bluuebunny/crossref_metadata_2025 huggingface dataset:
Positive and negative regulation of Easter, a member of the serine protease family that controls dorsal-ventral patterning in the Drosophila embryo
Authors: Misra Sima , Hecht Peter , Maeda Robert , Anderson Kathryn V.
Published: April 2021

The sequential activities of four members of the trypsin family of extracellular serine proteases are required for the production of the ventrally localized ligand that organizes the dorsal-ventral pattern of the Drosophila embryo. The last protease in this sequence is encoded by easter, which is a candidate to activate proteolytically the ligand encoded by spätzle. Here, we demonstrate biochemically that the zymogen form of Easter is processed in vivo by a proteolytic cleavage event that requires the three upstream proteases. Processed Easter is present in extremely low amounts in the early embryo because it is rapidly converted into a high molecular mass complex, which may contain a protease inhibitor. Easter zymogen activation is also controlled by a negative feedback loop from Dorsal, the transcription factor at the end of the signaling pathway. Each of these regulated biochemical processes is likely to be important in generating the ventral-to-dorsal gradient of Dorsal protein that organizes cell fates in the early embryo.
Nuclear-fallout, a Drosophila protein that cycles from the cytoplasm to the centrosomes, regulates cortical microfilament organization
Authors: Rothwell Wendy F. , Fogarty Patrick , Field Christine M. , Sullivan William
Published: April 2021

nuclear fallout (nuf) is a maternal effect mutation that specifically disrupts the cortical syncytial divisions during Drosophila embryogenesis. We show that the nuf gene encodes a highly phosphorylated novel protein of 502 amino acids with C-terminal regions predicted to form coiled-coils. During prophase of the late syncytial divisions, Nuf concentrates at the centrosomes and is generally cytoplasmic throughout the rest of the nuclear cycle. In nuf-derived embryos, the recruitment of actin from caps to furrows during prophase is disrupted. This results in incomplete metaphase furrows specifically in regions distant from the centrosomes. The nuf mutation does not disrupt anillin or peanut recruitment to the metaphase furrows indicating that Nuf is not involved in the signaling of metaphase furrow formation. These results also suggest that anillin and peanut localization are independent of actin localization to the metaphase furrows. nuf also disrupts the initial stages of cellularization and produces disruptions in cellularization furrows similar to those observed in the metaphase furrows. The localization of Nuf to centrosomal regions throughout cellularization suggests that it plays a similar role in the initial formation of both metaphase and cellularization furrows. A model is presented in which Nuf provides a functional link between centrosomes and microfilaments.
Characterization of the early development of specific hypaxial muscles from the ventrolateral myotome
Authors: Cinnamon Yuval , Kahane Nitza , Kalcheim Chaya
Published: April 2021

We have previously found that the myotome is formed by a first wave of pioneer cells generated along the medial epithelial somite and a second wave emanating from the dorsomedial lip (DML), rostral and caudal edges of the dermomyotome (Kahane, N., Cinnamon, Y. and Kalcheim, C. (1998a) Mech. Dev. 74, 59-73; Kahane, N., Cinnamon, Y. and Kalcheim, C. (1998b) Development 125, 4259-4271). In this study, we have addressed the development and precise fate of the ventrolateral lip (VLL) in non-limb regions of the axis. To this end, fluorescent vital dyes were iontophoretically injected in the center of the VLL and the translocation of labeled cells was followed by confocal microscopy. VLL-derived cells colonized the ventrolateral portion of the myotome. This occurred following an early longitudinal cell translocation along the medial boundary until reaching the rostral or caudal dermomyotome lips from which fibers emerged into the myotome. Thus, the behavior of VLL cells parallels that of their DML counterparts which colonize the opposite, dorsomedial portion of the myotome. To precisely understand the way the myotome expands,we addressed the early generation of hypaxial intercostal muscles. We found that intercostal muscles were formed by VLL-derived fibers that intermingled with fibers emerging from the ventrolateral aspect of both rostral and caudal edges of the dermomyotome. Notably, hypaxial intercostal muscles also contained pioneer myofibers (first wave) showing for the first time that lateral myotome-derived muscles contain a fundamental component of fibers generated in the medial domain of the somite. In addition, we show that during myotome growth and evolution into muscle, second-wave myofibers progressively intercalate between the pioneer fibers, suggesting a constant mode of myotomal expansion in its dorsomedial to ventrolateral extent. This further suggests that specific hypaxial muscles develop following a consistent ventral expansion of a ‘compound myotome’ into the somatopleure.
Dual role of the basic helix-loop-helix transcription factor scleraxis in mesoderm formation and chondrogenesis during mouse embryogenesis
Authors: Brown Doris , Wagner Dan , Li Xiang-qing , Richardson James A. , Olson Eric N.
Published: April 2021

