bluuebunny/crossref_metadata_2025 huggingface dataset:
Rescue of the parathyroid hormone-related protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development
Authors: Wysolmerski John J. , Philbrick William M. , Dunbar Maureen E. , Lanske Beate , Kronenberg Henry , Karaplis Andrew , Broadus Arthur E.
Published: April 2021

Parathyroid hormone-related protein (PTHrP) was originally discovered as a tumor product that causes humoral hypercalcemia of malignancy. PTHrP is now known to be widely expressed in normal tissues and growing evidence suggests that it is an important developmental regulatory molecule. We had previously reported that overexpression of PTHrP in the mammary glands of transgenic mice impaired branching morphogenesis during sexual maturity and early pregnancy. We now demonstrate that PTHrP plays a critical role in the epithelial-mesenchymal communications that guide the initial round of branching morphogenesis that occurs during the embryonic development of the mammary gland. We have rescued the PTHrP-knockout mice from neonatal death by transgenic expression of PTHrP targeted to chondrocytes. These rescued mice are devoid of mammary epithelial ducts. We show that disruption of the PTHrP gene leads to a failure of the initial round of branching growth that is responsible for transforming the mammary bud into the rudimentary mammary duct system. In the absence of PTHrP, the mammary epithelial cells degenerate and disappear. The ability of PTHrP to support embryonic mammary development is a function of amino-terminal PTHrP, acting via the PTH/PTHrP receptor, for ablation of the PTH/PTHrP receptor gene recapitulates the phenotype of PTHrP gene ablation. We have localized PTHrP expression to the embryonic mammary epithelial cells and PTH/PTHrP receptor expression to the mammary mesenchyme using in situ hybridization histochemistry. Finally, we have rescued mammary gland development in PTHrP-null animals by transgenic expression of PTHrP in embryonic mammary epithelial cells. We conclude that PTHrP is a critical epithelial signal received by the mammary mesenchyme and involved in supporting the initiation of branching morphogenesis.
The let-99 gene is required for proper spindle orientation during cleavage of the C. elegans embryo
Authors: Rose Lesilee S. , Kemphues Ken
Published: April 2021

The orientation of cell division is a critical aspect of development. In 2-cell C. elegans embryos, the spindle in the posterior cell is aligned along the long axis of the embryo and contributes to the unequal partitioning of cytoplasm, while the spindle in the anterior cell is oriented transverse to the long axis. Differing spindle alignments arise from blastomere-specific rotations of the nuclear-centrosome complex at prophase. We have found that mutations in the maternally expressed gene let-99 affect spindle orientation in all cells during the first three cleavages. During these divisions, the nuclear-centrosome complex appears unstable in position. In addition, in almost half of the mutant embryos, there are reversals of the normal pattern of spindle orientations at second cleavage: the spindle of the anterior cell is aligned with the long axis of the embryo and nuclear rotation fails in the posterior cell causing the spindle to form transverse to the long axis. In most of the remaining embryos, spindles in both cells are transverse at second cleavage. The distributions of several asymmetrically localized proteins, including P granules and PAR-3, are normal in early let-99 embryos, but are perturbed by the abnormal cell division orientations at second cleavage. The accumulation of actin and actin capping protein, which marks the site involved in nuclear rotation in 2-cell wild-type embryos, is abnormal but is not reversed in let-99 mutant embryos. Based on these data, we conclude that let-99(+) is required for the proper orientation of spindles after the establishment of polarity, and we postulate that let-99(+) plays a role in interactions between the astral microtubules and the cortical cytoskeleton.
A role for serum response factor in coronary smooth muscle differentiation from proepicardial cells
Authors: Landerholm Thomas E. , Dong Xiu-Rong , Lu Jun , Belaguli Narasimhaswamy S. , Schwartz Robert J. , Majesky Mark W.
Published: April 2021

Coronary artery smooth muscle (SM) cells originate from proepicardial cells that migrate over the surface of the heart, undergo epithelial to mesenchymal transformation and invade the subepicardial and cardiac matrix. Prior to contact with the heart, proepicardial cells exhibit no expression of smooth muscle markers including SMαactin, SM22α, calponin, SMγactin or SM-myosin heavy chain detectable by RT-PCR or by immunostaining. To identify factors required for coronary smooth muscle differentiation, we excised proepicardial cells from Hamburger-Hamilton stage-17 quail embryos and examined them ex vivo. Proepicardial cells initially formed an epithelial colony that was uniformly positive for cytokeratin, an epicardial marker. Transcripts for flk-1, Nkx 2.5, GATA4 or smooth muscle markers were undetectable, indicating an absence of endothelial, myocardial or preformed smooth muscle cells. By 24 hours, cytokeratin-positive cells became SMαactin-positive. Moreover, serum response factor, undetectable in freshly isolated proepicardial cells, became strongly expressed in virtually all epicardial cells. By 72 hours, a subset of epicardial cells exhibited a rearrangement of cytoskeletal actin, focal adhesion formation and acquisition of a motile phenotype. Coordinately with mesenchymal transformation, calponin, SM22α and SMγactin became expressed. By 5-10 days, SM-myosin heavy chain mRNA was found, by which time nearly all cells had become mesenchymal. RT-PCR showed that large increases in serum response factor expression coincide with smooth muscle differentiation in vitro. Two different dominant-negative serum response factor constructs prevented the appearance of calponin-, SM22α- and SMγactin-positive cells. By contrast, dominant-negative serum response factor did not block mesenchymal transformation nor significantly reduce the number of cytokeratin-positive cells. These results indicate that the stepwise differentiation of coronary smooth muscle cells from proepicardial cells requires transcriptionally active serum response factor.
Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in the regulation of floral induction
Authors: Mockler Todd C. , Guo Hongwei , Yang Hongyun , Duong Hien , Lin Chentao
Published: April 2021

