bluuebunny/crossref_metadata_2025 huggingface dataset:
Cubitus interruptus-independent transduction of the Hedgehog signal in Drosophila
Authors: Gallet Armel , Angelats Corinne , Kerridge Stephen , Thérond Pascal P.
Published: April 2021

The Hedgehog (Hh) family of secreted proteins are key factors that control pattern formation in invertebrates and vertebrates. The manner in which Hh molecules regulate a target cell remains poorly understood. In the Drosophila embryo, Hh is produced in identical stripes of cells in the posterior compartment of each segment. From these cells a Hh signal acts in both anterior and posterior directions. In the anterior cells, the target genes wingless and patched are activated whereas posterior cells respond to Hh by expressing rhomboid and patched. Here, we have examined the role of the transcription factor Cubitus interruptus (Ci) in this process.

So far, Ci has been thought to be the most downstream component of the Hh pathway capable of activating all Hh functions. However, our current study of a null ci allele, indicates that it is actually not required for all Hh functions. Whereas Hh and Ci are both required for patched expression, the target genes wingless and rhomboid have unequal requirements for Hh and Ci activity.

Hh is required for the maintenance of wingless expression before embryonic stage 11 whereas Ci is necessary only later during stage 11. For rhomboid expression Hh is required positively whereas Ci exhibits negative input. These results indicate that factors other than Ci are necessary for Hh target gene regulation. We present evidence that the zinc-finger protein Teashirt is one candidate for this activity. We show that it is required positively for rhomboid expression and that Teashirt and Ci act in a partially redundant manner before stage 11 to maintain wingless expression in the trunk.
Combinatorial signaling through BMP receptor IB and GDF5: shaping of the distal mouse limb and the genetics of distal limb diversity
Authors: Baur Scott T. , Mai Jia J. , Dymecki Susan M.
Published: April 2021

In this study, we use a mouse insertional mutant to delineate gene activities that shape the distal limb skeleton. A recessive mutation that results in brachydactyly was found in a lineage of transgenic mice. Sequences flanking the transgene insertion site were cloned, mapped to chromosome 3, and used to identify the brachydactyly gene as the type IB bone morphogenetic protein receptor, BmprIB (ALK6). Expression analyses in wild-type mice revealed two major classes of BmprIB transcripts. Rather than representing unique coding RNAs generated by alternative splicing of a single pro-mRNA transcribed from one promoter, the distinct isoforms reflect evolution of two BmprIB promoters: one located distally, driving expression in the developing limb skeleton, and one situated proximally, initiating transcription in neural epithelium. The distal promoter is deleted in the insertional mutant, resulting in a regulatory allele (BmprIBTg) lacking cis-sequences necessary for limb BmprIB expression. Mutants fail to generate digit cartilage, indicating that BMPRIB is the physiologic transducer for the formation of digit cartilage from the skeletal blastema. Expansion of BmprIB expression into the limb through acquisition of these distal cis-regulatory sequences appears, therefore, to be an important genetic component driving morphological diversity in distal extremities. GDF5 is a BMP-related signal, which is also required for proper digit formation. Analyses incorporating both Gdf5 and BmprIBTg alleles revealed that BMPRIB regulates chondrogenesis and segmentation through both GDF5-dependent and-independent processes, and that, reciprocally, GDF5 acts through both IB and other type I receptors. Together, these findings provide in vivo support for the concept of combinatorial BMP signaling, in which distinct outcomes result both from a single receptor being triggered by different ligands and from a single ligand binding to different receptors.
The bromodomain protein LIN-49 and trithorax-related protein LIN-59 affect development and gene expression in Caenorhabditis elegans
Authors: Chamberlin Helen M. , Thomas James H.
Published: April 2021

We have molecularly characterized the lin-49 and lin-59 genes in C. elegans, and found their products are related to Drosophila trithorax group (trx-G) proteins and other proteins implicated in chromatin remodelling. LIN-49 is structurally most similar to the human bromodomain protein BR140, and LIN-59 is most similar to the Drosophila trx-G protein ASH1. In C. elegans, lin-49 and lin-59 are required for the normal development of the mating structures of the adult male tail, for the normal morphology and function of hindgut (rectum) cells in both males and hermaphrodites and for the maintenance of structural integrity in the hindgut and egg-laying system in adults. Expression of the Hox genes egl-5 and mab-5 is reduced in lin-49 and lin-59 mutants, suggesting lin-49 and lin-59 regulate HOM-C gene expression in C. elegans as the trx-G genes do in Drosophila. lin-49 and lin-59 transgenes are expressed widely throughout C. elegans animals. Thus, in contrast to the C. elegans Polycomb group (Pc-G)-related genes mes-2 and mes-6 that function primarily in the germline, we propose lin-49 and lin-59 function in somatic development similar to the Drosophila trx-G genes.
Post-translational control of occludin membrane assembly in mouse trophectoderm: a mechanism to regulate timing of tight junction biogenesis and blastocyst formation
Authors: Sheth Bhavwanti , Moran Breda , Anderson James M. , Fleming Tom P.
Published: April 2021