Scleraxis is a basic helix-loop-helix (bHLH) transcription factor shown previously to be expressed in developing chondrogenic cell lineages during embryogenesis. To investigate its function in embryonic development, we produced scleraxis-null mice by gene targeting. Homozygous mutant embryos developed normally until the early egg cylinder stage (embryonic day 6.0), when they became growth-arrested and failed to gastrulate. Consistent with this early embryonic phenotype, scleraxis was found to be expressed throughout the embryo at the time of gastrulation before becoming restricted to chondrogenic precursor cells at embryonic day 9.5. At the time of developmental arrest, scleraxis-null embryos consisted of ectodermal and primitive endodermal cell layers, but lacked a primitive streak or recognizable mesoderm. Analysis of molecular markers of the three embryonic germ layers confirmed that scleraxis mutant embryos were unable to form mesoderm. By generating chimeric embryos, using lacZ-marked scleraxis-null and wild-type embryonic stem cells, we examined the ability of mutant cells to contribute to regions of the embryo beyond the time of lethality of homozygous mutants. Scleraxis-null cells were specifically excluded from the sclerotomal compartment of somites, which gives rise to the axial skeleton, and from developing ribs, but were able to contribute to most other regions of the embryo, including mesoderm-derived tissues. These results reveal an essential early role for scleraxis in mesoderm formation, as well as a later role in formation of somite-derived chondrogenic lineages, and suggest that scleraxis target genes mediate these processes.
The early-flowering mutant efs is involved in the autonomous promotion pathway of Arabidopsis thaliana
Authors: Soppe Wim J. J. , Bentsink Leónie , Koornneef Maarten
Published: April 2021

The transition to flowering is a crucial moment in a plant’s life cycle of which the mechanism has only been partly revealed. In a screen for early flowering, after mutagenesis of the late-flowering fwa mutant of Arabidopsis thaliana, the early flowering in short days (efs) mutant was identified. Under long-day light conditions, the recessive monogenic efs mutant flowers at the same time as wild type but, under short-day conditions, the mutant flowers much earlier. In addition to its early-flowering phenotype, efs has several pleiotropic effects such as a reduction in plant size, fertility and apical dominance. Double mutant analysis with several late-flowering mutants from the autonomous promotion (fca and fve) and the photoperiod promotion (co, fwa and gi) pathways of flowering showed that efs reduces the flowering time of all these mutants. However, efs is completely epistatic to fca and fve but additive to co, fwa and gi, indicating that EFS is an inhibitor of flowering specifically involved in the autonomous promotion pathway. A vernalisation treatment does not further reduce the flowering time of the efs mutant, suggesting that vernalisation promotes flowering through EFS. By comparing the length of the juvenile and adult phases of vegetative growth for wild-type, efs and the double mutant plants, it is apparent that efs mainly reduces the length of the adult phase.
Defining subregions of Hensen's node essential for caudalward movement, midline development and cell survival
Authors: Charrier J.B. , Teillet M.A. , Lapointe F. , Le Douarin N.M.
Published: April 2021

Hensen's node, also called the chordoneural hinge in the tail bud, is a group of cells that constitutes the organizer of the avian embryo and that expresses the gene HNF-3(β). During gastrulation and neurulation, it undergoes a rostral-to-caudal movement as the embryo elongates. Labeling of Hensen's node by the quail-chick chimera system has shown that, while moving caudally, Hensen's node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endodermal cells. In this work, we demonstrate that the node can be divided into functionally distinct subregions. Caudalward migration of the node depends on the presence of the most posterior region, which is closely apposed to the anterior portion of the primitive streak as defined by expression of the T-box gene Ch-Tbx6L. We call this region the axial-paraxial hinge because it corresponds to the junction of the presumptive midline axial structures (notochord and floor plate) and the paraxial mesoderm. We propose that the axial-paraxial hinge is the equivalent of the neuroenteric canal of other vertebrates such as Xenopus. Blocking the caudal movement of Hensen's node at the 5- to 6-somite stage by removing the axial-paraxial hinge deprives the embryo of midline structures caudal to the brachial level, but does not prevent formation of the neural tube and mesoderm located posteriorly. However, the whole embryonic region generated posterior to the level of Hensen's node arrest undergoes widespread apoptosis within the next 24 hours. Hensen's node-derived structures (notochord and floor plate) thus appear to produce maintenance factor(s) that ensures the survival and further development of adjacent tissues.
Spatial and temporal properties of ventral blood island induction in Xenopus laevis
Authors: Kumano Gaku , Belluzzi Lisa , Smith William C.
Published: April 2021

