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Development recent issues

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In vivo imaging: shining a light on stem cells in the living animal [SPOTLIGHT]


Phong Dang Nguyen and Peter David Currie

Stem cells are undifferentiated cells that play crucial roles during development, growth and regeneration. Traditionally, these cells have been primarily characterised by histology, cell sorting, cell culture and ex vivo methods. However, as stem cells interact in a complex environment within specific tissue niches, there has been increasing interest in examining their in vivo behaviours, particularly in response to injury. Advances in imaging technologies and genetic tools have converged to enable unprecedented access to the endogenous stem cell niche. In this Spotlight article, we highlight how in vivo imaging can probe a range of biological processes that relate to stem cell activity, behaviour and control.

The ontogeny, activation and function of the epicardium during heart development and regeneration [REVIEW]


Filipa C. Simoes and Paul R. Riley

The epicardium plays a key role during cardiac development, homeostasis and repair, and has thus emerged as a potential target in the treatment of cardiovascular disease. However, therapeutically manipulating the epicardium and epicardium-derived cells (EPDCs) requires insights into their developmental origin and the mechanisms driving their activation, recruitment and contribution to both the embryonic and adult injured heart. In recent years, studies of various model systems have provided us with a deeper understanding of the microenvironment in which EPDCs reside and emerge into, of the crosstalk between the multitude of cardiovascular cell types that influence the epicardium, and of the genetic programmes that orchestrate epicardial cell behaviour. Here, we review these discoveries and discuss how technological advances could further enhance our knowledge of epicardium-based repair mechanisms and ultimately influence potential therapeutic outcomes in cardiovascular regenerative medicine.

Head formation requires Dishevelled degradation that is mediated by March2 in concert with Dapper1 [RESEARCH ARTICLE]


Hyeyoon Lee, Seong-Moon Cheong, Wonhee Han, Youngmu Koo, Saet-Byeol Jo, Gun-Sik Cho, Jae-Seong Yang, Sanguk Kim, and Jin-Kwan Han

Dishevelled (Dvl/Dsh) is a key scaffold protein that propagates Wnt signaling essential for embryogenesis and homeostasis. However, whether the antagonism of Wnt signaling that is necessary for vertebrate head formation can be achieved through regulation of Dsh protein stability is unclear. Here, we show that membrane-associated RING-CH2 (March2), a RING-type E3 ubiquitin ligase, antagonizes Wnt signaling by regulating the turnover of Dsh protein via ubiquitin-mediated lysosomal degradation in the prospective head region of Xenopus. We further found that March2 acquires regional and functional specificities for head formation from the Dsh-interacting protein Dapper1 (Dpr1). Dpr1 stabilizes the interaction between March2 and Dsh in order to mediate ubiquitylation and the subsequent degradation of Dsh protein only in the dorso-animal region of Xenopus embryo. These results suggest that March2 restricts cytosolic pools of Dsh protein and reduces the need for Wnt signaling in precise vertebrate head development.

The heart tube forms and elongates through dynamic cell rearrangement coordinated with foregut extension [RESEARCH ARTICLE]


Hinako Kidokoro, Sayuri Yonei-Tamura, Koji Tamura, Gary C. Schoenwolf, and Yukio Saijoh

In the initiation of cardiogenesis, the heart primordia transform from bilateral flat sheets of mesoderm into an elongated midline tube. Here, we discover that this rapid architectural change is driven by actomyosin-based oriented cell rearrangement and resulting dynamic tissue reshaping (convergent extension, CE). By labeling clusters of cells spanning the entire heart primordia, we show that the heart primordia converge toward the midline to form a narrow tube, while extending perpendicularly to rapidly lengthen it. Our data for the first time visualize the process of early heart tube formation from both the medial (second) and lateral (first) heart fields, revealing that both fields form the early heart tube by essentially the same mechanism. Additionally, the adjacent endoderm coordinately forms the foregut through previously unrecognized movements that parallel those of the heart mesoderm and elongates by CE. In conclusion, our data illustrate how initially two-dimensional flat primordia rapidly change their shapes and construct the three-dimensional morphology of emerging organs in coordination with neighboring morphogenesis.

Xenopus ADAM19 regulates Wnt signaling and neural crest specification by stabilizing ADAM13 [RESEARCH ARTICLE]


Jiejing Li, Mark Perfetto, Russell Neuner, Harinath Bahudhanapati, Laura Christian, Ketan Mathavan, Lance C. Bridges, Dominique Alfandari, and Shuo Wei

During vertebrate gastrulation, canonical Wnt signaling induces the formation of neural plate border (NPB). Wnt is also thought to be required for the subsequent specification of neural crest (NC) lineage at the NPB, but the direct evidence is lacking. We found previously that the disintegrin metalloproteinase ADAM13 is required for Wnt activation and NC induction in Xenopus. Here, we report that knockdown of ADAM13 or its close paralog ADAM19 severely downregulates Wnt activity at the NPB, inhibiting NC specification without affecting earlier NPB formation. Surprisingly, ADAM19 functions nonproteolytically in NC specification by interacting with ADAM13 and inhibiting its proteasomal degradation. Ectopic expression of stabilized ADAM13 mutants that function independently of ADAM19 can induce the NC marker/specifier snail2 in the future epidermis via Wnt signaling. These results unveil the essential roles of a novel protease-protease interaction in regulating a distinct wave of Wnt signaling, which directly specifies the NC lineage.

Disrupting the three-dimensional regulatory topology of the Pitx1 locus results in overtly normal development [RESEARCH ARTICLE]


Richard Sarro, Acadia A. Kocher, Deena Emera, Severin Uebbing, Emily V. Dutrow, Scott D. Weatherbee, Timothy Nottoli, and James P. Noonan

Developmental gene expression patterns are orchestrated by thousands of distant-acting transcriptional enhancers. However, identifying enhancers essential for the expression of their target genes has proven challenging. Maps of long-range regulatory interactions may provide the means to identify enhancers crucial for developmental gene expression. To investigate this hypothesis, we used circular chromosome conformation capture coupled with interaction maps in the mouse limb to characterize the regulatory topology of Pitx1, which is essential for hindlimb development. We identified a robust hindlimb-specific interaction between Pitx1 and a putative hindlimb-specific enhancer. To interrogate the role of this interaction in Pitx1 regulation, we used genome editing to delete this enhancer in mouse. Although deletion of the enhancer completely disrupts the interaction, Pitx1 expression in the hindlimb is only mildly affected, without any detectable compensatory interactions between the Pitx1 promoter and potentially redundant enhancers. Pitx1 enhancer null mice did not exhibit any of the characteristic morphological defects of the Pitx1–/– mutant. Our results suggest that robust, tissue-specific physical interactions at essential developmental genes have limited predictive value for identifying enhancer mutations with strong loss-of-function phenotypes.