The mouse blastocyst forms during the 32-cell stage with the emergence of the blastocoelic cavity. This developmental transition is dependent upon the differentiation and transport function of the trophectoderm epithelium which forms the wall of the blastocyst and exhibits functional intercellular tight junctions (TJs) to maintain epithelial integrity during blastocoele expansion. To investigate mechanisms regulating the timing of blastocyst formation, we have examined the dynamics of expression of occludin, an integral membrane protein of the TJ. Confocal microscopy of intact embryos and synchronised cell clusters revealed that occludin first assembles at the apicolateral membrane contact site between nascent trophectoderm cells usually during the early 32-cell stage, just prior to the time of blastocoele cavitation. This is a late event in the assembly of TJ-associated proteins within trophectoderm which, from our previous data, spans from 8-to 32-cell stages. Occludin membrane assembly is dependent upon prior E-cadherin-mediated cell-cell adhesion and is sensitive to brefeldin A, an inhibitor of Golgi-to-membrane transport. Occludin is delivered to the TJ site in association with the TJ plaque protein, ZO-1α+, which we have shown previously is newly transcribed and translated during late cleavage. Immediately after assembly and before cavitation, occludin localised at the TJ site switches from a Triton X-100-soluble to -insoluble form indicative of actin cytoskeletal and/or membrane anchorage. Occludin mRNA and protein are detectable throughout cleavage by RT-PCR and immunoblotting, respectively, indicating that timing of membrane assembly is not controlled by expression alone. Rather, we have identified changes in the pattern of different occludin forms expressed during cleavage which, using phosphatase treatment of embryo lysates, include post-translational modifications. We propose that the phosphorylation of one form of occludin (band 2, 65-67 kDa) during late cleavage, which leads to its exclusive conversion from a Triton X-100-soluble to-insoluble pool, may regulate occludin association with ZO-1α+ and membrane assembly, and thereby act to control completion of TJ biogenesis and the timing of blastocyst formation.
Pax2 and homeodomain proteins cooperatively regulate a 435 bp enhancer of the mouse Pax5 gene at the midbrain-hindbrain boundary
Authors: Pfeffer Peter L. , Bouchard Maxime , Busslinger Meinrad
Published: April 2021

Pax and homeodomain transcription factors are essential for the formation of an organizing center at the midbrain-hindbrain boundary (mhb) which controls the genesis of the midbrain and cerebellum in the vertebrate embryo. Pax2 and Pax5 are sequentially activated in this brain region, with Pax2 expression preceding that of Pax5. Using a transgenic reporter assay, we have now identified a conserved 435 bp enhancer in the 5′ flanking region of mammalian Pax5 genes which directs lacZ expression in the correct temporal and spatial pattern at the mhb. This minimal enhancer is composed of two distinct elements, as shown by protein-binding assays with mhb-specific extracts. The proximal element contains overlapping consensus binding sites for members of the Pax2/5/8 and POU protein families, whereas a distal element is bound by homeodomain and zinc finger transcription factors. Expression analysis of transgenes carrying specific mutations in these recognition motifs identified the Pax-and homeodomain-binding sites as functional elements which cooperatively control the activity of the mhb enhancer. lacZ genes under the control of either the minimal enhancer or the endogenous Pax5 locus were normally expressed at the mhb in Pax5 mutant embryos, indicating that this enhancer does not depend on autoregulation by Pax5. In Pax2 mutant embryos, expression of the endogenous Pax5 gene was, however, delayed and severely reduced in lateral aspects of the neural plate which, on neural tube closure, becomes the dorsal mhb region. This cross-regulation by Pax2 is mediated by the Pax-binding site of the minimal enhancer which, upon specific mutation, resulted in severely reduced transgene expression in the dorsal part of the mhb. Together these data indicate that Pax2 and homeodomain proteins directly bind to and cooperatively regulate the mhb enhancer of Pax5.
Winged helix transcription factor Foxb1 is essential for access of mammillothalamic axons to the thalamus
Authors: Alvarez-Bolado Gonzalo , Zhou Xunlei , Voss Anne K. , Thomas Tim , Gruss Peter
Published: April 2021

Our aim was to study the mechanisms of brain histogenesis. As a model, we have used the role of winged helix transcription factor gene Foxb1 in the emergence of a very specific morphological trait of the diencephalon, the mammillary axonal complex. Foxb1 is expressed in a large hypothalamic neuronal group (the mammillary body), which gives origin to a major axonal bundle with branches to thalamus, tectum and tegmentum. We have generated mice carrying a targeted mutation of Foxb1 plus the tau-lacZ reporter. In these mutants, a subpopulation of dorsal thalamic ventricular cells (“thalamic palisade”) show abnormal persistence of Foxb1 transcriptional activity; the thalamic branch of the mammillary axonal complex is not able to grow past these cells and enter the thalamus. The other two branches of the mammillary axonal complex (to tectum and tegmentum) are unaffected by the mutation. Most of the neurons that originate the mammillothalamic axons suffer apoptosis after navigational failure. Analysis of chimeric brains with wild-type and Foxb1 mutant cells suggests that correct expression of Foxb1 in the thalamic palisade is sufficient to rescue the normal phenotype. Our results indicate that Foxb1 is essential for diencephalic histogenesis and that it exerts its effects by controlling access to the target by one particular axonal branch.
Different roles forPax6in the optic vesicle and facial epithelium mediate early morphogenesis of the murine eye
Authors: Collinson J. Martin , Hill Robert E. , West John D.
Published: April 2021