The Arabidopsis photoreceptors cry1, cry2 and phyB are known to play roles in the regulation of flowering time, for which the molecular mechanisms remain unclear. We have previously hypothesized that phyB mediates a red-light inhibition of floral initiation and cry2 mediates a blue-light inhibition of the phyB function. Studies of the cry2/phyB double mutant provide direct evidence in support of this hypothesis. The function of cryptochromes in floral induction was further investigated using the cry2/cry1 double mutants. The cry2/cry1 double mutants showed delayed flowering in monochromatic blue light, whereas neither monogenic cry1 nor cry2 mutant exhibited late flowering in blue light. This result suggests that, in addition to the phyB-dependent function, cry2 also acts redundantly with cry1 to promote floral initiation in a phyB-independent manner. To understand how photoreceptors regulate the transition from vegetative growth to reproductive development, we examined the effect of sequential illumination by blue light and red light on the flowering time of plants. We found that there was a light-quality-sensitive phase of plant development, during which the quality of light exerts a profound influence on flowering time. After this developmental stage, which is between approximately day-1 to day-7 post germination, plants are committed to floral initiation and the quality of light has little effect on the flowering time. Mutations in either the PHYB gene or both the CRY1 and CRY2 genes resulted in the loss of the light-quality-sensitive phase manifested during floral development. The commitment time of floral transition, defined by a plant’s sensitivity to light quality, coincides with the commitment time of inflorescence development revealed previously by a plant’s sensitivity to light quantity – the photoperiod. Therefore, the developmental mechanism resulting in the commitment to flowering appears to be the direct target of the antagonistic actions of the photoreceptors.
Zebrafish aussicht mutant embryos exhibit widespread overexpression of ace (fgf8) and coincident defects in CNS development
Authors: Heisenberg Carl-Philipp , Brennan Caroline , Wilson Stephen W.
Published: April 2021

During the development of the zebrafish nervous system both noi, a zebrafish pax2 homolog, and ace, a zebrafish fgf8 homolog, are required for development of the midbrain and cerebellum. Here we describe a dominant mutation, aussicht (aus), in which the expression of noi and ace is upregulated. In aus mutant embryos, ace is upregulated at many sites in the embryo, while noi expression is only upregulated in regions of the forebrain and midbrain which also express ace. Subsequent to the alterations in noi and ace expression, aus mutants exhibit defects in the differentiation of the forebrain, midbrain and eyes. Within the forebrain, the formation of the anterior and postoptic commissures is delayed and the expression of markers within the pretectal area is reduced. Within the midbrain, En and wnt1 expression is expanded. In heterozygous aus embryos, there is ectopic outgrowth of neural retina in the temporal half of the eyes, whereas in putative homozygous aus embryos, the ventral retina is reduced and the pigmented retinal epithelium is expanded towards the midline.

The observation that aus mutant embryos exhibit widespread upregulation of ace raised the possibility that aus might represent an allele of the ace gene itself. However, by crossing carriers for both aus and ace, we were able to generate homozygous ace mutant embryos that also exhibited the aus phenotype. This indicated that aus is not tightly linked to ace and is unlikely to be a mutation directly affecting the ace locus. However, increased Ace activity may underly many aspects of the aus phenotype and we show that the upregulation of noi in the forebrain of aus mutants is partially dependent upon functional Ace activity. Conversely, increased ace expression in the forebrain of aus mutants is not dependent upon functional Noi activity. We conclude that aus represents a mutation involving a locus normally required for the regulation of ace expression during embryogenesis.
Ectopic expression of individual E(spl) genes has differential effects on different cell fate decisions and underscores the biphasic requirement for Notch activity in wing margin establishment in Drosophila
Authors: Ligoxygakis Petros , Bray Sarah J. , Apidianakis Yiorgos , Delidakis Christos
Published: April 2021