The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye [RESEARCH ARTICLE]


Hong Duan, Luis F. de Navas, Fuqu Hu, Kailiang Sun, Yannis E. Mavromatakis, Kayla Viets, Cyrus Zhou, Joshua Kavaler, Robert J. Johnston, Andrew Tomlinson, and Eric C. Lai

Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling mediated by Epidermal growth factor receptor (EGFR) and the Sevenless (Sev) receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently derepresses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev, which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that involves substantial miRNA control.

Conserved functional control, but distinct regulation, of cell proliferation in rice and Arabidopsis leaves revealed by comparative analysis of GRF-INTERACTING FACTOR 1 orthologs [RESEARCH ARTICLE]


Satomi Shimano, Ken-ichiro Hibara, Tomoyuki Furuya, Shin-ichi Arimura, Hirokazu Tsukaya, and Jun-Ichi Itoh

Regulation of cell proliferation is crucial for establishing the shape of plant leaves. We have identified MAKIBA3 (MKB3), a loss-of-function mutant of which exhibits a narrowed- and rolled-leaf phenotype in rice. MKB3 was found to be an ortholog of Arabidopsis ANGUSTIFOLIA3 (AN3), which positively regulates cell proliferation. The reduced leaf size of mkb3 plants with enlarged cells and the increased size of MKB3-overexpressing leaves with normal-sized cells indicate that MKB3 is a positive regulator of leaf proliferation and that mkb3 mutation triggers a compensation syndrome, as does Arabidopsis an3. Expression analysis revealed that MKB3 is predominantly expressed on the epidermis of leaf primordia, which is different from the location of AN3. A protein movement assay demonstrated that MKB3 moves from an MKB3-expressing domain to a non-expressing domain, which is required for normal leaf development. Our results suggest that rice MKB3 and Arabidopsis AN3 have conserved functions and effects on leaf development. However, the expression pattern of MKB3 and direction of protein movement are different between rice and Arabidopsis, which might reflect differences in leaf primordia development in these two species.

Imprinted gene dysregulation in a Tet1 null mouse model is stochastic and variable in the germline and offspring [RESEARCH ARTICLE]


Jennifer M. SanMiguel, Lara K. Abramowitz, and Marisa S. Bartolomei

Imprinted genes are expressed from one parental allele and regulated by differential DNA methylation at imprinting control regions (ICRs). ICRs are reprogrammed in the germline through erasure and re-establishment of DNA methylation. Although much is known about DNA methylation establishment, DNA demethylation is less well understood. Recently, the Ten-Eleven Translocation proteins (TET1-3) have been shown to initiate DNA demethylation, with Tet1–/– mice exhibiting aberrant levels of imprinted gene expression and ICR methylation. Nevertheless, the role of TET1 in demethylating ICRs in the female germline and in controlling allele-specific expression remains unknown. Here, we examined ICR-specific DNA methylation in Tet1–/– germ cells and ascertained whether abnormal ICR methylation impacted imprinted gene expression in F1 hybrid somatic tissues derived from Tet1–/– eggs or sperm. We show that Tet1 deficiency is associated with hypermethylation of a subset of ICRs in germ cells. Moreover, ICRs with defective germline reprogramming exhibit aberrant DNA methylation and biallelic expression of linked imprinted genes in somatic tissues. Thus, we define a discrete set of genomic regions that require TET1 for germline reprogramming and discuss mechanisms for stochastic imprinting defects.

L(3)mbt and the LINT complex safeguard cellular identity in the Drosophila ovary [RESEARCH ARTICLE]


Remi-Xavier Coux, Felipe Karam Teixeira, and Ruth Lehmann

Maintenance of cellular identity is essential for tissue development and homeostasis. At the molecular level, cell identity is determined by the coordinated activation and repression of defined sets of genes. The tumor suppressor L(3)mbt has been shown to secure cellular identity in Drosophila larval brains by repressing germline-specific genes. Here, we interrogate the temporal and spatial requirements for L(3)mbt in the Drosophila ovary, and show that it safeguards the integrity of both somatic and germline tissues. l(3)mbt mutant ovaries exhibit multiple developmental defects, which we find to be largely caused by the inappropriate expression of a single gene, nanos, a key regulator of germline fate, in the somatic ovarian cells. In the female germline, we find that L(3)mbt represses testis-specific and neuronal genes. At the molecular level, we show that L(3)mbt function in the ovary is mediated through its co-factor Lint-1 but independently of the dREAM complex. Together, our work uncovers a more complex role for L(3)mbt than previously understood and demonstrates that L(3)mbt secures tissue identity by preventing the simultaneous expression of original identity markers and tissue-specific misexpression signatures.

Evolutionarily conserved anterior expansion of the central nervous system promoted by a common PcG-Hox program [RESEARCH ARTICLE]


Behzad Yaghmaeian Salmani, Ignacio Monedero Cobeta, Jonathan Rakar, Susanne Bauer, Jesus Rodriguez Curt, Annika Starkenberg, and Stefan Thor

A conserved feature of the central nervous system (CNS) is the prominent expansion of anterior regions (brain) compared with posterior (nerve cord). The cellular and regulatory processes driving anterior CNS expansion are not well understood in any bilaterian species. Here, we address this expansion in Drosophila and mouse. We find that, compared with the nerve cord, the brain displays extended progenitor proliferation, more elaborate daughter cell proliferation and more rapid cell cycle speed in both Drosophila and mouse. These features contribute to anterior CNS expansion in both species. With respect to genetic control, enhanced brain proliferation is severely reduced by ectopic Hox gene expression, by either Hox misexpression or by loss of Polycomb group (PcG) function. Strikingly, in PcG mutants, early CNS proliferation appears to be unaffected, whereas subsequent brain proliferation is severely reduced. Hence, a conserved PcG-Hox program promotes the anterior expansion of the CNS. The profound differences in proliferation and in the underlying genetic mechanisms between brain and nerve cord lend support to the emerging concept of separate evolutionary origins of these two CNS regions.

Polycomb group (PcG) proteins and Pax6 cooperate to inhibit in vivo reprogramming of the developing Drosophila eye [RESEARCH ARTICLE]


Jinjin Zhu, Alison J. Ordway, Lena Weber, Kasun Buddika, and Justin P. Kumar

How different cells and tissues commit to and determine their fates has been a central question in developmental biology since the seminal embryological experiments conducted by Wilhelm Roux and Hans Driesch in sea urchins and frogs. Here, we demonstrate that Polycomb group (PcG) proteins maintain Drosophila eye specification by suppressing the activation of alternative fate choices. The loss of PcG in the developing eye results in a cellular reprogramming event in which the eye is redirected to a wing fate. This fate transformation occurs with either the individual loss of Polycomb proteins or the simultaneous reduction of the Pleiohomeotic repressive complex and Pax6. Interestingly, the requirement for retinal selector genes is limited to Pax6, as the removal of more downstream members does not lead to the eye-wing transformation. We also show that distinct PcG complexes are required during different developmental windows throughout eye formation. These findings build on earlier observations that the eye can be reprogrammed to initiate head epidermis, antennal and leg development.