Chimaeric mice were made by aggregating Pax6−/−and wild-type mouse embryos, in order to study the interaction between the optic vesicle and the prospective lens epithelium during early stages of eye development. Histological analysis of the distribution of homozygous mutant cells in the chimaeras showed that the cell- autonomous removal of Pax6−/−cells from the lens, shown previously at E12.5, is nearly complete by E9.5. Most mutant cells are eliminated from an area of facial epithelium wider than, but including, the developing lens placode. This result suggests a role for Pax6 in maintaining a region of the facial epithelium that has the tissue competence to undergo lens differentiation.

Segregation of wild-type and Pax6−/−cells occurs in the optic vesicle at E9.5 and is most likely a result of different adhesive properties of wild-type and mutant cells. Also, proximo-distal specification of the optic vesicle (as assayed by the elimination of Pax6−/−cells distally), is disrupted in the presence of a high proportion of mutant cells. This suggests that Pax6 operates during the establishment of patterning along the proximo-distal axis of the vesicle.

Examination of chimaeras with a high proportion of mutant cells showed that Pax6 is required in the optic vesicle for maintenance of contact with the overlying lens epithelium. This may explain why Pax6−/−optic vesicles are inefficient at inducing a lens placode. Contact is preferentially maintained when the lens epithelium is also wild-type.

Together, these results demonstrate requirements for functional Pax6 in both the optic vesicle and surface epithelia in order to mediate the interactions between the two tissues during the earliest stages of eye development.
Endodermal Nodal-related signals and mesoderm induction in Xenopus
Authors: Agius Eric , Oelgeschläger Michael , Wessely Oliver , Kemp Caroline , Robertis E. M. De
Published: April 2021

In Xenopus, mesoderm induction by endoderm at the blastula stage is well documented, but the molecular nature of the endogenous inductive signals remains unknown. The carboxy-terminal fragment of Cerberus, designated Cer-S, provides a specific secreted antagonist of mesoderm-inducing Xenopus Nodal-Related (Xnr) factors. Cer-S does not inhibit signalling by other mesoderm inducers such as Activin, Derrière, Vg1 and BMP4, nor by the neural inducer Xnr3. In the present study we show that Cer-S blocks the induction of both dorsal and ventral mesoderm in animal-vegetal Nieuwkoop-type recombinants. During blastula stages Xnr1, Xnr2 and Xnr4 are expressed in a dorsal to ventral gradient in endodermal cells. Dose-response experiments using cer-S mRNA injections support the existence of an endogenous activity gradient of Xnrs. Xnr expression at blastula can be activated by the vegetal determinants VegT and Vg1 acting in synergy with dorsal β-catenin. The data support a modified model for mesoderm induction in Xenopus, in which mesoderm induction is mediated by a gradient of multiple Nodal-related signals released by endoderm at the blastula stage.
Regulation of the early expression of the Xenopus nodal-related 1 gene, Xnr1
Authors: Hyde Cheryl E. , Old Robert W.
Published: April 2021

The Xenopus nodal related-1 (Xnr1) gene has a complex expression pattern in embryos, with two temporal phases. In the first phase, transcripts are first detected in perinuclear sites in the vegetal region of the blastula. During gastrulation, this expression disappears and transcripts become localised to the dorsal marginal zone. Expression stops and then restarts in a second phase at neurula and tailbud stages, firstly in two symmetric patches near the posterior end of the notochord, and then asymmetrically in a large domain in the left lateral plate mesoderm. In this study, we have investigated the regulation of the early phase of expression of Xnr1. We show that the T-box transcription factor VegT can induce Xnr1. It had previously been shown that Xnr1 can induce VegT in ectoderm cells and we show that the early expression of Xnr1 is regulated by an autoregulatory loop. By inspection of the Xnr1 promoter sequence, we have identified two non-palindromic T-box-binding sites, which are 10 bp apart. Using mutational analysis, we have shown that these elements are required for the VegT induction of Xnr1. The Xnr1 promoter shows striking homologies with the Xnr3 promoter. In particular, two elements that are required for Wnt signaling are conserved between these two promoters, but the two T-box sites are not conserved, and Xnr3 is not induced by VegT. A region of the promoter containing the T-box sites and the Wnt sites is sufficient to drive expression of a reporter gene in a dorsal domain in transgenic Xenopus at the gastrula stage. We show that this pattern of expression of the transgene in gastrulae is not dependent on the T-box sites.
Distinct effects of XBF-1 in regulating the cell cycle inhibitor p27XIC1 and imparting a neural fate
Authors: Hardcastle Zoë , Papalopulu Nancy
Published: April 2021