Questions of dorsoventral axis determination and patterning in Xenopus seek to uncover the mechanisms by which particular mesodermal fates, for example somite, are specified in the dorsal pole of the axis while other mesoderm fates, for example, ventral blood island (VBI), are specified at the ventral pole. We report here that the genes Xvent-1, Xvent-2, and Xwnt-8 do not appear to be in the pathway of VBI induction, contrary to previous reports. Results from the selective inhibition of bone morphogenetic protein (BMP) activity, a key regulator of VBI induction, by ectopic Noggin, Chordin, or dominant negative BMP ligands and receptors suggest an alternative route of VBI induction. Injection of noggin or chordin RNA into animal pole blastomeres effectively inhibited VBI development, while marginal zone injection had no effect. Cell autonomous inhibition of BMP activity in epidermis with dominant negative ligand dramatically reduced the amount of αT3 globin expression. These results indicate that signaling activity from the Spemann Organizer alone may not be sufficient for dorsoventral patterning in the marginal zone and that an inductive interaction between presumptive VBIs and ectoderm late in gastrulation may be crucial. In agreement with these observations, other results show that in explanted blastula-stage marginal zones a distinct pattern develops with a restricted VBI-forming region at the vegetal pole that is independent of the patterning activity of the Spemann Organizer.
Regulation of Hoxa2 in cranial neural crest cells involves members of the AP-2 family
Authors: Maconochie Mark , Krishnamurthy Ramachandran , Nonchev Stefan , Meier Pascal , Manzanares Miguel , Mitchell Pamela J. , Krumlauf Robb
Published: April 2021

Hoxa2 is expressed in cranial neural crest cells that migrate into the second branchial arch and is essential for proper patterning of neural-crest-derived structures in this region. We have used transgenic analysis to begin to address the regulatory mechanisms which underlie neural-crest-specific expression of Hoxa2. By performing a deletion analysis on an enhancer from the Hoxa2 gene that is capable of mediating expression in neural crest cells in a manner similar to the endogenous gene, we demonstrated that multiple cis-acting elements are required for neural-crest-specific activity. One of these elements consists of a sequence that binds to the three transcription factor AP-2 family members. Mutation or deletion of this site in the Hoxa2 enhancer abrogates reporter expression in cranial neural crest cells but not in the hindbrain. In both cell culture co-transfection assays and transgenic embryos AP-2 family members are able to trans-activate reporter expression, showing that this enhancer functions as an AP-2-responsive element in vivo. Reporter expression is not abolished in an AP-2α null mutant embryos, suggesting redundancy with other AP-2 family members for activation of the Hoxa2 enhancer. Other cis-elements identified in this study critical for neural-crest-specific expression include an element that influences levels of expression and a conserved sequence, which when multimerized directs expression in a broad subset of neural crest cells. These elements work together to co-ordinate and restrict neural crest expression to the second branchial arch and more posterior regions. Our findings have identified the cis-components that allow Hoxa2 to be regulated independently in rhombomeres and cranial neural crest cells.
The flat-top gene is required for the expansion and regionalization of the telencephalic primordium
Authors: Hentges Kathryn , Thompson Kevin , Peterson Andrew
Published: April 2021

The telencephalic vesicles form in the mouse embryo by the expansion of precursor regions in the anterior neural tube. Once the vesicles have formed, discrete dorsal and ventral territories can be recognized that later give rise to cortical and subcortical structures, respectively. To investigate the mechanisms that regulate the expansion and regionalization of the telencephalon, we have carried out a screen to identify recessive mutations that disrupt these events. We isolated a mouse mutant in which an early and critical step in development of the telencephalic vesicles is disrupted. Telencephalic primordia are present in flat-top embryos but they fail to progress to form the telencephalic vesicles. An increased rate of proliferation in the forebrain neurectoderm that accompanies telencephalic expansion in wild-type embryos fails to occur in flat-top embryos. Regionalization events that would normally take place during expansion of the primordia also fail to occur. Thus the phenotype of the flat-top mouse reveals that outgrowth of the telencephalic vesicles and their regionalization are coupled processes.
A multigene locus containing the Manx and bobcat genes is required for development of chordate features in the ascidian tadpole larva
Authors: Swalla Billie J. , Just Margaret A. , Pederson Erica L. , Jeffery William R.
Published: April 2021

The Manx gene is required for the development of the tail and other chordate features in the ascidian tadpole larva. To determine the structure of the Manx gene, we isolated and sequenced genomic clones from the tailed ascidian Molgula oculata. The Manx gene contains 9 exons and encodes both major and minor Manx mRNAs, which differ in the length of their 5′ untranslated regions. The coding region of the single-copy bobcat gene, which encodes a DEAD-box RNA helicase, is embedded within the first Manx intron. The organization of the bobcat and Manx transcription units was determined by comparing genomic and cDNA clones. The Manx-bobcat gene locus has an unusual organization in which a non-coding first exon is alternatively spliced at the 5′ end of two different mRNAs. The bobcat and Manx genes are expressed coordinately during oogenesis and embryogenesis, but not during spermatogenesis, in which bobcat Mrna accumulates independently of Manx mRNA. Similar to Manx, zygotic bobcat transcripts accumulate in the embryonic primordia responsible for generating chordate features, including the dorsal neural tube and notochord, are downregulated during embryogenesis in the tailless species Molgula occulta and are upregulated in M. occulta ×M. oculata hybrids, which restore these chordate features. Antisense experiments indicate that zygotic bobcat expression is required for development of the same suite of chordate features as Manx. The results show that the Manx-bobcat gene complex has a role in the development of chordate features in ascidian tadpole larvae.
Asymmetric cell division of thoracic neuroblast 6-4 to bifurcate glial and neuronal lineage in Drosophila
Authors: Akiyama-Oda Yasuko , Hosoya Toshihiko , Hotta Yoshiki
Published: April 2021