A common consequence of Notch signalling in Drosophila is the transcriptional activation of seven Enhancer of split [E(spl)] genes, which encode a family of closely related basic-helix-loop-helix transcriptional repressors. Different E(spl) proteins can functionally substitute for each other, hampering loss-of-function genetic analysis and raising the question of whether any specialization exists within the family. We expressed each individual E(spl) gene using the GAL4-UAS system in order to analyse their effect in a number of cell fate decisions taking place in the wing imaginal disk. We focussed on sensory organ precursor determination, wing vein determination and wing pattern formation. All of the E(spl) proteins affect the first two processes in the same way, namely they antagonize neural precursor and vein fates. Yet, the efficacy of this antagonism is quite distinct: E(spl)mβ has the strongest vein suppression effect, whereas E(spl)m8 and E(spl)m7 are the most active bristle suppressors. During wing patterning, Notch activity orchestrates a complex sequence of events that define the dorsoventral boundary of the wing. We have discerned two phases within this process based on the sensitivity of N loss-of-function phenotypes to concomitant expression of E(spl) genes. E(spl) proteins are initially involved in repression of the vg quadrant enhancer, whereas later they appear to relay the Notch signal that triggers activation of cut expression. Of the seven proteins, E(spl)mγ is most active in both of these processes. In conclusion, E(spl) proteins have partially redundant functions, yet they have evolved distinct preferences in implementing different cell fate decisions, which closely match their individual normal expression patterns.
Post-transcriptional regulation of Xwnt-8 expression is required for normal myogenesis during vertebrate embryonic development
Authors: Tian Qi , Nakayama Takuya , Dixon Michael P. , Christian Jan L.
Published: April 2021

The Xenopus Wnt-8 gene is transiently expressed in ventral and lateral mesoderm during gastrulation and plays a critical role in patterning these tissues. In the current study, we show that the spatial and temporal pattern of expression of endogenous Xwnt-8 is regulated, in part, at a post-transcriptional level. We have identified a novel sequence element in the 3′ untranslated region of the Xwnt-8 RNA that controls the polyadenylation status of reporter and endogenous Xwnt-8 RNAs, directs rapid RNA degradation beginning precisely at the early gastrula stage, and represses translation of transcripts throughout development. Expression of endogenous Xwnt-8 is normally downregulated within lateral (presomitic) mesoderm following gastrulation. We demonstrate that rapid degradation of Xwnt-8 transcripts, mediated by these regulatory elements in the 3′ untranslated region, is essential to this process and that downregulation is required to prevent overcommitment of somitic cells to a myogenic fate. These studies demonstrate a role for post-transcriptional regulation of zygotic gene expression in vertebrate embryonic patterning.
Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development
Authors: Parichy David M. , Rawls John F. , Pratt Stephen J. , Whitfield Tanya T. , Johnson Stephen L.
Published: April 2021

The relative roles of the Kit receptor in promoting the migration and survival of amniote melanocytes are unresolved. We show that, in the zebrafish, Danio rerio, the pigment pattern mutation sparse corresponds to an orthologue of c-kit. This finding allows us to further elucidate morphogenetic roles for this c-kit-related gene in melanocyte morphogenesis. Our analyses of zebrafish melanocyte development demonstrate that the c-kit orthologue identified in this study is required both for normal migration and for survival of embryonic melanocytes. We also find that, in contrast to mouse, the zebrafish c-kit gene that we have identified is not essential for hematopoiesis or primordial germ cell development. These unexpected differences may reflect evolutionary divergence in c-kit functions following gene duplication events in teleosts.
MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube
Authors: Saga Yumiko , Miyagawa-Tomita Sachiko , Takagi Atsuya , Kitajima Satoshi , Miyazaki Jun-ichi , Inoue Tohru
Published: April 2021

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a β-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by β-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the β-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.
Huckebein repressor activity in Drosophila terminal patterning is mediated by Groucho
Authors: Goldstein Robert E. , Jiménez Gerardo , Cook Orna , Gur Devorah , Paroush Ze’ev
Published: April 2021

The Groucho corepressor mediates negative transcriptional regulation in association with various DNA-binding proteins in diverse developmental contexts. We have previously implicated Groucho in Drosophila embryonic terminal patterning, showing that it is required to confine tailless and huckebein terminal gap gene expression to the pole regions of the embryo. Here we reveal an additional requirement for Groucho in this developmental process by establishing that Groucho mediates repressor activity of the Huckebein protein. Putative Huckebein target genes are derepressed in embryos lacking maternal groucho activity and biochemical experiments demonstrate that Huckebein physically interacts with Groucho. Using an in vivo repression assay, we identify a functional repressor domain in Huckebein that contains an FRPW tetrapeptide, similar to the WRPW Groucho-recruitment domain found in Hairy-related repressor proteins. Mutations in Huckebein’s FRPW motif abolish Groucho binding and in vivo repression activity, indicating that binding of Groucho through the FRPW motif is required for the repressor function of Huckebein. Taken together with our earlier results, these findings show that Groucho-repression regulates sequential aspects of terminal patterning in Drosophila.
SDQR migrations in Caenorhabditis elegans are controlled by multiple guidance cues and changing responses to netrin UNC-6
Authors: Kim Seonhee , Ren Xing-Cong , Fox Eileen , Wadsworth William G.
Published: April 2021