PDGF-A signaling is required for secondary alveolar septation and controls epithelial proliferation in the developing lung [RESEARCH ARTICLE]


Leonor Gouveia, Christer Betsholtz, and Johanna Andrae

Platelet-derived growth factor A (PDGF-A) signaling through PDGF receptor α is essential for alveogenesis. Previous studies have shown that Pdgfa–/– mouse lungs have enlarged alveolar airspace with absence of secondary septation, both distinctive features of bronchopulmonary dysplasia. To study how PDGF-A signaling is involved in alveogenesis, we generated lung-specific Pdgfa knockout mice (Pdgfafl/–; Spc-cre) and characterized their phenotype postnatally. Histological differences between mutant mice and littermate controls were visible after the onset of alveogenesis and maintained until adulthood. Additionally, we generated Pdgfafl/–; Spc-cre; PdgfraGFP/+ mice in which Pdgfra+ cells exhibit nuclear GFP expression. In the absence of PDGF-A, the number of PdgfraGFP+ cells was significantly decreased. In addition, proliferation of PdgfraGFP+ cells was reduced. During alveogenesis, PdgfraGFP+ myofibroblasts failed to form the α-smooth muscle actin rings necessary for alveolar secondary septation. These results indicate that PDGF-A signaling is involved in myofibroblast proliferation and migration. In addition, we show an increase in both the number and proliferation of alveolar type II cells in Pdgfafl/–; Spc-cre lungs, suggesting that the increased alveolar airspace is not caused solely by deficient myofibroblast function.

Evolutionary divergence of the sex-determining gene MID uncoupled from the transition to anisogamy in volvocine algae [RESEARCH ARTICLE]


Sa Geng, Ayano Miyagi, and James G. Umen

Volvocine algae constitute a unique comparative model for investigating the evolution of oogamy from isogamous mating types. The sex- or mating type-determining gene MID encodes a conserved RWP-RK transcription factor found in either the MT– or male mating locus of dioecious volvocine species. We previously found that MID from the isogamous species Chlamydomonas reinhardtii (CrMID) could not induce ectopic spermatogenesis when expressed heterologously in Volvox carteri females, suggesting coevolution of Mid function with gamete dimorphism. Here we found that ectopic expression of MID from the anisogamous species Pleodorina starrii (PsMID) could efficiently induce spermatogenesis when expressed in V. carteri females and, unexpectedly, that GpMID from the isogamous species Gonium pectorale was also able to induce V. carteri spermatogenesis. Neither VcMID nor GpMID could complement a C. reinhardtii mid mutant, at least partly owing to instability of heterologous Mid proteins. Our data show that Mid divergence was not a major contributor to the transition between isogamy and anisogamy/oogamy in volvocine algae, and instead implicate changes in cis-regulatory interactions and/or trans-acting factors of the Mid network in the evolution of sexual dimorphism.

Regulation and function of H3K36 di-methylation by the trithorax-group protein complex AMC [RESEARCH ARTICLE]


Sigrun Schmähling, Arno Meiler, Yoonjung Lee, Arif Mohammed, Katja Finkl, Katharina Tauscher, Lars Israel, Marc Wirth, Julia Philippou-Massier, Helmut Blum, Bianca Habermann, Axel Imhof, Ji-Joon Song, and Jürg Müller

The Drosophila Ash1 protein is a trithorax-group (trxG) regulator that antagonizes Polycomb repression at HOX genes. Ash1 di-methylates lysine 36 in histone H3 (H3K36me2) but how this activity is controlled and at which genes it functions is not well understood. We show that Ash1 protein purified from Drosophila exists in a complex with MRG15 and Caf1 that we named AMC. In Drosophila and human AMC, MRG15 binds a conserved FxLP motif near the Ash1 SET domain and stimulates H3K36 di-methylation on nucleosomes. Drosophila MRG15-null and ash1 catalytic mutants show remarkably specific trxG phenotypes: stochastic loss of HOX gene expression and homeotic transformations in adults. In mutants lacking AMC, H3K36me2 bulk levels appear undiminished but H3K36me2 is reduced in the chromatin of HOX and other AMC-regulated genes. AMC therefore appears to act on top of the H3K36me2/me3 landscape generated by the major H3K36 methyltransferases NSD and Set2. Our analyses suggest that H3K36 di-methylation at HOX genes is the crucial physiological function of AMC and the mechanism by which the complex antagonizes Polycomb repression at these genes.

PUF-8 facilitates homologous chromosome pairing by promoting proteasome activity during meiotic entry in C. elegans [RESEARCH ARTICLE]


Ganga Anil Kumar and Kuppuswamy Subramaniam

Pairing of homologous chromosomes is essential for genetic recombination during gametogenesis. In many organisms, chromosome ends are attached to cytoplasmic dynein, and dynein-driven chromosomal movements facilitate the pairing process. Factors that promote or control the cytoskeletal tethering of chromosomes are largely unknown. Here, we show that the conserved RNA-binding protein PUF-8 facilitates the tethering and pairing processes in the C. elegans germline by promoting proteasome activity. We have isolated a hypomorphic allele of pas-1, which encodes a proteasome core subunit, and find that the homologous chromosomes fail to pair in the puf-8; pas-1 double mutant due to failure of chromosome tethering. Our results reveal that the puf-8; pas-1 meiotic defects are caused by the loss of proteasome activity. The axis component HTP-3 accumulates prematurely in the double mutant, and reduction of its activity partially suppresses some of the puf-8; pas-1 meiotic defects, suggesting that HTP-3 might be an important target of the proteasome in promoting early meiotic events. In summary, our results reveal a role for the proteasome in chromosome tethering and identify PUF-8 as a regulator of proteasome activity during early meiosis.

A new Editor-in-Chief for Development [EDITORIAL]


Sarah Bray, Kate Storey, and Katherine Brown

An interview with Susan Strome [SPOTLIGHT]


Aidan Maartens

Susan Strome is Distinguished Professor of Molecular, Cell and Developmental Biology at the University of California, Santa Cruz, USA. Recently appointed an editor at Development, her lab studies the regulation of germ cell development in C. elegans, with a particular focus on the epigenetic transmission of chromatin states. We caught up with Susan to discuss her early career switch from prokaryotes to worms, her experiences of small and big science, and why teaching is so important to her.