In the development of the Drosophila central nervous system, some of the neuroblasts designated as neuroglioblasts generate both glia and neurons. Little is known about how neuroglioblasts produce these different cell types. NB6-4 in the thoracic segment (NB6-4T) is a neuroglioblast, although the corresponding cell in the abdominal segment (NB6-4A) produces only glia. Here, we describe the cell divisions in the NB6-4T lineage, following changes in cell number and cell arrangement. We also examined successive changes in the expression of glial cells missing (gcm) mRNA and protein, activity of which is known to direct glial fate from the neuronal default state. The first cell division of NB6-4T occurred in the medial-lateral orientation, and was found to bifurcate the glial and neuronal lineage. After division, the medial daughter cell expressed GCM protein to produce three glial cells, while the lateral daughter cell with no GCM expression produced ganglion mother cells, secondary precursors of neurons. Although gcm mRNA was present evenly in the cytoplasm of NB6-4T before the first cell division, it became detected asymmetrically in the cell during mitosis and eventually only in the medial daughter cell. In contrast, NB6-4A showed a symmetrical distribution of gcm mRNA and GCM protein through division. Our observations suggest that mechanisms regulating gcm mRNA expression and its translation play an important role in glial and neuronal lineage bifurcation that results from asymmetric cell division.
The zebrafish unplugged gene controls motor axon pathway selection
Authors: Zhang Jing , Granato Michael
Published: April 2021

En route to their targets, motor axons encounter choice points at which they select their future path. Experimental studies predict that at each choice point specialized cells provide local guidance to pathfinding motor axons, however, the identity of these cells and their signals is unknown. Here, we identify the zebrafish unplugged gene as a key component for choice point navigation of pioneering motor axons. We show that in unplugged mutant embryos, motor neuron growth cones reach the choice point but make inappropriate pathway decisions. Analysis of chimeric embryos demonstrates that unplugged activity is produced by a selective group of mesodermal cells located adjacent to the choice point. As the first motor growth cones approach the choice point, these mesodermal cells migrate away, suggesting that unplugged activity influences growth cones by a contact-independent mechanism. These data suggest that unplugged defines a somite-derived signal that elicits differential guidance decisions in motor growth cones.
A transitional stage in the commitment of mesoderm to hematopoiesis requiring the transcription factor SCL/tal-1
Authors: Robertson Scott M. , Kennedy Marion , Shannon John M. , Keller Gordon
Published: April 2021

In this report, we describe the identification and characterization of an early embryoid body-derived colony, termed the transitional colony, which contains cell populations undergoing the commitment of mesoderm to the hematopoietic and endothelial lineages. Analysis of individual transitional colonies indicated that they express Brachyury as well as flk-1, SCL/tal-1, GATA-1, βH1 and βmajor reflecting the combination of mesodermal, hematopoietic and endothelial populations. This pattern differs from that found in the previously described hemangioblast-derived blast cell colonies in that they typically lacked Brachyury expression, consistent with their post-mesodermal stage of development (Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S., Kabrun, N. and Keller, G. (1997) Nature 386, 488-493). Replating studies demonstrated that transitional colonies contain low numbers of primitive erythroid precursors as well as a subset of precursors associated with early stage definitive hematopoiesis. Blast cell colonies contain higher numbers and a broader spectrum of definitive precursors than found in the transitional colonies. ES cells homozygous null for the SCL/tal-1 gene, a transcription factor known to be essential for development of the primitive and definitive hematopoietic systems, were not able to form blast colonies but did form transitional colonies. Together these findings suggest that the transitional colony represents a stage of development earlier than the blast cell colony and one that uniquely defines the requirement for a functional SCL/tal-1 gene for the progression to hematopoietic commitment.
The SCARFACE gene is required for cotyledon and leaf vein patterning
Authors: Deyholos Michael K. , Cordner Glen , Beebe Dwight , Sieburth Leslie E.
Published: April 2021

Mechanisms controlling vein patterning are poorly understood. We describe a recessive Arabidopsis mutant, scarface (sfc), which maps to chromosome 5. sfc mutants have vein pattern defects in cotyledons, leaves, sepals and petals. In contrast to the wild type, in which these organs all have linear veins that are continuous with at least one other vein, in sfc mutants these organs’ secondary and tertiary veins are largely replaced by small segments of discontinuous veins, which we call vascular islands. Patterning defects are manifest in cotyledon provascular tissue, suggesting that the patterning defect occurs early in organogenesis. sfc mutants have exaggerated responses to exogenous auxin. Analysis of monopteros (mpT370) sfc-1 double mutants suggested that SFC has partially overlapping functions with MP in patterning of both primary and secondary veins.
Dual origin and segmental organisation of the avian scapula
Authors: Huang Ruijin , Zhi Qixia , Patel Ketan , Wilting Jörg , Christ Bodo
Published: April 2021