The netrin guidance cue, UNC-6, and the netrin receptors, UNC-5 and UNC-40, guide SDQR cell and axon migrations in C. elegans. In wild-type larvae, SDQR migrations are away from ventral UNC-6-expressing cells, suggesting that UNC-6 repels SDQR. In unc-6 null larvae, SDQR migrations are towards the ventral midline, indicating a response to other guidance cues that directs the migrations ventrally. Although ectopic UNC-6 expression dorsal to the SDQR cell body would be predicted to cause ventral SDQR migrations in unc-6 null larvae, in fact, more migrations are directed dorsally, suggesting that SDQR is not always repelled from the dorsal source of UNC-6. UNC-5 is required for dorsal SDQR migrations, but not for the ventral migrations in unc-6 null larvae. UNC-40 appears to moderate both the response to UNC-6 and to the other cues. Our results show that SDQR responds to multiple guidance cues and they suggest that, besides UNC-6, other factors influence whether an UNC-6 responsive cell migrates toward or away from an UNC-6 source in vivo. We propose that multiple signals elicited by the guidance cues are integrated and interpreted by SDQR and that the response to UNC-6 can change depending on the combination of cues encountered during migration. These responses determine the final dorsoventral position of the SDQR cell and axon.
Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development
Authors: Weaver Molly , Yingling Jonathan M. , Dunn N. Ray , Bellusci Savério , Hogan Brigid L. M.
Published: April 2021

In the mature mouse lung, the proximal-distal (P-D) axis is delineated by two distinct epithelial subpopulations: the proximal bronchiolar epithelium and the distal respiratory epithelium. Little is known about the signaling molecules that pattern the lung along the P-D axis. One candidate is Bone Morphogenetic Protein 4 (Bmp4), which is expressed in a dynamic pattern in the epithelial cells in the tips of growing lung buds. Previous studies in which Bmp4 was overexpressed in the lung endoderm (Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. M. (1996) Development 122, 1693-1702) suggested that this factor plays an important role in lung morphogenesis. To further investigate this question, two complementary approaches were utilized to inhibit Bmp signaling in vivo. The Bmp antagonist Xnoggin and, independently, a dominant negative Bmp receptor (dnAlk6), were overexpressed using the surfactant protein C (Sp-C) promoter/enhancer. Inhibiting Bmp signaling results in a severe reduction in distal epithelial cell types and a concurrent increase in proximal cell types, as indicated by morphology and expression of marker genes, including the proximally expressed hepatocyte nuclear factor/forkhead homologue 4 (Hfh4) and Clara cell marker CC10, and the distal marker Sp-C. In addition, electron microscopy demonstrates the presence of ciliated cells, a proximal cell type, in the most peripheral regions of the transgenic lungs. We propose a model in which Bmp4 is a component of an apical signaling center controlling P-D patterning. Endodermal cells at the periphery of the lung, which are exposed to high levels of Bmp4, maintain or adopt a distal character, while cells receiving little or no Bmp4 signal initiate a proximal differentiation program.
Nurr1, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain
Authors: Sakurada Kazuhiro , Ohshima-Sakurada Mikiko , Palmer Theo D. , Gage Fred H.
Published: April 2021

Adult rat-derived hippocampal progenitor cells express many of the molecules implicated in midbrain dopaminergic determination, including FGF receptors 1, 2 and 3, the sonic hedgehog receptor components Smo and Ptc, and the region-specific transcription factors Ptx3 and Nurr1. Here we use undifferentiated progenitors to probe the events leading to the dopaminergic phenotype and find that the influences of Nurr1 can be temporally and mechanistically uncoupled from the patterning influences of sonic hedgehog and FGF-8 or the more generic process of neuronal differentiation itself. In gain-of-function experiments, Nurr1 is able to activate transcription of the tyrosine hydroxylase gene by binding a response element within a region of the tyrosine hydroxylase promoter necessary for midbrain-specific expression. This activation is mediated through a retinoid X receptor independent mechanism and occurs in all precursors, regardless of differentiation status. Overexpression of Nurr1 does not affect proliferation or stimulate neuronal differentiation and has no influence on the expression of other dopaminergic markers. This uncoupling of tyrosine hydroxylase expression from other dopaminergic markers suggests that the midbrain dopaminergic identity is dictated by a combination of pan-dopaminergic (e.g., Shh/FGF-8) and region-specific (Nurr1) mechanisms.
Loss of p27Kip1 function results in increased proliferative capacity of oligodendrocyte progenitors but unaltered timing of differentiation
Authors: Casaccia-Bonnefil P. , Hardy R. J. , Teng K. K. , Levine J. M. , Koff A. , Chao M. V.
Published: April 2021