XBF-1 is an anterior neural plate-specific, winged helix transcription factor that affects neural development in a concentration-dependent manner. A high concentration of XBF-1 results in suppression of endogenous neuronal differentiation and an expansion of undifferentiated neuroectoderm. Here we investigate the mechanism by which this expansion is achieved. Our findings suggest that XBF-1 converts ectoderm to a neural fate and it does so independently of any effects on the mesoderm. In addition, we show that a high dose of XBF-1 promotes the proliferation of neuroectodermal cells while a low dose inhibits ectodermal proliferation. Thus, the neural expansion observed after high dose XBF-1 misexpression is due both to an increase in the number of ectodermal cells devoted to a neural fate and an increase in their proliferation. We show that the effect on cell proliferation is likely to be mediated by p27XIC1, a cyclin-dependent kinase (cdk) inhibitor. We show that p27XIC1 is expressed in a spatially restricted pattern in the embryo, including the anterior neural plate, and when misexpressed it is sufficient to block the cell cycle in vivo. We find that p27XIC1 is transcriptionally regulated by XBF-1 in a dose-dependent manner such that it is suppressed or ectopically induced by a high or low dose of XBF-1, respectively. However, while a low dose of XBF-1 induces ectopic p27XIC1 and ectopic neurons, misexpression of p27XIC1 does not induce ectopic neurons, suggesting that the effects of XBF-1 on cell fate and cell proliferation are distinct. Finally, we show that p27XIC1 is suppressed by XBF-1 in the absence of protein synthesis, suggesting that at least one component of p27XIC1 regulation by XBF-1 may be direct. Thus, XBF-1 is a neural-specific transcription factor that can independently affect both the cell fate choice and the proliferative status of the cells in which it is expressed.
Morphogenetic furrow initiation and progression during eye development in Drosophila: the roles of decapentaplegic, hedgehog and eyes absent
Authors: Curtiss Jennifer , Mlodzik Marek
Published: April 2021

The Drosophila signaling factor decapentaplegic (dpp) mediates the effects of hedgehog (hh) in tissue patterning by regulating the expression of tissue-specific genes. In the eye disc, the transcription factors eyeless (ey), eyes absent (eya), sine oculis (so) and dachshund (dac) participate with these signaling molecules in a complex regulatory network that results in the initiation of eye development. Our analysis of functional relationships in the early eye disc indicates that hh and dpp play no role in regulating ey, but are required for eya, so and dac expression. We show that restoring expression of eya in loss-of-function dpp mutant backgrounds is sufficient to induce so and dac expression and to rescue eye development. Thus, once expressed, eya can carry out its functions in the absence of dpp. These experiments indicate that dpp functions downstream of or in parallel with ey, but upstream of eya, so and dac. Additional control is provided by a feedback loop that maintains expression of eya and so and includes dpp. The fact that exogenous overexpression of ey, eya, so and dac interferes with wild-type eye development demonstrates the importance of such a complicated mechanism for maintaining proper levels of these factors during early eye development. Whereas initiation of eye development fails in either Hh or Dpp signaling mutants, the subsequent progression of the morphogenetic furrow is only slowed down. However, we find that clones that are simultaneously mutant for Hh and Dpp signaling components completely block furrow progression and eye differentiation, suggesting that Hh and Dpp serve partially redundant functions in this process. Interestingly, furrow-associated expression of eya, so and dac is not affected by double mutant tissue, suggesting that some other factor(s) regulates their expression during furrow progression.
A model for anteroposterior patterning of the vertebrate limb based on sequential long- and short-range Shh signalling and Bmp signalling
Authors: Drossopoulou G. , Lewis K. E. , Sanz-Ezquerro J. J. , Nikbakht N. , McMahon A. P. , Hofmann C. , Tickle C.
Published: April 2021

It has been proposed that digit identity in chick limb bud is specified in a dose-dependent fashion by a long-range morphogen, produced by the polarising region. One candidate is Sonic hedgehog (Shh) protein, but it is not clear whether Shh acts long or short range or via Bmps. Here we dissect the relationship between Shh and Bmp signalling. We show that Shh is necessary not only for initiating bmp2 expression but also for sustaining its expression during the period when additional digits are being specified. We also show that we can reproduce much of the effect of Shh during this period by applying only Bmp2. We further demonstrate that it is Bmps that are responsible for digit specification by transiently adding Noggin or Bmp antibodies to limbs treated with Shh. In such limbs, multiple additional digits still form but they all have the same identity. We also explored time dependency and range of Shh signalling by examining ptc expression. We show that high-level ptc expression is induced rapidly when either Shh beads or polarising regions are grafted to a host limb. Furthermore, we find that high-level ptc expression is first widespread but later more restricted. All these data lead us to propose a new model for digit patterning. We suggest that Shh initially acts long range to prime the region of the limb competent to form digits and thus control digit number. Then later, Shh acts short range to induce expression of Bmps, whose morphogenetic action specifies digit identity.
A developmentally regulated GAGA box-binding factor and Sp1 are required for transcription of the hsp70.1 gene at the onset of mouse zygotic genome activation
Authors: Bevilacqua Arturo , Fiorenza Maria Teresa , Mangia Franco
Published: April 2021