Bones of the postcranial skeleton of higher vertebrates originate from either somitic mesoderm or somatopleural layer of the lateral plate mesoderm. Controversy surrounds the origin of the scapula, a major component of the shoulder girdle, with both somitic and lateral plate origins being proposed. Abnormal scapular development has been described in the naturally occurring undulated series of mouse mutants, which has implicated Pax1 in the formation of this bone. Here we addressed the development of the scapula, firstly, by analysing the relationship between Pax1 expression and chondrogenesis and, secondly, by determining the developmental origin of the scapula using chick quail chimeric analysis. We show the following. (1) The scapula develops in a rostral-to-caudal direction and overt chondrification is preceded by an accumulation of Pax1-expressing cells. (2) The scapular head and neck are of lateral plate mesodermal origin. (3) In contrast, the scapular blade is composed of somitic cells. (4) Unlike the Pax1-positive cells of the vertebral column, which are of sclerotomal origin, the Pax1-positive cells of the scapular blade originate from the dermomyotome. (5) Finally, we show that cells of the scapular blade are organised into spatially restricted domains along its rostrocaudal axis in the same order as the somites from which they originated. Our results imply that the scapular blade is an ossifying muscular insertion rather than an original skeletal element, and that the scapular head and neck are homologous to the ‘true coracoid’ of higher vertebrates.
The combgap locus encodes a zinc-finger protein that regulates cubitus interruptus during limb development in Drosophila melanogaster
Authors: Svendsen Pia C. , Marshall Sean D. G. , Kyba Michael , Brook William J.
Published: April 2021

The combgap locus, first described by C. B. Bridges in 1925, is a gene required for proper anteroposterior pattern formation in the limbs of Drosophila melanogaster. The development of the anteroposterior axis of fly limbs is initiated by hedgehog signaling from cells of the posterior half to cells of the anterior half of the limb primordium. Hedgehog signaling requires the anterior-specific expression of the gene cubitus interruptus to establish posterior-specific hedgehog secretion and anterior-specific competence to respond to hedgehog. We have cloned combgap and find that it encodes a chromosomal protein with 11 C2H2 zinc fingers. Limb defects found in combgap mutants consist of either loss or duplication of pattern elements in the anteroposterior axis and can be explained through the inappropriate expression of cubitus interruptus and its downstream target genes. In combgap mutants, cubitus interruptus is ectopically expressed in the posterior compartments of wing imaginal discs and is downregulated in the anterior compartment of legs, wings and antennae. We are able to rescue anterior compartment combgap phenotypes by expressing additional cubitus interruptus using the Gal4/UAS system. Dominant alleles of cubitus interruptus, which result in posterior expression, phenocopy combgap posterior compartment phenotypes. Finally, we find that the combgap protein binds to polytene chromosomes at many sites including the cubitus interruptus locus, suggesting that it could be a direct regulator of cubitus interruptus transcription.
X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation
Authors: Okamoto Ikuhiro , Tan Seong-Seng , Takagi Nobuo
Published: April 2021

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6.5.
Control of tracheal tubulogenesis by Wingless signaling
Authors: Chihara Takahiro , Hayashi Shigeo
Published: April 2021

The tubular epithelium of the Drosophila tracheal system forms a network with a stereotyped pattern consisting of cells and branches with distinct identity. The tracheal primordium undergoes primary branching induced by the FGF homolog Branchless, differentiates cells with specialized functions such as fusion cells, which perform target recognition and adhesion during branch fusion, and extends branches toward specific targets. Specification of a unique identity for each primary branch is essential for directed migration, as a defect in either the EGFR or the Dpp pathway leads to a loss of branch identity and the misguidance of tracheal cell migration. Here, we investigate the role of Wingless signaling in the specification of cell and branch identity in the tracheal system. Wingless and its intracellular signal transducer, Armadillo, have multiple functions, including specifying the dorsal trunk through activation of Spalt expression and inducing differentiation of fusion cells in all fusion branches. Moreover, we show that Wingless signaling regulates Notch signaling by stimulating Delta expression at the tip of primary branches. These activities of Wingless signaling together specify the shape of the dorsal trunk and other fusion branches.
Brain derived neurotrophic factor is an endothelial cell survival factor required for intramyocardial vessel stabilization
Authors: Donovan Michael J. , Lin Michelle I. , Wiegn Phi , Ringstedt Thomas , Kraemer Rosemary , Hahn Rebecca , Wang Shiyang , Ibañez Carlos F. , Rafii Shahin , Hempstead Barbara L.
Published: April 2021