In many tissues, progenitor cells permanently withdraw from the cell cycle prior to commitment towards a differentiated phenotype. In the oligodendrocyte lineage a counting mechanism has been proposed, linking the number of cell divisions to growth arrest and differentiation. A direct prediction of this model is that an increase in the number of cell divisions would result in a delayed onset of differentiation. Since the cell cycle inhibitor p27Kip1 is an essential component of the machinery leading to oligodendrocyte progenitor growth arrest, we examined the temporal relationship between cell cycle withdrawal and expression of late differentiation markers in vivo, in mice carrying a targeted deletion in the p27Kip1 gene. Using bromodeoxyuridine to label proliferating cells, quaking (QKI) to identify embryonic glial progenitors, NG2 to identify neonatal oligodendrocyte progenitors, and myelin basic protein to label differentiated oligodendrocytes, we found an increased number of proliferating QKI- and NG2-positive cells in germinal zones of p27Kip1−⁄− mice at the peak of gliogenesis. However, no delay was observed in these mice in the appearance of the late differentiation marker myelin basic protein in the developing corpus callosum and cerebellum. Significantly, a decrease in cyclin E levels was observed in the brain of p27Kip1 null mice coincident with oligodendrocyte growth arrest. We conclude that two distinct modalities of growth arrest occur in the oligodendrocyte lineage: a p27Kip1-dependent mechanism of growth arrest affecting proliferation in early phases of gliogenesis, and a p27Kip1-independent event leading to withdrawal from the cell cycle and differentiation.
Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast
Authors: Lee Tzumin , Lee Arthur , Luo Liqun
Published: April 2021

The mushroom bodies (MBs) are prominent structures in the Drosophila brain that are essential for olfactory learning and memory. Characterization of the development and projection patterns of individual MB neurons will be important for elucidating their functions. Using mosaic analysis with a repressible cell marker (Lee, T. and Luo, L. (1999) Neuron 22, 451-461), we have positively marked the axons and dendrites of multicellular and single-cell mushroom body clones at specific developmental stages. Systematic clonal analysis demonstrates that a single mushroom body neuroblast sequentially generates at least three types of morphologically distinct neurons. Neurons projecting into the γ lobe of the adult MB are born first, prior to the mid-3rd instar larval stage. Neurons projecting into the α’ and β’ lobes are born between the mid-3rd instar larval stage and puparium formation. Finally, neurons projecting into the α and β lobes are born after puparium formation. Visualization of individual MB neurons has also revealed how different neurons acquire their characteristic axon projections. During the larval stage, axons of all MB neurons bifurcate into both the dorsal and medial lobes. Shortly after puparium formation, larval MB neurons are selectively pruned according to birthdays. Degeneration of axon branches makes early-born (γ) neurons retain only their main processes in the peduncle, which then project into the adult γ lobe without bifurcation. In contrast, the basic axon projections of the later-born (α’/β’) larval neurons are preserved during metamorphosis. This study illustrates the cellular organization of mushroom bodies and the development of different MB neurons at the single cell level. It allows for future studies on the molecular mechanisms of mushroom body development.
Mutual antagonism between signals secreted by adjacent Wingless and Engrailed cells leads to specification of complementary regions of the Drosophila parasegment
Authors: Gritzan Uwe , Hatini Victor , DiNardo Stephen
Published: April 2021

Specialized groups of cells known as organizers govern the establishment of cell type diversity across cellular fields. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for organizer function. Here cells differentiate into smooth cuticle or distinct denticle types. At parasegment boundaries, cells expressing Wingless confront cells co-expressing Engrailed and Hedgehog. While Wingless is essential for smooth cell fates, the signals that establish denticle diversity are unknown. We show that wg mutants have residual mirror-symmetric pattern that is due to an Engrailed-dependent signal specifying anterior denticle fates. The Engrailed-dependent signal acts unidirectionally and Wg activity imposes this asymmetry. Reciprocally, the Engrailed/Hedgehog interface imposes asymmetry on Wg signaling. Thus, a bipartite organizer, with each signal acting essentially unidirectionally, specifies segmental pattern.
Sprouty is a general inhibitor of receptor tyrosine kinase signaling
Authors: Reich Aderet , Sapir Amir , Shilo Ben-Zion
Published: April 2021

Sprouty was originally identified as an inhibitor of Drosophila FGF receptor signaling during tracheal development. By following the capacity of ectopic Sprouty to abolish the pattern of activated MAP kinase in embryos, we show that Sprouty can inhibit other receptor tyrosine kinase (RTK) signaling pathways, namely the Heartless FGF receptor and the EGF receptor. Similarly, in wing imaginal discs, ectopic Sprouty abolishes activated MAP kinase induced by the EGF receptor pathway. Sprouty expression is induced by the EGFR pathway in some, but not all, tissues in which EGFR is activated, most notably in follicle cells of the ovary, the wing imaginal disc and the eye disc. In the ovary, induction of sprouty expression follows the pattern of EGFR activation in the follicle cells. Generation of homozygous sprouty mutant follicle-cell clones demonstrates an essential role for Sprouty in restricting EGFR activation throughout oogenesis. At the stage when dorso-ventral polarity of the follicle cells is established, Sprouty limits the ventral expansion of the activating Gurken signal. Later, when dorsal appendage fates are determined, reduction of signaling by Sprouty facilitates the induction of inter-appendage cell fates. The capacity of Sprouty to reduce or eliminate accumulation of activated MAP kinase indicates that in vivo it intersects with the pathway upstream to MAP kinase. The ability of ectopic Sprouty to rescue lethality caused by activated Raf suggests that it may impinge upon the pathway by interacting with Raf or downstream to it.
Building the heart piece by piece: modularity of cis-elements regulating Nkx2-5 transcription
Authors: Schwartz Robert J. , Olson Eric N.
Published: April 2021