An interview with James Briscoe [SPOTLIGHT]


Katherine Brown

James Briscoe is a group leader at The Francis Crick Institute in London. His lab's research focusses on the developing vertebrate spinal cord, with a particular interest in how sonic hedgehog gradients, and the downstream signal transduction and transcriptional networks, regulate the development of this tissue. In September 2018, James will become the new Editor-in-Chief of Development. We met with James to discuss his career and research interests, the importance of interdisciplinary thinking in developmental biology, and his views on the current state and future opportunities in scientific publishing.

Developing in 3D: the role of CTCF in cell differentiation [REVIEW]


Rodrigo G. Arzate-Mejia, Felix Recillas-Targa, and Victor G. Corces

CTCF is a highly conserved zinc-finger DNA-binding protein that mediates interactions between distant sequences in the genome. As a consequence, CTCF regulates enhancer-promoter interactions and contributes to the three-dimensional organization of the genome. Recent studies indicate that CTCF is developmentally regulated, suggesting that it plays a role in cell type-specific genome organization. Here, we review these studies and discuss how CTCF functions during the development of various cell and tissue types, ranging from embryonic stem cells and gametes, to neural, muscle and cardiac cells. We propose that the lineage-specific control of CTCF levels, and its partnership with lineage-specific transcription factors, allows for the control of cell type-specific gene expression via chromatin looping.

The principles that govern transcription factor network functions in stem cells [REVIEW]


Hitoshi Niwa

Tissue-specific transcription factors primarily act to define the phenotype of the cell. The power of a single transcription factor to alter cell fate is often minimal, as seen in gain-of-function analyses, but when multiple transcription factors cooperate synergistically it potentiates their ability to induce changes in cell fate. By contrast, transcription factor function is often dispensable in the maintenance of cell phenotype, as is evident in loss-of-function assays. Why does this phenomenon, commonly known as redundancy, occur? Here, I discuss the role that transcription factor networks play in collaboratively regulating stem cell fate and differentiation by providing multiple explanations for their functional redundancy.

Delayed male germ cell sex-specification permits transition into embryonal carcinoma cells with features of primed pluripotency [RESEARCH ARTICLE]


Emily P. Dawson, Denise G. Lanza, Nicholas J. Webster, Susan M. Benton, Isao Suetake, and Jason D. Heaney

Testicular teratomas result from anomalies in embryonic germ cell development. In 129 inbred mice, teratoma initiation coincides with germ cell sex-specific differentiation and the mitotic-meiotic switch: XX and XY germ cells repress pluripotency, XX germ cells initiate meiosis, and XY germ cells activate male-specific differentiation and mitotic arrest. Here, we report that expression of Nanos2, a gene that is crucial to male sex specification, is delayed in teratoma-susceptible germ cells. Decreased expression of Nanos2 was found to be due, in part, to the Nanos2 allele present in 129 mice. In teratoma-susceptible germ cells, diminished expression of genes downstream of Nanos2 disrupted processes that were crucial to male germ cell differentiation. Deficiency for Nanos2 increased teratoma incidence in 129 mice and induced developmental abnormalities associated with tumor initiation in teratoma-resistant germ cells. Finally, in the absence of commitment to the male germ cell fate, we discovered that a subpopulation of teratoma-susceptible germ cells transition into embryonal carcinoma (EC) cells with primed pluripotent features. We conclude that delayed male germ cell sex-specification facilitates the transformation of germ cells with naïve pluripotent features into primed pluripotent EC cells.

PER2 regulation of mammary gland development [RESEARCH ARTICLE]


Cole M. McQueen, Emily E. Schmitt, Tapasree R. Sarkar, Jessica Elswood, Richard P. Metz, David Earnest, Monique Rijnkels, and Weston W. Porter

The molecular clock plays key roles in daily physiological functions, development and cancer. Period 2 (PER2) is a repressive element, which inhibits transcription activated by positive clock elements, resulting in diurnal cycling of genes. However, there are gaps in our understanding of the role of the clock in normal development outside of its time-keeping function. Here, we show that PER2 has a noncircadian function that is crucial to mammalian mammary gland development. Virgin Per2-deficient mice, Per2–/–, have underdeveloped glands, containing fewer bifurcations and terminal ducts than glands of wild-type mice. Using a transplantation model, we show that these changes are intrinsic to the gland and further identify changes in cell fate commitment. Per2–/– mouse mammary glands have a dual luminal/basal phenotypic character in cells of the ductal epithelium. We identified colocalization of E-cadherin and keratin 14 in luminal cells. Similar results were demonstrated using MCF10A and shPER2 MCF10A human cell lines. Collectively this study reveals a crucial noncircadian function of PER2 in mammalian mammary gland development, validates the Per2–/– model, and describes a potential role for PER2 in breast cancer.

Polar cell fate stimulates Wolbachia intracellular growth [RESEARCH ARTICLE]


Ajit D. Kamath, Mark A. Deehan, and Horacio M. Frydman

Bacteria are crucial partners in the development and evolution of vertebrates and invertebrates. A large fraction of insects harbor Wolbachia, bacterial endosymbionts that manipulate host reproduction to favor their spreading. Because they are maternally inherited, Wolbachia are under selective pressure to reach the female germline and infect the offspring. However, Wolbachia infection is not limited to the germline. Somatic cell types, including stem cell niches, have higher Wolbachia loads compared with the surrounding tissue. Here, we show a novel Wolbachia tropism to polar cells (PCs), specialized somatic cells in the Drosophila ovary. During oogenesis, all stages of PC development are easily visualized, facilitating the investigation of the kinetics of Wolbachia intracellular growth. Wolbachia accumulation is triggered by particular events of PC morphogenesis, including differentiation from progenitors and between stages 8 and 9 of oogenesis. Moreover, induction of ectopic PC fate is sufficient to promote Wolbachia accumulation. We found that Wolbachia PC tropism is evolutionarily conserved across most Drosophila species, but not in Culex mosquitos. These findings highlight the coordination of endosymbiont tropism with host development and cell differentiation.

Microtubule organization in presynaptic boutons relies on the formin DAAM [RESEARCH ARTICLE]


Ede Migh, Torsten Götz, Istvan Földi, Szilard Szikora, Rita Gombos, Zsuzsanna Darula, Katalin F. Medzihradszky, Jozsef Maleth, Peter Hegyi, Stephan Sigrist, and Jozsef Mihaly

Regulation of the cytoskeleton is fundamental to the development and function of synaptic terminals, such as neuromuscular junctions. Despite the identification of numerous proteins that regulate synaptic actin and microtubule dynamics, the mechanisms of cytoskeletal control during terminal arbor formation have remained largely elusive. Here, we show that DAAM, a member of the formin family of cytoskeleton organizing factors, is an important presynaptic regulator of neuromuscular junction development in Drosophila. We demonstrate that the actin filament assembly activity of DAAM plays a negligible role in terminal formation; rather, DAAM is necessary for synaptic microtubule organization. Genetic interaction studies consistently link DAAM with the Wg/Ank2/Futsch module of microtubule regulation and bouton formation. Finally, we provide evidence that DAAM is tightly associated with the synaptic active zone scaffold, and electrophysiological data point to a role in the modulation of synaptic vesicle release. Based on these results, we propose that DAAM is an important cytoskeletal effector element of the Wg/Ank2 pathway involved in the determination of basic synaptic structures, and, additionally, that DAAM may couple the active zone scaffold to the presynaptic cytoskeleton.