We have investigated the onset of zygotic genome transcription in early two-cell mouse embryos by analyzing the regulation of hsp70.1, one of the first genes expressed after fertilization. The transcriptional activation of both an episomic hsp70 promoter and the endogenous hsp70.1 gene requires the contiguity of the GC box proximal to the TATA box with a GAGA box and involves GC box- and GAGA box-binding factors. In vivo transcription factor titrations with double-stranded oligodeoxyribonucleotides and antibodies pinpoint these factors as Sp1 and a novel murine GAGA box-binding factor, which is structurally related to the Drosophila GAGA factor and acts as transcriptional coactivator/potentiator of Sp1. Mouse unfertilized eggs and one-cell and two-cell embryos display a GAGA box-binding activity of maternal origin that disappears at the four-cell stage and is also abundant in the gonads, but is barely detectable in other adult tissues. In light of the well-established nucleosome-disruption role of the Drosophila GAGA factor, these findings suggest a novel mechanism of enhancer-independent gene derepression in early mouse embryos.
Retinoid signaling is essential for patterning the endoderm of the third and fourth pharyngeal arches
Authors: Wendling Olivia , Dennefeld Christine , and Pierre Chambon , Mark Manuel
Published: April 2021

The requirement of retinoic acid (RA) in the initial formation of the pharyngeal arches was investigated by treating headfold-stage mouse embryos with a pan-RAR antagonist in vitro and in vivo. This results in a complete absence of mesenchyme, arteries, nerves and epibranchial placodes of the 3rd and 4th pharyngeal arches, complete agenesis of the 3rd and 4th pouches and consistent lack of the 6th arch artery. Mesodermally derived endothelial cells are absent from the 3rd and 4th pharyngeal arch region and the distribution domain of EphA2 transcripts in mesodermal cells is shifted caudally. In situ hybridization with CRABPI, kreisler and EphA4 probes and the pattern of expression of a Wnt1-lacZ transgene show that neural crest cells (NCC) normally destined to the 3rd and 4th arches migrate ectopically. Most interestingly, the appearance of the 3rd and 4th arches is prevented by the antagonist only during a very narrow window of time, which does not correspond to the period of post-otic NCC migration. Both the timing of appearance and the nature of the defects in RAR antagonist-treated embryos indicate that migrating NCC and mesodermal cells destined to the caudal pharyngeal arches do not represent primary targets of RA action. Alterations in the endodermal expression pattern of Hoxa1, Hoxb1, Pax1, Pax9, Fgf3 and Fgf8 in response to the antagonist-induced block in RA signal transduction demonstrate for the first time that RA signaling is indispensable for the specification of the pharyngeal endoderm and suggest that this signaling is necessary to provide a permissive environment locally for the migration of NCC and mesodermal cells. Our study also indicates that the formation of the 2nd pharyngeal arch and that of the 3rd and 4th pharyngeal arches probably involve distinct RA-dependent developmental processes.
A S/M DNA replication checkpoint prevents nuclear and cytoplasmic events of cell division including centrosomal axis alignment and inhibits activation of cyclin-dependent kinase-like proteins in fucoid zygotes
Authors: Corellou Florence , Bisgrove Sherryl R. , Kropf Darryl L. , Meijer Laurent , Kloareg Bernard , Bouget François-Yves
Published: April 2021

S/M checkpoints prevent various aspects of cell division when DNA has not been replicated. Such checkpoints are stringent in yeast and animal somatic cells but are usually partial or not present in animal embryos. Because little is known about S/M checkpoints in plant cells and embryos, we have investigated the effect of aphidicolin, a specific inhibitor of DNA polymerases α and δ, on cell division and morphogenesis in Fucus and Pelvetia zygotes. Both DNA replication and cell division were inhibited by aphidicolin, indicating the presence, in fucoid zygotes, of a S/M checkpoint. This checkpoint prevents chromatin condensation, spindle formation, centrosomal alignment with the growth axis and cytokinesis but has no effect on germination or rhizoid elongation. This S/M checkpoint also prevents tyrosine dephosphorylation of cyclin-dependent kinase-like proteins at the onset of mitosis. The kinase activity is restored in extracts upon incubation with cdc25A phosphatase. When added in S phase, olomoucine, a specific inhibitor of cyclin-dependent kinases, has similar effects as aphidicolin on cell division although alignment of the centrosomal axis still occurs. We propose a model involving the inactivation of CDK-like proteins to account for the S/M DNA replication checkpoint in fucoid zygotes and embryos.
POLTERGEIST functions to regulate meristem development downstream of the CLAVATA loci
Authors: Yu Lita P. , Simon Ephraim J. , Trotochaud Amy E. , Clark Steven E.
Published: April 2021