Heart formation in Drosophila is dependent on the homeobox gene tinman. The homeobox gene Nkx2-5 is closely related to tinman and is the earliest known marker for cardiogenesis in vertebrate embryos. Recent studies of cis-regulatory elements required for Nkx2-5 expression in the developing mouse heart have revealed an extraordinary array of independent cardiac enhancers, and associated negative regulatory elements, that direct transcription in distinct regions of the embryonic heart. These studies demonstrate the modularity in cardiac transcription, in which different regulatory elements respond to distinct sets of transcription factors to control gene expression in different compartments of the developing heart. We consider the potential mechanisms underlying such transcriptional complexity, its possible significance for cardiac function, and the implications for evolution of the multichambered heart.
Engrailed-1 and Netrin-1 regulate axon pathfinding by association interneurons that project to motor neurons
Authors: Saueressig Harald , Burrill John , Goulding Martyn
Published: April 2021

During early development, multiple classes of interneurons are generated in the spinal cord including association interneurons that synapse with motor neurons and regulate their activity. Very little is known about the molecular mechanisms that generate these interneuron cell types, nor is it known how axons from association interneurons are guided toward somatic motor neurons. By targeting the axonal reporter gene tau-lacZ to the En1 locus, we show the cell-type-specific transcription factor Engrailed-1 (EN1) defines a population of association neurons that project locally to somatic motor neurons. These EN1 interneurons are born early and their axons pioneer an ipsilateral longitudinal projection in the ventral spinal cord. The EN1 interneurons extend axons in a stereotypic manner, first ventrally, then rostrally for one to two segments where their axons terminate close to motor neurons. We show that the growth of EN1 axons along a ventrolateral pathway toward motor neurons is dependent on netrin-1 signaling. In addition, we demonstrate that En1 regulates pathfinding and fasciculation during the second phase of EN1 axon growth in the ventrolateral funiculus (VLF); however, En1 is not required for the early specification of ventral interneuron cell types in the embryonic spinal cord.
Head versus trunk patterning in the Drosophila embryo; collier requirement for formation of the intercalary segment
Authors: Crozatier Michèle , Valle Denise , Dubois Laurence , Ibnsouda Saad , Vincent Alain
Published: April 2021

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. In this paper, we report the phenotypic analysis of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. The parasegmental register of col activation is controlled by the combined activities of the head-gap genes buttonhead and empty spiracles and the pair-rule gene even skipped; it therefore integrates inputs from both the head and trunk segmentation systems, which were previously considered as being essentially independent. After gastrulation, positive autoregulation of col is limited to cells of anterior PS0. Conversely, heat-pulse induced ubiquitous expression of Col leads to disruption of the head skeleton. Together, these results indicate that col is required for establishment of the PS(-1)/PS0 parasegmental border and formation of the intercalary segment. Our data support neither a simple combinatorial model for segmental patterning of the head nor a direct activation of segment polarity gene expression by head-gap genes, but rather argue for the existence of parasegment-specific second order regulators acting in the head, at a level similar to that of pair-rule genes in the trunk.
The role of the rhombic lip in avian cerebellum development
Authors: Wingate Richard J. T. , Hatten Mary E.
Published: April 2021

We have used a combination of quail-chick fate-mapping techniques and dye labelling to investigate the development of the avian cerebellum. Using Hoxa2 as a guide for the microsurgical construction of quail-chick chimaeras, we show that the caudal boundary of the presumptive cerebellum at E6 maps to the caudal boundary of rhombomere 1. By fate mapping the dorsoventral axis of rhombomere 1, we demonstrate that granule cell precursors are generated at the rhombic lip together with neurons of the lateral pontine nucleus. DiI-labelling of cerebellum explants reveals that external germinal layer precursors have a characteristic unipolar morphology and undergo an orientated, active migration away from the rhombic lip, which is apparently independent of either glial or axon guidance or ‘chain’ formation.
Growth cones stall and collapse during axon outgrowth in Caenorhabditis elegans
Authors: Knobel Karla M. , Jorgensen Erik M. , Bastiani Michael J.
Published: April 2021