The histone acetyltransferase inhibitor Nir regulates epidermis development [RESEARCH ARTICLE]


Delphine Duteil, Yves Tourrette, Adrien Eberlin, Dominica Willmann, Dharmeshkumar Patel, Nicolaus Friedrichs, Judith M. Müller, and Roland Schüle

In addition to its function as an inhibitor of histone acetyltransferases, Nir (Noc2l) binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, resulting in perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation are increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates the expression of essential factors in epidermis development, such as keratins, integrins and laminins. Furthermore, Nir binds to and controls the expression of p63 and limits H3K18ac at the p63 promoter. Corroborating the stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development.

Fat body glycogen serves as a metabolic safeguard for the maintenance of sugar levels in Drosophila [RESEARCH ARTICLE]


Takayuki Yamada, Okiko Habara, Hitomi Kubo, and Takashi Nishimura

Adapting to changes in food availability is a central challenge for survival. Glucose is an important resource for energy production, and therefore many organisms synthesize and retain sugar storage molecules. In insects, glucose is stored in two different forms: the disaccharide trehalose and the branched polymer glycogen. Glycogen is synthesized and stored in several tissues, including in muscle and the fat body. Despite the major role of the fat body as a center for energy metabolism, the importance of its glycogen content remains unclear. Here, we show that glycogen metabolism is regulated in a tissue-specific manner under starvation conditions in the fruit fly Drosophila. The mobilization of fat body glycogen in larvae is independent of Adipokinetic hormone (Akh, the glucagon homolog) but is regulated by sugar availability in a tissue-autonomous manner. Fat body glycogen plays a crucial role in the maintenance of circulating sugars, including trehalose, under fasting conditions. These results demonstrate the importance of fat body glycogen as a metabolic safeguard in Drosophila.

Polycystin 1 loss of function is directly linked to an imbalance in G-protein signaling in the kidney [RESEARCH ARTICLE]


Bo Zhang, Uyen Tran, and Oliver Wessely

The development of the kidney relies on the establishment and maintenance of a precise tubular diameter of its functional units, the nephrons. This process is disrupted in polycystic kidney disease (PKD), resulting in dilations of the nephron and renal cyst formation. In the course of exploring G-protein-coupled signaling in the Xenopus pronephric kidney, we discovered that loss of the G-protein α subunit, Gnas, results in a PKD phenotype. Polycystin 1, one of the genes mutated in human PKD, encodes a protein resembling a G-protein-coupled receptor. Furthermore, deletion of the G-protein-binding domain present in the intracellular C terminus of polycystin 1 impacts functionality. A comprehensive analysis of all the G-protein α subunits expressed in the Xenopus pronephric kidney demonstrates that polycystin 1 recruits a select subset of G-protein α subunits and that their knockdown – as in the case of Gnas – results in a PKD phenotype. Mechanistically, the phenotype is caused by increased endogenous G-protein β/ signaling and can be reversed by pharmacological inhibitors as well as knocking down Gnb1. Together, our data support the hypothesis that G proteins are recruited to the intracellular domain of PKD1 and that this interaction is crucial for its function in the kidney.

Loss of Mob1a/b in mice results in chondrodysplasia due to YAP1/TAZ-TEAD-dependent repression of SOX9 [RESEARCH ARTICLE]


Hiroki Goto, Miki Nishio, Yoko To, Tatsuya Oishi, Yosuke Miyachi, Tomohiko Maehama, Hiroshi Nishina, Haruhiko Akiyama, Tak Wah Mak, Yuma Makii, Taku Saito, Akihiro Yasoda, Noriyuki Tsumaki, and Akira Suzuki

Hippo signaling is modulated in response to cell density, external mechanical forces, and rigidity of the extracellular matrix (ECM). The Mps one binder kinase activator (MOB) adaptor proteins are core components of Hippo signaling and influence Yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ), which are potent transcriptional regulators. YAP1/TAZ are key contributors to cartilage and bone development but the molecular mechanisms by which the Hippo pathway controls chondrogenesis are largely unknown. Cartilage is rich in ECM and also subject to strong external forces – two upstream factors regulating Hippo signaling. Chondrogenesis and endochondral ossification are tightly controlled by growth factors, morphogens, hormones, and transcriptional factors that engage in crosstalk with Hippo-YAP1/TAZ signaling. Here, we generated tamoxifen-inducible, chondrocyte-specific Mob1a/b-deficient mice and show that hyperactivation of endogenous YAP1/TAZ impairs chondrocyte proliferation and differentiation/maturation, leading to chondrodysplasia. These defects were linked to suppression of SOX9, a master regulator of chondrogenesis, the expression of which is mediated by TEAD transcription factors. Our data indicate that a MOB1-dependent YAP1/TAZ-TEAD complex functions as a transcriptional repressor of SOX9 and thereby negatively regulates chondrogenesis.

Prothoracicotropic hormone modulates environmental adaptive plasticity through the control of developmental timing [RESEARCH ARTICLE]


MaryJane Shimell, Xueyang Pan, Francisco A. Martin, Arpan C. Ghosh, Pierre Leopold, Michael B. O'Connor, and Nuria M. Romero

Adult size and fitness are controlled by a combination of genetics and environmental cues. In Drosophila, growth is confined to the larval phase and final body size is impacted by the duration of this phase, which is under neuroendocrine control. The neuropeptide prothoracicotropic hormone (PTTH) has been proposed to play a central role in controlling the length of the larval phase through regulation of ecdysone production, a steroid hormone that initiates larval molting and metamorphosis. Here, we test this by examining the consequences of null mutations in the Ptth gene for Drosophila development. Loss of Ptth causes several developmental defects, including a delay in developmental timing, increase in critical weight, loss of coordination between body and imaginal disc growth, and reduced adult survival in suboptimal environmental conditions such as nutritional deprivation or high population density. These defects are caused by a decrease in ecdysone production associated with altered transcription of ecdysone biosynthetic genes. Therefore, the PTTH signal contributes to coordination between environmental cues and the developmental program to ensure individual fitness and survival.