Mutations at the CLAVATA loci (CLV1, CLV2 and CLV3) result in the accumulation of undifferentiated cells at the shoot and floral meristems. We have isolated three mutant alleles of a novel locus, POLTERGEIST (POL), as suppressors of clv1, clv2 and clv3 phenotypes. All pol mutants were nearly indistinguishable from wild-type plants; however, pol mutations provided recessive, partial suppression of meristem defects in strong clv1 and clv3 mutants, and nearly complete suppression of weak clv1 mutants. pol mutations partially suppressed clv2 floral and pedicel defects in a dominant fashion, and almost completely suppressed clv2 phenotypes in a recessive manner. These observations, along with dominant interactions observed between the pol and wuschel (wus) mutations, indicate that POL functions as a critical regulator of meristem development downstream of the CLV loci and redundantly with WUS. Consistent with this, pol mutations do not suppress clv3 phenotypes by altering CLV1 receptor activation.
The regulation of proliferation and differentiation in oligodendrocyte progenitor cells by αv integrins
Authors: Blaschuk K. L. , Frost E. E. , ffrench-Constant C.
Published: April 2021

We have previously shown that oligodendrocyte progenitor cells exhibit developmental switching between αv-associated β integrin subunits to sequentially express αvβ1, αvβ3 and αvβ5 integrins during differentiation in vitro. To understand the role that αvβ3 integrin may play in regulating oligodendrocyte progenitor cell behaviour, cells of the rat cell line, CG-4, were genetically engineered to constitutively express αvβ3 integrin by transfection with full-length human β3 integrin subunit cDNA. Time-lapse videomicroscopy showed no effect of β3 expression on cell migration but revealed enhanced proliferation on vitronectin substrata. Comparison of mitotic indices, as measured by 5-bromo-2′-deoxyuridine incorporation, confirmed that human β3 integrin-expressing cells exhibited enhanced proliferation, as compared to both vector-only transfected, and wild-type CG-4 cells when switched to differentiation medium from growth medium, but only in cultures grown on vitronectin and not on poly-D-lysine. The effects on proliferation were inhibited by a function-blocking antibody specifically directed against the human β3 integrin subunit. Human β3 integrin-expressing cells also exhibited reduced differentiation. This differentiation could be reduced still further by a function-blocking monoclonal antibody against αvβ5 integrin, as could differentiation in the wild-type CG-4 cells. Taken together, these results suggest that αvβ3 integrin may regulate oligodendroglial cell proliferation and that both downregulation of αvβ3 integrin expression and signalling through αvβ5 integrin may be critical to continued differentiation in vitro.
The Arabidopsis floral homeotic gene APETALA3 differentially regulates intercellular signaling required for petal and stamen development
Authors: Jenik Pablo D. , Irish Vivian F.
Published: April 2021

Cell-cell signaling is crucial for the coordination of cell division and differentiation during plant organogenesis. We have developed a novel mosaic analysis method for Arabidopsis, based on the maize Ac/Ds transposable element system, to assess the requirements of individual genes in intercellular signaling. Using this strategy, we have shown that the floral homeotic APETALA3 (AP3) gene has distinct roles in regulating intercellular signaling in different tissues. In petals, AP3 acts primarily in a cell-autonomous fashion to regulate cell type differentiation, but its function is also required in a non-cell-autonomous fashion to regulate organ shape. In contrast, AP3-regulated intercellular interactions are required for conferring both cell type identity and organ shape and size in the stamens. Using antibodies raised against AP3, we have shown that the AP3 protein does not traffic between cells. These observations imply that AP3 acts by differentially regulating the production of intercellular signals in a whorl-specific manner.
mosaic eyes: a zebrafish gene required in pigmented epithelium for apical localization of retinal cell division and lamination
Authors: Jensen Abbie M. , Walker Charline , Westerfield Monte
Published: April 2021

For proper function of the retina, the correct proportions of retinal cell types must be generated, they must be organized into cell-specific laminae, and appropriate synaptic connections must be made. To understand the genetic regulation of retinal development, we have analyzed mutations in the mosaic eyes gene that disrupt retinal lamination, the localization of retinal cell divisions to the retinal pigmented epithelial surface and retinal pigmented epithelial development. Although retinal organization is severely disrupted in mosaic eyes mutants, surprisingly, retinal cell differentiation occurs. The positions of dividing cells and neurons in the brain appear normal in mosaic eyes mutants, suggesting that wild-type mosaic eyes function is specifically required for normal retinal development. We demonstrate that mosaic eyes function is required within the retinal pigmented epithelium, rather than in dividing retinal cells. This analysis reveals an interaction between the retinal pigmented epithelium and the retina that is required for retinal patterning. We suggest that wild-type mosaic eyes function is required for the retinal pigmented epithelium to signal properly to the retina.
Monofocal origin of telencephalic oligodendrocytes in the anterior entopeduncular area of the chick embryo
Authors: Olivier Christelle , Cobos Immaculada , Villegas Eva Maria Perez , Spassky Nathalie , Zalc Bernard , Martinez Salvador , Thomas Jean-Léon
Published: April 2021