During nervous system development, neurons form synaptic contacts with distant target cells. These connections are formed by the extension of axonal processes along predetermined pathways. Axon outgrowth is directed by growth cones located at the tips of these neuronal processes. Although the behavior of growth cones has been well-characterized in vitro, it is difficult to observe growth cones in vivo. We have observed motor neuron growth cones migrating in living Caenorhabditis elegans larvae using time-lapse confocal microscopy. Specifically, we observed the VD motor neurons extend axons from the ventral to dorsal nerve cord during the L2 stage. The growth cones of these neurons are round and migrate rapidly across the epidermis if they are unobstructed. When they contact axons of the lateral nerve fascicles, growth cones stall and spread out along the fascicle to form anvil-shaped structures. After pausing for a few minutes, they extend lamellipodia beyond the fascicle and resume migration toward the dorsal nerve cord. Growth cones stall again when they contact the body wall muscles. These muscles are tightly attached to the epidermis by narrowly spaced circumferential attachment structures. Stalled growth cones extend fingers dorsally between these hypodermal attachment structures. When a single finger has projected through the body wall muscle quadrant, the growth cone located on the ventral side of the muscle collapses and a new growth cone forms at the dorsal tip of the predominating finger. Thus, we observe that complete growth cone collapse occurs in vivo and not just in culture assays. In contrast to studies indicating that collapse occurs upon contact with repulsive substrata, collapse of the VD growth cones may result from an intrinsic signal that serves to maintain growth cone primacy and conserve cellular material.
Interaction of the TEK and TIE receptor tyrosine kinases during cardiovascular development
Authors: Puri Mira C. , Partanen Juha , Rossant Janet , Bernstein Alan
Published: April 2021

TEK (TIE2) and TIE (TIE1) are structurally related receptor tyrosine kinases expressed in endothelial cells and their precursors. Genetic studies in the mouse have revealed essential functions of both receptors in angiogenic expansion of the vasculature during development. As previously shown, mouse embryos homozygous for a disrupted Tek allele die by day 10.5 of embryogenesis due to endocardial defects, hemorrhaging, and impaired vascular network formation. Furthermore, TIE is required cell autonomously for endothelial cell survival and extension of the vascular network during late embryogenesis. Here we have investigated possible redundancy in the TEK and TIE signalling pathways during vascular development. Vasculogenesis proceeds normally in embryos lacking both TEK and TIE, although such embryos die early in gestation of multiple cardiovascular defects. Mosaic analysis revealed an absolute requirement for TEK in the endocardium at E10.5, whereas TEK and TIE are dispensable for the initial assembly of the rest of the vasculature. In contrast, both receptors are required in the microvasculature during late organogenesis and in essentially all blood vessels of the adult. This analysis demonstrates essential functions for TEK and TIE in maintaining the integrity of the mature vasculature.
Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila
Authors: Buszczak Michael , Freeman Marc R. , Carlson John R. , Bender Michael , Cooley Lynn , Segraves William A.
Published: April 2021

The steroid hormone ecdysone regulates larval development and metamorphosis in Drosophila melanogaster through a complex genetic hierarchy that begins with a small set of early response genes. Here, we present data indicating that the ecdysone response hierarchy also mediates egg chamber maturation during mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific manner while EcR expression is ubiquitous throughout oogenesis. Decreasing or increasing the ovarian ecdysone titer using a temperature-sensitive mutation or exogenous ecdysone results in corresponding changes in early gene expression. The stage 10 follicle cell expression of E75 in wild-type, K10 and EGF receptor (Egfr) mutant egg chambers reveals regulation of E75 by both the Egfr and ecdysone signaling pathways. Genetic analysis indicates a germline requirement for ecdysone-responsive gene expression. Germline clones of E75 mutations arrest and degenerate during mid-oogenesis and EcR germline clones exhibit a similar phenotype, demonstrating a functional requirement for ecdysone responsiveness during the vitellogenic phase of oogenesis. Finally, the expression of Drosophila Adrenodoxin Reductase increases during mid-oogenesis and clonal analysis confirms that this steroidogenic enzyme is required in the germline for egg chamber development. Together these data suggest that the temporal expression profile of E75, E74 and BR-C may be a functional reflection of ecdysone levels and that ecdysone provides temporal signals regulating the progression of oogenesis and proper specification of dorsal follicle cell fates.
The dominant hemimelia mutation uncouples epithelial-mesenchymal interactions and disrupts anterior mesenchyme formation in mouse hindlimbs
Authors: Lettice Laura , Hecksher-Sørensen Jacob , Hill Robert E.
Published: April 2021