Regulatory integration of Hox factor activity with T-box factors in limb development [RESEARCH ARTICLE]


Deepak Jain, Stephen Nemec, Maëva Luxey, Yves Gauthier, Amandine Bemmo, Aurelio Balsalobre, and Jacques Drouin

In tetrapods, Tbx4, Tbx5 and Hox cluster genes are crucial for forelimb and hindlimb development and mutations in these genes are responsible for congenital limb defects. The molecular basis of their integrated mechanisms of action in the context of limb development remains poorly understood. We studied Tbx4 and Hoxc10 owing to their overlapping loss-of-function phenotypes and colocalized expression in mouse hindlimb buds. We report an extensive overlap between Tbx4 and Hoxc10 genome occupancy and their putative target genes. Tbx4 and Hoxc10 interact directly with each other, have the ability to bind to a previously unrecognized T-box–Hox composite DNA motif and show synergistic activity when acting on reporter genes. Pitx1, the master regulator for hindlimb specification, also shows extensive genomic colocalization with Tbx4 and Hoxc10. Genome occupancy by Tbx4 in hindlimb buds is similar to Tbx5 occupancy in forelimbs. By contrast, another Hox factor, Hoxd13, also interacts with Tbx4/Tbx5 but antagonizes Tbx4/Tbx5-dependent transcriptional activity. Collectively, the modulation of Tbx-dependent activity by Hox factors acting on common DNA targets may integrate different developmental processes for the balanced formation of proportionate limbs.

Hormonal control of growth in the wing imaginal disks of Junonia coenia: the relative contributions of insulin and ecdysone [RESEARCH ARTICLE]


H. Frederik Nijhout, Emily Laub, and Laura W. Grunert

The wing imaginal disks of Lepidoptera can be grown in tissue culture, but require both insulin and ecdysone to grow normally. Here, we investigate the contributions the two hormones make to growth. Ecdysone is required to maintain mitoses, whereas in the presence of insulin alone mitoses stop. Both ecdysone and insulin stimulate protein synthesis, but only ecdysone stimulates DNA synthesis. Insulin stimulates primarily cytoplasmic growth and an increase in cell size, whereas ecdysone, by virtue of its stimulation of DNA synthesis and mitosis, stimulates growth by an increase in cell number. Although both hormones stimulate protein synthesis, they do so in different spatial patterns. Both hormones stimulate protein synthesis in the inter-vein regions, but ecdysone stimulates synthesis more strongly in the veins and in the margin of the wing disk. We propose that the balance of insulin and ecdysone signaling must be regulated to maintain normal growth, and when growth appears to be due primarily to an increase in cell number, or an increase in cell size, this may indicate growth occurred under conditions that favored a stronger role for ecdysone, or insulin, respectively.

Lineage- and stage-specific expressed CYCD7;1 coordinates the single symmetric division that creates stomatal guard cells [RESEARCH ARTICLE]


Annika K. Weimer, Juliana L. Matos, Nidhi Sharma, Farah Patell, James A. H. Murray, Walter Dewitte, and Dominique C. Bergmann

Plants, with cells fixed in place by rigid walls, often utilize spatial and temporally distinct cell division programs to organize and maintain organs. This leads to the question of how developmental regulators interact with the cell cycle machinery to link cell division events with particular developmental trajectories. In Arabidopsis leaves, the development of stomata, two-celled epidermal valves that mediate plant-atmosphere gas exchange, relies on a series of oriented stem cell-like asymmetric divisions followed by a single symmetric division. The stomatal lineage is embedded in a tissue in which other cells transition from proliferation to postmitotic differentiation earlier, necessitating stomatal lineage-specific factors to prolong competence to divide. We show that the D-type cyclin, CYCD7;1, is specifically expressed just prior to the symmetric guard cell-forming division, and that it is limiting for this division. Further, we find that CYCD7;1 is capable of promoting divisions in multiple contexts, likely through RBR1-dependent promotion of the G1/S transition, but that CYCD7;1 is regulated at the transcriptional level by cell type-specific transcription factors that confine its expression to the appropriate developmental window.

Dynamics of chromatin marks and the role of JMJD3 during pancreatic endocrine cell fate commitment [RESEARCH ARTICLE]


Xin-Xin Yu, Wei-Lin Qiu, Liu Yang, Lin-Chen Li, Yu-Wei Zhang, and Cheng-Ran Xu

Pancreatic endocrine lineages are derived from pancreatic progenitors that undergo a cell fate transition requiring a switch from low to high Ngn3 expression. However, the underlying chromatin regulatory mechanisms are unclear. Here, we performed epigenomic analysis of gene regulatory loci featuring histone marks in cells with low or high level of Ngn3 expression. In combination with transcriptomic analysis, we discovered that in Ngn3-high cells, the removal of H3K27me3 was associated with the activation of key transcription factors and the establishment of primed and active enhancers. Deletion of Jmjd3, a histone demethylase for H3K27me3, at the pancreatic progenitor stage impaired the efficiency of endocrine cell fate transition and thereafter islet formation. Curiously, single-cell RNA-seq revealed that the transcriptome and developmental pathway of Ngn3-high cells were not affected by the deletion of Jmjd3. Our study indicates sequential chromatin events and identifies a crucial role for Jmjd3 in regulating the efficiency of the transition from Ngn3-low to Ngn3-high cells.

Centrosomal protein Dzip1l binds Cby, promotes ciliary bud formation, and acts redundantly with Bromi to regulate ciliogenesis in the mouse [RESEARCH ARTICLE]


Chengbing Wang, Jia Li, Ken-Ichi Takemaru, Xiaogang Jiang, Guoqiang Xu, and Baolin Wang

The primary cilium is a microtubule-based organelle required for Hedgehog (Hh) signaling and consists of a basal body, a ciliary axoneme and a compartment between the first two structures, called the transition zone (TZ). The TZ serves as a gatekeeper to control protein composition in cilia, but less is known about its role in ciliary bud formation. Here, we show that centrosomal protein Dzip1l is required for Hh signaling between Smoothened and Sufu. Dzip1l colocalizes with basal body appendage proteins and Rpgrip1l, a TZ protein. Loss of Dzip1l results in reduced ciliogenesis and dysmorphic cilia in vivo. Dzip1l interacts with, and acts upstream of, Cby, an appendage protein, in ciliogenesis. Dzip1l also has overlapping functions with Bromi (Tbc1d32) in ciliogenesis, cilia morphogenesis and neural tube patterning. Loss of Dzip1l arrests ciliogenesis at the stage of ciliary bud formation from the TZ. Consistent with this, Dzip1l mutant cells fail to remove the capping protein Cp110 (Ccp110) from the distal end of mother centrioles and to recruit Rpgrip1l to the TZ. Therefore, Dzip1l promotes ciliary bud formation and is required for the integrity of the TZ.

Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond [SPOTLIGHT]


Chun Liu, Angelos Oikonomopoulos, Nazish Sayed, and Joseph C. Wu

The advent of human induced pluripotent stem cells (iPSCs) presents unprecedented opportunities to model human diseases. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers have proven to be a relatively simple tool for exploring disease pathogenesis and underlying mechanisms. In this Spotlight article, we discuss the progress and limitations of the current 2D iPSC disease-modeling platform, as well as recent advancements in the development of human iPSC models that mimic in vivo tissues and organs at the three-dimensional (3D) level. Recent bioengineering approaches have begun to combine different 3D organoid types into a single ‘4D multi-organ system’. We summarize the advantages of this approach and speculate on the future role of 4D multi-organ systems in human disease modeling.

On the nature and function of organizers [REVIEW]


Alfonso Martinez Arias and Ben Steventon

Organizers, which comprise groups of cells with the ability to instruct adjacent cells into specific states, represent a key principle in developmental biology. The concept was first introduced by Spemann and Mangold, who showed that there is a cellular population in the newt embryo that elicits the development of a secondary axis from adjacent cells. Similar experiments in chicken and rabbit embryos subsequently revealed groups of cells with similar instructive potential. In birds and mammals, organizer activity is often associated with a structure known as the node, which has thus been considered a functional homologue of Spemann's organizer. Here, we take an in-depth look at the structure and function of organizers across species and note that, whereas the amphibian organizer is a contingent collection of elements, each performing a specific function, the elements of organizers in other species are dispersed in time and space. This observation urges us to reconsider the universality and meaning of the organizer concept.

Reduced expression of the Nodal co-receptor Oep causes loss of mesendodermal competence in zebrafish [RESEARCH REPORT]


Pavel Vopalensky, Sabrina Pralow, and Nadine L. Vastenhouw

The activation of specific gene expression programs depends on the presence of the appropriate signals and the competence of cells to respond to those signals. Although it is well established that cellular competence is regulated in space and time, the molecular mechanisms underlying the loss of competence remain largely unknown. Here, we determine the time window during which zebrafish prospective ectoderm loses its ability to respond to Nodal signals, and show that this coincides with a decrease in the levels of the Nodal co-receptor One-eyed pinhead (Oep). Bypassing Oep using a photoactivatable receptor, or an Oep-independent ligand, allows activation of Nodal target genes for an extended period of time. These results suggest that the reduced expression of Oep causes the loss of responsiveness to Nodal signals in the prospective ectoderm. Indeed, extending the presence of Oep prolongs the window of competence to respond to Nodal signals. Our findings suggest a simple mechanism in which the decreasing level of one component of the Nodal signaling pathway regulates the loss of mesendodermal competence in the prospective ectoderm.

Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from degradation by Pacman [RESEARCH REPORT]


Kenichi Nishioka, Xian-Feng Wang, Hitomi Miyazaki, Hidenobu Soejima, and Susumu Hirose

Under stress conditions, the coactivator Multiprotein bridging factor 1 (Mbf1) translocates from the cytoplasm into the nucleus to induce stress-response genes. However, its role in the cytoplasm, where it is mainly located, has remained elusive. Here, we show that Drosophila Mbf1 associates with E(z) mRNA and protects it from degradation by the exoribonuclease Pacman (Pcm), thereby ensuring Polycomb silencing. In genetic studies, loss of mbf1 function enhanced a Polycomb phenotype in Polycomb group mutants, and was accompanied by a significant reduction in E(z) mRNA expression. Furthermore, a pcm mutation suppressed the Polycomb phenotype and restored the expression level of E(z) mRNA, while pcm overexpression exhibited the Polycomb phenotype in the mbf1 mutant but not in the wild-type background. In vitro, Mbf1 protected E(z) RNA from Pcm activity. Our results suggest that Mbf1 buffers fluctuations in Pcm activity to maintain an E(z) mRNA expression level sufficient for Polycomb silencing.

Precise spatial restriction of BMP signaling in developing joints is perturbed upon loss of embryo movement [RESEARCH ARTICLE]


Pratik Narendra Pratap Singh, Claire A. Shea, Shashank Kumar Sonker, Rebecca A. Rolfe, Ayan Ray, Sandeep Kumar, Pankaj Gupta, Paula Murphy, and Amitabha Bandyopadhyay

Dynamic mechanical loading of synovial joints is necessary for normal joint development, as evidenced in certain clinical conditions, congenital disorders and animal models where dynamic muscle contractions are reduced or absent. Although the importance of mechanical forces on joint development is unequivocal, little is known about the molecular mechanisms involved. Here, using chick and mouse embryos, we observed that molecular changes in expression of multiple genes analyzed in the absence of mechanical stimulation are consistent across species. Our results suggest that abnormal joint development in immobilized embryos involves inappropriate regulation of Wnt and BMP signaling during definition of the emerging joint territories, i.e. reduced β-catenin activation and concomitant upregulation of pSMAD1/5/8 signaling. Moreover, dynamic mechanical loading of the developing knee joint activates Smurf1 expression; our data suggest that Smurf1 insulates the joint region from pSMAD1/5/8 signaling and is essential for maintenance of joint progenitor cell fate.

An evolutionarily conserved NIMA-related kinase directs rhizoid tip growth in the basal land plant Marchantia polymorpha [RESEARCH ARTICLE]


Kento Otani, Kimitsune Ishizaki, Ryuichi Nishihama, Shogo Takatani, Takayuki Kohchi, Taku Takahashi, and Hiroyasu Motose

Tip growth is driven by turgor pressure and mediated by the polarized accumulation of cellular materials. How a single polarized growth site is established and maintained is unclear. Here, we analyzed the function of NIMA-related protein kinase 1 (MpNEK1) in the liverwort Marchantia polymorpha. In the wild type, rhizoid cells differentiate from the ventral epidermis and elongate through tip growth to form hair-like protrusions. In Mpnek1 knockout mutants, rhizoids underwent frequent changes in growth direction, resulting in a twisted and/or spiral morphology. The functional MpNEK1-Citrine protein fusion localized to microtubule foci in the apical growing region of rhizoids. Mpnek1 knockouts exhibited increases in both microtubule density and bundling in the apical dome of rhizoids. Treatment with the microtubule-stabilizing drug taxol phenocopied the Mpnek1 knockout. These results suggest that MpNEK1 directs tip growth in rhizoids through microtubule organization. Furthermore, MpNEK1 expression rescued ectopic outgrowth of epidermal cells in the Arabidopsis thaliana nek6 mutant, strongly supporting an evolutionarily conserved NEK-dependent mechanism of directional growth. It is possible that such a mechanism contributed to the evolution of the early rooting system in land plants.

