ability to induce new WNT-ligand production. Our results reveal how variables external to the pathway, including differentiation status and crosstalk with other pathways, dramatically alter WNT/{beta}-catenin dynamics.

developmental biology

Reverse genetic screen reveals that Il34 facilitates yolk sac macrophage distribution and seeding of the brain

Microglia are brain resident macrophages, which have specialized functions important in brain development and in disease. They colonize the brain in early embryonic stages, but few factors that drive the migration of yolk sac macrophages into the embryonic brain, or regulate their acquisition of specialized properties are currently known.\n\nHere, we present a CRISPR/Cas9-based in vivo reverse genetic screening pipeline to identify new regulators important for microglia development using zebrafish. Zebrafish larvae are particularly suitable due to their external development, transparency, high fecundity and conserved microglia features. We targeted putative microglia regulators, including signature genes and non-cell autonomous factors, by Cas9/gRNA-complex injections, followed by neutral red-based visualization of microglia. Microglia were quantified automatically in 3-day-old larvae using a software tool we called SpotNGlia. We identified that loss of function of the zebrafish homolog of the colony stimulating factor 1 receptor (CSF1R) ligand IL34, caused strongly reduced microglia numbers in early development. Previous studies on the role of the IL34 on microglia development in vivo were ambiguous. Our data, and a concurrent paper, show that in zebrafish, il34 is required during the earliest seeding of the brain by microglia progenitors. Our data also indicate that Il34 is required for distribution to other organs.\n\nPreviously, we showed that csf1ra and csf1rb double mutant zebrafish have no microglia. As there is a moderate effect of il34 on microglia development, relative to the effect csf1r, additional Csf1r-dependent signalling may be needed for establishment of the microglia network. In all, we identified il34 as a modifier of microglia colonization, by affecting distribution of yolk sac macrophages to target organs, validating our reverse genetic screening pipeline in zebrafish which can be used for the identification of additional regulators of microglia development.

developmental biology

Draxin alters laminin expression during basement membrane reorganization to control cranial neural crest EMT

Premigratory neural crest cells arise within the dorsal neural tube and subsequently undergo an epithelial-to-mesenchymal transition (EMT) to leave the neuroepithelium and initiate migration. Draxin is a Wnt modulator that has been shown to control the timing of cranial neural crest EMT. Here we show that this process is accompanied by three stages of remodeling of the basement membrane protein laminin, from regression to expansion and channel formation. Loss of Draxin results in blocking laminin remodeling at the regression stage, whereas ectopic maintenance of Draxin blocks remodeling at the expansion stage. The latter effect is rescued by addition of Snail2, previously shown to be downstream of Draxin. Our results demonstrate an essential function for the Wnt modulator Draxin in regulating basement membrane remodeling during cranial neural crest EMT.\n\nHIGHLIGHTSO_LICranial neural crest migrate through a laminin-rich basement membrane channel\nC_LIO_LIPerturbation of Draxin, a Wnt antagonist, alters laminin channel formation\nC_LIO_LIDraxins effect on laminin channel formation is largely mediated by Snail2\nC_LI

developmental biology

The Ciliopathy Gene Ftm/Rpgrip1l Controls Mouse Forebrain Patterning Via Region-Specific Modulation Of Hedgehog/Gli Signaling.

Primary cilia are essential for central nervous system development. In the mouse, they play a critical role in patterning the spinal cord and telencephalon via the regulation of Hedgehog/Gli signaling. However, despite the frequent disruption of this signaling pathway in human forebrain malformations, the role of primary cilia in forebrain morphogenesis has been little investigated outside the telencephalon. Here we studied development of the diencephalon, hypothalamus and eyes in mutant mice in which the Ftm/Rgprip1l ciliopathy gene is disrupted. At the end of gestation, Ftm-/- fetuses displayed anophthalmia, a reduction of the ventral hypothalamus and a disorganization of diencephalic nuclei and axonal tracts. In Ftm-/- embryos, we found that the ventral forebrain structures and the rostral thalamus were missing. Optic vesicles formed but lacked the optic cups. We analyzed the molecular causes of these defects. In Ftm-/- embryos, Sonic hedgehog (Shh) expression was lost in the ventral forebrain but maintained in the zona limitans intrathalamica (ZLI), the mid-diencephalic organizer. In the diencephalon, Gli activity was dampened in regions adjacent to the Shh-expressing ZLI but displayed a higher Hh-independent ground level in the other regions. Our data uncover a complex role of cilia in development of the diencephalon, hypothalamus and eyes via the region-specific control of the ratio of activator and repressor forms of the Gli transcription factors. They call for a closer examination of forebrain defects in severe ciliopathies and for a search for ciliopathy genes as modifiers in other human conditions with forebrain defects.