Brain derived neurotrophic factor, BDNF, is a neurotrophin best characterized for its survival and differentiative effects on neurons expressing the trk B receptor tyrosine kinase. Although many of these neurons are lost in the BDNF−/− mouse, the early postnatal lethality of these animals suggests a wider function for this growth factor. Here, we demonstrate that deficient expression of BDNF impairs the survival of endothelial cells in intramyocardial arteries and capillaries in the early postnatal period, although the embryonic vasculature can remodel into arteries, capillaries and veins. BDNF deficiency results in a reduction in endothelial cell-cell contacts and in endothelial cell apoptosis, leading to intraventricular wall hemorrhage, depressed cardiac contractility and early postnatal death. Vascular hemorrhage is restricted to cardiac vessels, reflecting the localized expression of BDNF and trk B by capillaries and arterioles in this vascular bed. Conversely, ectopic BDNF overexpression in midgestational mouse hearts results in an increase in capillary density. Moreover, BDNF activation of endogenous trk B receptors supports the survival of cardiac microvascular endothelial cells cultured from neonatal mice. These results establish an essential role for BDNF in maintaining vessel stability in the heart through direct angiogenic actions on endothelial cells.
Two homeobox genes define the domain of EphA3 expression in the developing chick retina
Authors: Schulte Dorothea , Cepko Constance L.
Published: April 2021

Graded expression of the Eph receptor EphA3 in the retina and its two ligands, ephrin A2 and ephrin A5 in the optic tectum, the primary target of retinal axons, have been implicated in the formation of the retinotectal projection map. Two homeobox containing genes, SOHo1 and GH6, are expressed in a nasal-high, temporal-low pattern during early retinal development, and thus in opposing gradients to EphA3. Retroviral misexpression of SOHo1 or GH6 completely and specifically repressed EphA3 expression in the neural retina, but not in other parts of the central nervous system, such as the optic tectum. Under these conditions, some temporal ganglion cell axons overshot their expected termination zones in the rostral optic tectum, terminating aberrantly at more posterior locations. However, the majority of ganglion cell axons mapped to the appropriate rostrocaudal locations, although they formed somewhat more diffuse termination zones. These findings indicate that other mechanisms, in addition to differential EphA3 expression in the neural retina, are required for retinal ganglion axons to map to the appropriate rostrocaudal locations in the optic tectum. They further suggest that the control of topographic specificity along the retinal nasal-temporal axis is split into several independent pathways already at a very early time in development.
Early myotome specification regulates PDGFA expression and axial skeleton development
Authors: Tallquist Michelle D. , Weismann Karin E. , Hellström Mats , Soriano Philippe
Published: April 2021

Reciprocal defects in signaling between the myotome and the sclerotome compartments of the somites in PDGFRαand Myf5 mutant embryos lead to alterations in the formation of the vertebrae and the ribs. To investigate the significance of these observations, we have examined the role of PDGF signaling in the developing somite. PDGFA ligand expression was not detected in the myotome of Myf5 null mutant embryos and PDGFA promoter activity was regulated by Myf5 in vitro. PDGFA stimulated chondrogenesis in somite micromass cultures as well as in embryos when PDGFA was knocked into the Myf5 locus, resulting in increased vertebral and rib development. PDGFA expression in the myotome was fully restored in embryos in which MyoD has been introduced at the Myf5 locus but to a lesser extent in similar myogenin knock-in embryos. These results underscore the importance of growth factor signaling within the developing somite and suggest an important role for myogenic determination factors in orchestrating normal development of the axial skeleton.
The zebrafish colourless gene regulates development of non-ectomesenchymal neural crest derivatives
Authors: Kelsh Robert N. , Eisen Judith S.
Published: April 2021

Neural crest forms four major categories of derivatives: pigment cells, peripheral neurons, peripheral glia, and ectomesenchymal cells. Some early neural crest cells generate progeny of several fates. How specific cell fates become specified is still poorly understood. Here we show that zebrafish embryos with mutations in the colourless gene have severe defects in most crest-derived cell types, including pigment cells, neurons and specific glia. In contrast, craniofacial skeleton and medial fin mesenchyme are normal. These observations suggest that colourless has a key role in development of non-ectomesenchymal neural crest fates, but not in development of ectomesenchymal fates. Thus, the cls mutant phenotype reveals a segregation of ectomesenchymal and non-ectomesenchymal fates during zebrafish neural crest development. The combination of pigmentation and enteric nervous system defects makes colourless mutations a model for two human neurocristopathies, Waardenburg-Shah syndrome and Hirschsprung’s disease.
N-terminal fatty-acylation of sonic hedgehog enhances the induction of rodent ventral forebrain neurons
Authors: Kohtz Jhumku D. , Lee Hae Young , Gaiano Nicholas , Segal Joshua , Ng Evan , Larson Theresa , Baker Darren P. , Garber Ellen A. , Williams Kevin P. , Fishell Gord
Published: April 2021