Epithelial-mesenchymal interactions are essential for both limb outgrowth and pattern formation in the limb. Molecules capable of communication between these two tissues are known and include the signaling molecules SHH and FGF4, FGF8 and FGF10. Evidence suggests that the pattern and maintenance of expression of these genes are dependent on a number of factors including regulatory loops between genes expressed in the AER and those in the underlying mesenchyme. We show here that the mouse mutation dominant hemimelia (Dh) alters the pattern of gene expression in the AER such that Fgf4, which is normally expressed in a posterior domain, and Fgf8, which is expressed throughout are expressed in anterior patterns. We show that maintenance of Shh expression in the posterior mesenchyme is not dependent on either expression of Fgf4 or normal levels of Fgf8 in the overlying AER. Conversely, AER expression of Fgf4 is not directly dependent on Shh expression. Also the reciprocal regulatory loop proposed for Fgf8 in the AER and Fgf10 in the underlying mesenchyme is also uncoupled by this mutation. Early during the process of limb initiation, Dh is involved in regulating the width of the limb bud, the mutation resulting in selective loss of anterior mesenchyme. The Dh gene functions in the initial stages of limb development and we suggest that these initial roles are linked to mechanisms that pattern gene expression in the AER.
Regulation of the onset of neural crest migration by coordinated activity of BMP4 and Noggin in the dorsal neural tube
Authors: Sela-Donenfeld Dalit , Kalcheim Chaya
Published: April 2021

For neural crest cells to engage in migration, it is necessary that epithelial premigratory crest cells convert into mesenchyme. The mechanisms that trigger cell delamination from the dorsal neural tube remain poorly understood. We find that, in 15-to 40-somite-stage avian embryos, BMP4 mRNA is homogeneously distributed along the longitudinal extent of the dorsal neural tube, whereas its specific inhibitor noggin exists in a gradient of expression that decreases caudorostrally. This rostralward reduction in signal intensity coincides with the onset of emigration of neural crest cells. Hence, we hypothesized that an interplay between Noggin and BMP4 in the dorsal tube generates graded concentrations of the latter that in turn triggers the delamination of neural crest progenitors. Consistent with this suggestion, disruption of the gradient by grafting Noggin-producing cells dorsal to the neural tube at levels opposite the segmental plate or newly formed somites, inhibited emigration of HNK-1-positive crest cells, which instead accumulated within the dorsal tube. Similar results were obtained with explanted neural tubes from the same somitic levels exposed to Noggin. Exposure to Follistatin, however, had no effect. The Noggin-dependent inhibition was overcome by concomitant treatment with BMP4, which when added alone, also accelerated cell emigration compared to untreated controls. Furthermore, the observed inhibition of neural crest emigration in vivo was preceded by a partial or total reduction in the expression of cadherin-6B and rhoB but not in the expression of slug mRNA or protein. Altogether, these results suggest that a coordinated activity of Noggin and BMP4 in the dorsal neural tube triggers delamination of specified, slug-expressing neural crest cells. Thus, BMPs play multiple and discernible roles at sequential stages of neural crest ontogeny, from specification through delamination and later differentiation of specific neural crest derivatives.
A requirement for neuropilin-1 in embryonic vessel formation
Authors: Kawasaki Takahiko , Kitsukawa Takashi , Bekku Yoko , Matsuda Yoichi , Sanbo Makoto , Yagi Takeshi , Fujisawa Hajime
Published: April 2021

Neuropilin-1 is a membrane protein that is expressed in developing neurons and functions as a receptor or a component of the receptor complex for the class 3 semaphorins, which are inhibitory axon guidance signals. Targeted inactivation of the neuropilin-1 gene in mice induced disorganization of the pathway and projection of nerve fibers, suggesting that neuropilin-1 mediates semaphorin-elicited signals and regulates nerve fiber guidance in embryogenesis. Neuropilin-1 is also expressed in endothelial cells and shown to bind vascular endothelial growth factor (VEGF), a potent regulator for vasculogenesis and angiogenesis. However, the roles of neuropilin-1 in vascular formation have been unclear. This paper reported that the neuropilin-1 mutant mouse embryos exhibited various types of vascular defects, including impairment in neural vascularization, agenesis and transposition of great vessels, insufficient aorticoplumonary truncus (persistent truncus arteriosus), and disorganized and insufficient development of vascular networks in the yolk sac. The vascular defects induced by neuropilin-1 deficiency in mouse embryos suggest that neuropilin-1 plays roles in embryonic vessel formation, as well as nerve fiber guidance.
The dorsal-open group gene raw is required for restricted DJNK signaling during closure
Authors: Byars Cherie Lynne , Bates Katherine L. , Letsou Anthea
Published: April 2021

During dorsal closure in Drosophila melanogaster, cells of the lateral epidermis migrate over the amnioserosa to encase the embryo. At least three classes of dorsal-open group gene products are necessary for this morphogenetic movement. Class I genes code for structural proteins that effect changes in epidermal cell shape and motility. Class II and III genes code for regulatory components of closure: Class II genes encode Drosophila Jun amino (N)- terminal kinase (DJNK) signaling molecules and Class III genes encode Decapentaplegic-mediated signaling molecules. All characterized dorsal-open group gene products function in the epidermis.

Here we report a molecular and genetic characterization of raw, a newly defined member of the Class II dorsal-open group genes. We show that the novel protein encoded by raw is required for restriction of DJNK signaling to leading edge epidermal cells as well as for proper development of the amnioserosa. Taken together, our results demonstrate a role for Raw in restriction of epidermal signaling during closure and suggest that this effect may be mediated via the amnioserosa.