developmental biology

Specific detection of cell-free DNA derived from intestinal epithelial cells using methylation patterns

Epithelial cells of the intestine undergo rapid turnover and are thought to be cleared via stool. Disruption of tissue architecture, as occurs in colorectal cancer (CRC), results in the release of material from dying intestinal epithelial cells to blood. This phenomenon could be utilized for diagnosis and monitoring of intestinal diseases, if circulating cell-free DNA (cfDNA) derived from intestinal cells could be specifically identified. Here we describe two genomic loci that are unmethylated specifically in intestinal epithelial cells, allowing for sensitive and specific detection of DNA derived from such cells. As expected, intestinal DNA is found in stool, but not in plasma, of healthy individuals. Patients with inflammatory bowel disease (IBD) have minimal amounts of intestinal cfDNA in the plasma, whereas patients with advanced CRC show a strong signal. The intestinal markers are not elevated in plasma samples from patients with pancreatic ductal adenocarcinoma (PDAC), and a combination of intestine- and pancreas-specific markers allowed for robust differentiation between plasma cfDNA derived from CRC and PDAC patients. Intestinal DNA markers provide a mutation-independent tool for monitoring intestinal dynamics in health and disease.

developmental biology

Loss of TLE3 Promotes Mitochondrial Program in Beige Adipocytes and Improves Glucose Metabolism

Prolonged cold exposure stimulates the recruitment of beige adipocytes within white adipose tissue. Beige adipocytes depend on mitochondrial oxidative phosphorylation to drive thermogenesis. The transcriptional mechanisms that promote remodeling in adipose tissue are not well understood. Here we demonstrate that the transcriptional coregulator TLE3 is induced with aging and inhibits mitochondrial gene expression in beige adipocytes. Conditional deletion of TLE3 in adipocytes prevents age- and diet-induced weight gain by promoting mitochondrial oxidative metabolism and increasing energy expenditure, thereby improving glucose control. Using chromatin immunoprecipitation and deep sequencing we found that TLE3 occupies distal enhancers in proximity to nuclear-encoded mitochondrial genes and that many of these enhancers are also enriched for EBF transcription factors. TLE3 interacts with EBF2 and blocks its ability to promote the thermogenic transcriptional program. Collectively, these studies demonstrate that TLE3 mediates age-dependent beige adipose thermogenic decline through inhibition of EBF2 transcriptional activity. Inhibition of TLE3 may provide a novel therapeutic approach for obesity and diabetes.

developmental biology

Maternal Circulating MicroRNAs Control the Placental Response to Alcohol

Prenatal Alcohol exposure (PAE), like other pregnancy complications, can result in placental insufficiency and fetal growth restriction, though the linking causal mechanisms are unclear. We previously identified 11 gestationally-elevated maternal circulating miRNAs that predicted infant growth deficits following PAE. Here, we investigated whether these HEamiRNAs contribute to the pathology of PAE, by inhibiting trophoblast epithelial-mesenchymal transition (EMT), a pathway critical for placental development. We now report for the first time, that PAE inhibits expression of placental pro-EMT pathway members in both rodents and primates, and that HEamiRNAs collectively, but not individually, mediate placental EMT inhibition. HEamiRNAs collectively, but not individually, also inhibited cell proliferation and the EMT pathway in cultured trophoblasts, while inducing cell stress, and following trophoblast syncytialization, aberrant endocrine maturation. Moreover, a single intra-vascular administration of the pooled murine-expressed HEamiRNAs, to pregnant mice, decreased placental and fetal growth and inhibited expression of pro-EMT transcripts in placenta. Our data suggests that HEamiRNAs collectively interfere with placental development, contributing to the pathology of PAE, and perhaps also, to other causes of fetal growth restriction. SummaryMaternal gestational circulating microRNAs, predictive of adverse infant outcomes including growth deficits, following prenatal alcohol exposure, contribute to placental pathology by impairing the EMT pathway in trophoblasts.

developmental biology