Oligodendrocytes are the myelin-forming cells in the central nervous system. In the brain, oligodendrocyte precursors arise in multiple restricted foci, distributed along the caudorostral axis of the ventricular neuroepithelium. In chick embryonic hind-, mid- and caudal forebrain, oligodendrocytes have a basoventral origin, while in the rostral fore-brain oligodendrocytes emerge from alar territories (Perez Villegas, E. M., Olivier, C., Spassky, N., Poncet, C., Cochard, P., Zalc, B., Thomas, J. L. and Martinez, S. (1999) Dev. Biol. 216, 98-113). To investigate the respective territories colonized by oligodendrocyte progenitor cells that originate from either the basoventral or alar foci, we have created a series of quail-chick chimeras. Homotopic chimeras demonstrate clearly that, during embryonic development, oligodendrocyte progenitors that emerge from the alar anterior entopeduncular area migrate tangentially to invade the entire telencephalon, whereas those from the basal rhombomeric foci show a restricted rostrocaudal distribution and colonize only their rhombomere of origin. Heterotopic chimeras indicate that differences in the migratory properties of oligodendroglial cells do not depend on their basoventral or alar ventricular origin. Irrespective of their origin (basal or alar), oligodendrocytes migrate only short distances in the hindbrain and long distances in the prosencephalon. Furthermore, we provide evidence that, in the developing chick brain, all telencephalic oligodendrocytes originate from the anterior entopeduncular area and that the prominent role of anterior entopeduncular area in telencephalic oligodendrogenesis is conserved between birds and mammals.
Genetic dissection ofnodalfunction in patterning the mouse embryo
Authors: Lowe Linda A. , Yamada Satoru , Kuehn Michael R.
Published: April 2021

Loss-of-function analysis has shown that the transforming growth factor-like signaling molecule nodal is essential for mouse mesoderm development. However, definitive proof of nodal function in other developmental processes in the mouse embryo has been lacking because the null mutation blocks gastrulation. We describe the generation and analysis of a hypomorphic nodal allele. Mouse embryos heterozygous for the hypomorphic allele and a null allele undergo gastrulation but then display abnormalities that fall into three distinct mutant phenotypic classes, which may result from expression levels falling below critical thresholds in one or more domains of nodal expression. Our analysis of each of these classes provides conclusive evidence for nodal-mediated regulation of several developmental processes in the mouse embryo, beyond its role in mesoderm formation. We find that nodal signaling is required for correct positioning of the anteroposterior axis, normal anterior and midline patterning, and the left- right asymmetric development of the heart, vasculature, lungs and stomach.
Centrosome migration into the Drosophila oocyte is independent of BicD and egl, and of the organisation of the microtubule cytoskeleton
Authors: Bolívar Jorge , Huynh ean-René , López-Schier Hernán , González Cayetano , Johnston Daniel St , González-Reyes Acaimo
Published: April 2021

During early Drosophila oogenesis, one cell from a cyst of 16 germ cells is selected to become the oocyte, and accumulates oocyte-specific proteins and the centrosomes from the other 15 cells. Here we show that the microtubule cytoskeleton and the centrosomes follow the same stepwise restriction to one cell as other oocyte markers. Surprisingly, the centrosomes still localise to one cell after colcemid treatment, and in BicD and egl mutants, which abolish the localisation of all other oocyte markers and the polarisation of the microtubule cytoskeleton. In contrast, the centrosomes fail to migrate in cysts mutant for Dynein heavy chain 64C, which disrupts the fusome. Thus, centrosome migration is independent of the organisation of the microtubule cytoskeleton, and seems to depend instead on the polarity of the fusome.
even skippedis required to produce atrans-acting signal for larval neuroblast proliferation that can be mimicked by ecdysone
Authors: Park Youngji , Fujioka Miki , Kobayashi Masatomo , Jaynes James B. , Datta Sumana
Published: April 2021

Development of a multicellular organism requires precise coordination of cell division and cell type determination. The selector homeoprotein Even skipped (Eve) plays a very specific role in determining cell identity in the Drosophila embryo, both during segmentation and in neuronal development. However, studies of gene expression in eve mutant embryos suggest that eve regulates the embryonic expression of the vast majority of genes. We present here genetic interaction and phenotypic analysis showing that eve functions in the trol pathway to regulate the onset of neuroblast division in the larval CNS. Surprisingly, Eve is not detected in the regulated neuroblasts, and culture experiments reveal that Eve is required in the body, not the CNS. Furthermore, the effect of an eve mutation can be rescued both in vivo and in culture by the hormone ecdysone. These results suggest that eve is required to produce a trans-acting factor that stimulates cell division in the larval brain.
Overexpression of Snail family members highlights their ability to promote chick neural crest formation
Authors: del Barrio Marta G. , Nieto M. Angela
Published: April 2021

The Snail gene family of transcription factors plays crucial roles in different morphogenetic processes during the development of vertebrate and invertebrate embryos. In previous studies of function interference for one of the family members, Slug, we showed its involvement and neural crest formation in the chick embryo. Now we have carried out a series of gain-of-function experiments in which we show that Slug overexpression in the neural tube of the chick embryo induces an increase in neural crest production. The analysis of electroporated embryos shows that Slug can induce the expression of rhoB and an increase in the number of HNK-1-positive migratory cells, indicating that it lies upstream of them in the genetic cascade of neural crest development. The increase in neural crest production after Slug overexpression was confined to the cranial region, indicating that the mechanisms of crest induction somehow differ between head and trunk.