Face morphogenesis is promoted by Pbx-dependent EMT via regulation of Snail1 during frontonasal prominence fusion [RESEARCH ARTICLE]


Marta Losa, Maurizio Risolino, Bingsi Li, James Hart, Laura Quintana, Irina Grishina, Hui Yang, Irene F. Choi, Patrick Lewicki, Sameer Khan, Robert Aho, Jennifer Feenstra, C. Theresa Vincent, Anthony M. C. Brown, Elisabetta Ferretti, Trevor Williams, and Licia Selleri

Human cleft lip with or without cleft palate (CL/P) is a common craniofacial abnormality caused by impaired fusion of the facial prominences. We have previously reported that, in the mouse embryo, epithelial apoptosis mediates fusion at the seam where the prominences coalesce. Here, we show that apoptosis alone is not sufficient to remove the epithelial layers. We observed morphological changes in the seam epithelia, intermingling of cells of epithelial descent into the mesenchyme and molecular signatures of epithelial-mesenchymal transition (EMT). Utilizing mouse lines with cephalic epithelium-specific Pbx loss exhibiting CL/P, we demonstrate that these cellular behaviors are Pbx dependent, as is the transcriptional regulation of the EMT driver Snail1. Furthermore, in the embryo, the majority of epithelial cells expressing high levels of Snail1 do not undergo apoptosis. Pbx1 loss- and gain-of-function in a tractable epithelial culture system revealed that Pbx1 is both necessary and sufficient for EMT induction. This study establishes that Pbx-dependent EMT programs mediate murine upper lip/primary palate morphogenesis and fusion via regulation of Snail1. Of note, the EMT signatures observed in the embryo are mirrored in the epithelial culture system.

A molecular framework controlling style morphology in Brassicaceae [RESEARCH ARTICLE]


Sara Simonini, Pauline Stephenson, and Lars Ostergaard

Organ formation in multicellular organisms depends on the coordinated activities of regulatory components that integrate developmental and hormonal cues to control gene expression and mediate cell-type specification. For example, development of the Arabidopsis gynoecium is tightly controlled by distribution and synthesis of the plant hormone auxin. The functions of several transcription factors (TFs) have been linked with auxin dynamics during gynoecium development; yet how their activities are coordinated is not known. Here, we show that five such TFs function together to ensure polarity establishment at the gynoecium apex. The auxin response factor ETTIN (ARF3; herein, ETT) is a central component of this framework. Interaction of ETT with TF partners is sensitive to the presence of auxin and our results suggest that ETT forms part of a repressive gene-regulatory complex. We show that this function is conserved between members of the Brassicaceae family and that variation in an ETT subdomain affects interaction strengths and gynoecium morphology. These results suggest that variation in affinities between conserved TFs can lead to morphological differences and thus contribute to the evolution of diverse organ shapes.

Nesprins and opposing microtubule motors generate a point force that drives directional nuclear motion in migrating neurons [RESEARCH ARTICLE]


You Kure Wu, Hiroki Umeshima, Junko Kurisu, and Mineko Kengaku

Nuclear migration of newly born neurons is essential for cortex formation in the brain. The nucleus is translocated by actin and microtubules, yet the actual force generated by the interplay of these cytoskeletons remains elusive. High-resolution time-lapse observation of migrating murine cerebellar granule cells revealed that the nucleus actively rotates along the direction of its translocation, independently of centrosome motion. Pharmacological and molecular perturbation indicated that spin torque is primarily generated by microtubule motors through the LINC complex in the absence of actomyosin contractility. In contrast to the prevailing view that microtubules are uniformly oriented around the nucleus, we observed that the perinuclear microtubule arrays are of mixed polarity and both cytoplasmic dynein complex and kinesin-1 are required for nuclear rotation. Kinesin-1 can exert a point force on the nuclear envelope via association with nesprins, and loss of kinesin-1 causes failure in neuronal migration in vivo. Thus, microtubules steer the nucleus and drive its rotation and translocation via a dynamic, focal interaction of nesprins with kinesin-1 and dynein, and this is necessary for neuronal migration during brain development.

Mechanical strain regulates the Hippo pathway in Drosophila [RESEARCH ARTICLE]


Georgina C. Fletcher, Maria-del-Carmen Diaz-de-la-Loza, Nerea Borreguero-Munoz, Maxine Holder, Mario Aguilar-Aragon, and Barry J. Thompson

Animal cells are thought to sense mechanical forces via the transcriptional co-activators YAP (or YAP1) and TAZ (or WWTR1), the sole Drosophila homolog of which is named Yorkie (Yki). In mammalian cells in culture, artificial mechanical forces induce nuclear translocation of YAP and TAZ. Here, we show that physiological mechanical strain can also drive nuclear localisation of Yki and activation of Yki target genes in the Drosophila follicular epithelium. Mechanical strain activates Yki by stretching the apical domain, reducing the concentration of apical Crumbs, Expanded, Kibra and Merlin, and reducing apical Hippo kinase dimerisation. Overexpressing Hippo kinase to induce ectopic activation in the cytoplasm is sufficient to prevent Yki nuclear localisation even in flattened follicle cells. Conversely, blocking Hippo signalling in warts clones causes Yki nuclear localisation even in columnar follicle cells. We find no evidence for involvement of other pathways, such as Src42A kinase, in regulation of Yki. Finally, our results in follicle cells appear generally applicable to other tissues, as nuclear translocation of Yki is also readily detectable in other flattened epithelial cells such as the peripodial epithelium of the wing imaginal disc, where it promotes cell flattening.

Phosphorylation states change Otx2 activity for cell proliferation and patterning in the Xenopus embryo [RESEARCH ARTICLE]


Yumeko Satou, Kohei Minami, Erina Hosono, Hajime Okada, Yuuri Yasuoka, Takashi Shibano, Toshiaki Tanaka, and Masanori Taira

The homeodomain transcription factor Otx2 has essential roles in head and eye formation via the negative and positive regulation of its target genes, but it remains elusive how this dual activity of Otx2 affects cellular functions. In the current study, we first demonstrated that both exogenous and endogenous Otx2 are phosphorylated at multiple sites. Using Xenopus embryos, we identified three possible cyclin-dependent kinase (Cdk) sites and one Akt site, and analyzed the biological activities of phosphomimetic (4E) and nonphosphorylatable (4A) mutants for those sites. In the neuroectoderm, the 4E but not the 4A mutant downregulated the Cdk inhibitor gene p27xic1 (cdknx) and posterior genes, and promoted cell proliferation, possibly forming a positive-feedback loop consisting of Cdk, Otx2 and p27xic1 for cell proliferation, together with anteriorization. Conversely, the 4A mutant functioned as an activator on its own and upregulated the expression of eye marker genes, resulting in enlarged eyes. Consistent with these results, the interaction of Otx2 with the corepressor Tle1 is suggested to be phosphorylation dependent. These data suggest that Otx2 orchestrates cell proliferation, anteroposterior patterning and eye formation via its phosphorylation state.