The adult basal ganglia arise from the medial and lateral ganglionic eminences, morphologically distinct structures found in the embryonic telencephalon. We have previously shown that temporal changes in sonic hedgehog (Shh) responsiveness determine the sequential induction of embryonic neurons that populate the medial and lateral ganglionic eminences. In this report, we show that Shh-mediated differentiation of neurons that populate the lateral ganglionic eminence express different combinations of the homeobox-containing transcription factors Dlx, Mash1 and Islet 1/2. Furthermore, we show that N-terminal fatty-acylation of Shh significantly enhances its ability to induce the differentiation of rat E11 telencephalic neurons expressing Dlx, Islet 1/2 or Mash1. Recent evidence indicates that in utero injection of the E9.5 mouse forebrain with retroviruses encoding wild-type Shh induces the ectopic expression of Dlx2 and severe deformities in the brain. In this report, we show that Shh containing a mutation at the site of acylation prevents either of these phenotypes. These results suggest that N-terminal fatty-acylation of Shh may play an important role in Shh-dependent signaling during rodent ventral forebrain formation.
Emx2directs the development of diencephalon in cooperation withOtx2
Authors: Suda Yoko , Hossain Zakir M. , Kobayashi Chiyoko , Hatano Osamu , Yoshida Michio , Matsuo Isao , Aizawa Shinichi
Published: April 2021

The vertebrate brain is among the most complex biological structures of which the organization remains unclear. Increasing numbers of studies have accumulated on the molecular basis of midbrain/hindbrain development, yet relatively little is known about forebrain organization. Nested expression among Otx and Emx genes has implicated their roles in rostral brain regionalization, but single mutant phenotypes of these genes have not provided sufficient information. In order to genetically determine the interaction between Emx and Otx genes in forebrain development, we have examined Emx2−/−Otx2+/− double mutants and Emx2 knock-in mutants into the Otx2 locus (Otx2+/Emx2). Emx2−/−Otx2+/− double mutants did not develop diencephalic structures such as ventral thalamus, dorsal thalamus/epithalamus and anterior pretectum. The defects were attributed to the loss of the Emx2-positive region at the three- to four-somite stage, when its expression occurs in the laterocaudal forebrain primordia. Ventral structures such as the hypothalamus, mammillary region and tegmentum developed normally. Moreover, dorsally the posterior pretectum and posterior commissure were also present in the double mutants. In contrast, Otx2+/Emx2 knock-in mutants displayed the majority of these diencephalic structures; however, the posterior pretectum and posterior commissure were specifically absent. Consequently, development of the dorsal and ventral thalamus and anterior pretectum requires cooperation between Emx2 and Otx2, whereas Emx2 expression is incompatible with development of the commissural region of the pretectum.
Patterning of fast and slow fibers within embryonic muscles is established independently of signals from the surrounding mesenchyme
Authors: Nikovits William , Cann Gordon M. , Huang Ruijin , Christ Bodo , Stockdale Frank E.
Published: April 2021

During embryonic development, and before functional innervation, a highly stereotypic pattern of slow- and fast-contracting primary muscle fibers is established within individual muscles of the limbs, from distinct populations of myoblasts. A difference between the fiber-type pattern found within chicken and quail pectoral muscles was exploited to investigate the contributions of somite-derived myogenic precursors and lateral plate-derived mesenchymal stroma to the establishment of muscle fiber-type patterns. Chimeric chicken/quail embryos were constructed by reciprocal transplantation of somites or lateral plate mesoderm at stages prior to muscle formation. Muscle fibers derived from quail myogenic precursors that had migrated into chicken stroma showed a quail pattern of mixed fast- and slow-contracting muscle fibers. Conversely, chicken myogenic precursors that had migrated into quail stroma showed a chicken pattern of nearly exclusive fast muscle fiber formation. These results demonstrate in vivo an intrinsic commitment to fiber-type on the part of the myoblast, independent of extrinsic signals it receives from the mesenchymal stroma in which it differentiates.
Developmental regulation of the heat shock response by nuclear transport factor karyopherin-α3
Authors: Fang Xiang-dong , Chen Tianxin , Tran Kim , Parker Carl S.
Published: April 2021

During early stages of Drosophila development the heat-shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a Drosophila nuclear transport protein karyopherin-α3 (dKap-α3). Biochemical methods demonstrate that the dKap-α3 protein binds specifically to the dHSF’s nuclear localization sequence (NLS). Furthermore, the dKap-α3 protein does not associate with NLSs that contain point mutations, which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-α3. Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-α3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.
Regionalisation of anterior neuroectoderm and its competence in responding to forebrain and midbrain inducing activities depend on mutual antagonism between OTX2 and GBX2
Authors: Martinez-Barbera Juan Pedro , Signore Massimo , Boyl Pietro Pilo , Puelles Eduardo , Acampora Dario , Gogoi Robin , Schubert Frank , Lumsden Andrew , Simeone Antonio
Published: April 2021