The expression of the two vertebrate family members, Slug and Snail, is peculiar with respect to the neural crest. Slug is not expressed in the premigratory crest in the mouse, whereas it is expressed in this cell population in the chick and the opposite is true for Snail(Sefton, M., Sánchez, S. and Nieto M. A. (1998) Development125, 3111-3121). This raises the question of whether they can be functionally equivalent. To test this hypothesis both intra- and interspecies, we have performed a series of ectopic expression experiments by electroporating chick and mouse Snail in the chick embryo hindbrain. We observe that both genes elicit the same responses in the neural tube. Our results indicate that they can be functionally equivalent, although the embryos show a higher response to the endogenous gene, chick Slug.
spiel ohne grenzen/pou2is required for zebrafish hindbrain segmentation
Authors: Hauptmann Giselbert , Belting Heinz-Georg , Wolke Uta , Lunde Karen , Söll Iris , Abdelilah-Seyfried Salim , Prince Victoria , Driever Wolfgang
Published: April 2021

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories.
Inhibition of Rho family GTPases by Rho GDP dissociation inhibitor disrupts cardiac morphogenesis and inhibits cardiomyocyte proliferation
Authors: Wei Lei , Imanaka-Yoshida Kyoko , Wang Lu , Zhan Song , Schneider Michael D. , DeMayo Francesco J. , Schwartz Robert J.
Published: April 2021

Studies of Rho GTPases in Drosophila and Xenopus suggest that Rho family proteins may play an important role in embryogenesis. A reverse genetic approach was employed to explore the role of Rho GTPases in murine cardiac development. Cardiac-specific inhibition of Rho family protein activities was achieved by expressing Rho GDIα, a specific GDP dissociation inhibitor for Rho family proteins, using the α-myosin heavy chain promoter, active at embryonic day (E)8.0 during morphogenesis of the linear heart tube. RhoA, Rac1 and Cdc42 activities were significantly inhibited, as shown by decreased membrane translocation of these proteins in the transgenic hearts. Transgenic F1 mice for each of two independent lines expressing the highest levels of the transgene, died around E10.5. Homozygotes of the middle copy-number lines, in which Rho GDIα expression was increased four-fold over normal levels, were also embryonic lethal. Cardiac morphogenesis in these embryos was disrupted, with incomplete looping, lack of chamber demarcation, hypocellularity and lack of trabeculation. Cell proliferation was inhibited in the transgenic hearts, as shown by immunostaining with anti-phosphohistone H3, a marker of mitosis. In addition, ventricular hypoplasia was associated with up-regulation of p21, an inhibitor of cyclin-dependent kinases, and with down-regulation of cyclin A, while cell survival was not affected. These results reveal new biological functions for Rho family proteins as essential determinants of cell proliferation signals at looping and chamber maturation stages in mammalian cardiac development.
Spatial patterns of ecdysteroid receptor activation during the onset ofDrosophilametamorphosis
Authors: Kozlova Tatiana , Thummel Carl S.
Published: April 2021

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo we established transgenic animals that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of β-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. We show that the patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. We also show that GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, we observe no GAL4-LBD activation in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. We conclude that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. Finally, we show that GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development.
PAG-3, a Zn-finger transcription factor, determines neuroblast fate inC. elegans
Authors: Cameron Scott , Clark Scott G. , McDermott Joan B. , Aamodt Eric , Horvitz H. Robert
Published: April 2021

During Caenorhabditis elegans development, the patterns of cell divisions, cell fates and programmed cell deaths are reproducible from animal to animal. In a search for mutants with abnormal patterns of programmed cell deaths in the ventral nerve cord, we identified mutations in the gene pag-3, which encodes a zinc-finger transcription factor similar to the mammalian Gfi-1 and Drosophila Senseless proteins. In pag-3 mutants, specific neuroblasts express the pattern of divisions normally associated with their mother cells, producing with each reiteration an abnormal anterior daughter neuroblast and an extra posterior daughter cell that either terminally differentiates or undergoes programmed cell death, which accounts for the extra cell corpses seen in pag-3 mutants. In addition, some neurons do not adopt their normal fates in pag-3 mutants. The phenotype of pag-3 mutants and the expression pattern of the PAG-3 protein suggest that in some lineages pag-3 couples the determination of neuroblast cell fate to subsequent neuronal differentiation. We propose that pag-3 counterparts in other organisms determine blast cell identity and for this reason may lead to cell lineage defects and cell proliferation when mutated.