The anterior neural ridge (ANR), and the isthmic organiser (IsO) represent two signalling centres possessing organising properties necessary for forebrain (ANR) as well as midbrain and rostral hindbrain (IsO) development. An important mediator of ANR and IsO organising property is the signalling molecule FGF8. Previous work has indicated that correct positioning of the IsO and Fgf8 expression in this domain is controlled by the transcription factors Otx2 and Gbx2. In order to provide novel insights into the roles of Otx2 and Gbx2, we have studied mutant embryos carrying different dosages of Otx2, Otx1 and Gbx2. Embryos deficient for both OTX2 and GBX2 proteins (hOtx12/hOtx12; Gbx2–/–) show abnormal patterning of the anterior neural tissue, which is evident at the presomite-early somite stage prior to the onset of Fgf8 neuroectodermal expression. Indeed, hOtx12/hOtx12; Gbx2–/– embryos exhibit broad co-expression of early forebrain, midbrain and rostral hindbrain markers such as hOtx1, Gbx2, Pax2, En1 and Wnt1 and subsequently fail to activate forebrain and midbrain-specific gene expression. In this genetic context, Fgf8 is expressed throughout the entire anterior neural plate, thus indicating that its activation is independent of both OTX2 and GBX2 function. Analysis of hOtx12/hOtx12; Gbx2–/– and Otx1+/–; Otx2+/– mutant embryos also suggests that FGF8 cannot repress Otx2 without the participation of GBX2. Finally, we report that embryos carrying a single strong hypomorphic Otx2 allele (Otx2λ) in an Otx2 and Gbx2 null background (Otx2λ/–; Gbx2–/–) recover both the headless phenotype exhibited by Otx2λ/– embryos and forebrain- and midbrain-specific gene expression that is not observed in hOtx12/hOtx12; Gbx2–/– mutants. Together, these data provide novel genetic evidence indicating that OTX2 and GBX2 are required for proper segregation of early regional identities anterior and posterior to the mid-hindbrain boundary (MHB) and for conferring competence to the anterior neuroectoderm in responding to forebrain-, midbrain- and rostral hindbrain-inducing activities.
Overlapping functions of lysosomal acid phosphatase (LAP) and tartrate-resistant acid phosphatase (Acp5) revealed by doubly deficient mice
Authors: Suter Anke , Everts Vincent , Boyde Alan , Jones Sheila J. , Lüllmann-Rauch Renate , Hartmann Dieter , Hayman Alison R. , Cox Timothy M. , Evans Martin J. , Meister Tobias , von Figura Kurt , Saftig Paul
Published: April 2021

To date, two lysosomal acid phosphatases are known to be expressed in cells of the monocyte/phagocyte lineage: the ubiquitously expressed lysosomal acid phosphatase (LAP) and the tartrate-resistant acid phosphatase-type 5 (Acp5). Deficiency of either acid phosphatase results in relatively mild phenotypes, suggesting that these enzymes may be capable of mutual complementation. This prompted us to generate LAP/Acp5 doubly deficient mice. LAP/Acp5 doubly deficient mice are viable and fertile but display marked alterations in soft and mineralised tissues. They are characterised by a progressive hepatosplenomegaly, gait disturbances and exaggerated foreshortening of long bones. Histologically, these animals are distinguished by an excessive lysosomal storage in macrophages of the liver, spleen, bone marrow, kidney and by altered growth plates. Microscopic analyses showed an accumulation of osteopontin adjacent to actively resorbing osteoclasts of Acp5- and LAP/Acp5-deficient mice. In osteoclasts of phosphatase-deficient mice, vacuoles were frequently found which contained fine filamentous material. The vacuoles in Acp5- and LAP/Acp5 doubly-deficient osteoclasts also contained crystallite-like features, as well as osteopontin, suggesting that Acp5 is important for processing of this protein. This is further supported by biochemical analyses that demonstrate strongly reduced dephosphorylation of osteopontin incubated with LAP/Acp5-deficient bone extracts. Fibroblasts derived from LAP/Acp5 deficient embryos were still able to dephosphorylate mannose 6-phosphate residues of endocytosed arylsulfatase A. We conclude that for several substrates LAP and Acp5 can substitute for each other and that these acid phosphatases are essential for processing of non-collagenous proteins, including osteopontin, by osteoclasts.
Slit proteins are not dominant chemorepellents for olfactory tract and spinal motor axons
Authors: Patel Kalpana , Nash Julia A. B. , Itoh Akira , Liu Zhe , Sundaresan Vasi , Pini Adrian
Published: April 2021

Members of the Slit family are large extracellular glycoproteins that may function as chemorepellents in axon guidance and neuronal cell migration. Their actions are mediated through members of the Robo family that act as their receptors. In vertebrates, Slit causes chemorepulsion of embryonic olfactory tract, spinal motor, hippocampal and retinal ganglion cell axons. Since Slits are expressed in the septum and floor plate during the period when these tissues cause chemorepulsion of olfactory tract and spinal motor axons respectively, it has been proposed that Slits function as guidance cues. We have tested this hypothesis in collagen gel co-cultures using soluble Robo/Fc chimeras, as competitive inhibitors, to disrupt Slit interactions. We find that the addition of soluble Robo/Fc has no effect on chemorepulsion of olfactory tract and spinal motor axons when co-cultured with septum or floor plate respectively. Thus, we conclude that although Slits are expressed in the septum and floor plate, their proteins do not contribute to the major chemorepulsive activities emanating from these tissues which cause repulsion of olfactory tract and spinal motor axons.