bluuebunny/crossref_metadata_2025 huggingface dataset:
Modifications of cell fate specification in equal-cleaving nemertean embryos: alternate patterns of spiralian development
Authors: Martindale Mark Q. , Henry Jonathan Q.
Published: April 2021

The nemerteans belong to a phylum of coelomate worms that display a highly conserved pattern of cell divisions referred to as spiral cleavage. It has recently been shown that the fates of the four embryonic cell quadrants in two species of nemerteans are not homologous to those in other spiralian embryos, such as the annelids and molluscs (Henry, J. Q. and Martindale, M. Q. (1994a) Develop. Genetics 15, 64-78). Equal-cleaving molluscs utilize inductive interactions to establish quadrant-specific cell fates and embryonic symmetry properties following fifth cleavage. In order to elucidate the manner in which cell fates are established in nemertean embryos, we have conducted cell isolation and deletion experiments to examine the developmental potential of the early cleavage blastomeres of two equal-cleaving nemerteans, Nemertopsis bivittata and Cerebratulus lacteus. These two species display different modes of development: N. bivittata develops directly via a non-feeding larvae, while C. lacteus develops to form a feeding pilidium larva which undergoes a radical metamorphosis to give rise to the juvenile worm. By examining the development of certain structures and cell types characteristic of quadrant-specific fates for each of these species, we have shown that isolated blastomeres of the indirect-developing nemertean, C. lacteus, are capable of generating cell fates that are not a consequence of that cell’s normal developmental program. For instance, dorsal blastomeres can form muscle fibers when cultured in isolation. In contrast, isolated blastomeres of the directdeveloping species, N. bivittata do not regulate their development to the same extent. Some cell fates are specified in a precocious manner in this species, such as those that give rise to the eyes. Thus, these findings indicate that equalcleaving spiralian embryos can utilize different mechanisms of cell fate and axis specification. The implications of these patterns of nemertean development are discussed in relation to experimental work in other spiralian embryos, and a model is presented that accounts for possible evolutionary changes in cell lineage and the process of cell fate specification amongst these protostome phyla.
Formation of lateral root meristems is a two-stage process
Authors: Laskowski Marta J. , Williams Mary E. , Nusbaum H. Chad , Sussex Ian M.
Published: April 2021

In both radish and Arabidopsis, lateral root initiation involves a series of rapid divisions in pericycle cells located on the xylem radius of the root. In Arabidopsis, the number of pericycle cells that divide to form a primordium was estimated to be about 11. To determine the stage at which primordia are able to function as root meristems, primordia of different stages were excised and cultured without added hormones. Under these conditions, primordia that consist of 2 cell layers fail to develop while primordia that consist of at least 3-5 cell layers develop as lateral roots. We hypothesize that meristem formation is a two-step process involving an initial period during which a population of rapidly dividing, approximately isodiametric cells that constitutes the primordium is formed, and a sub-sequent stage during which meristem organization takes place within the primordium.
DPY-30, a nuclear protein essential early in embryogenesis for Caenorhabditis elegans dosage compensation
Authors: Hsu David R. , Chuang Pao-Tien , Meyer Barbara J.
Published: April 2021

DPY-30 is an essential component of the C. elegans dosage compensation machinery that reduces X chromosome transcript levels in hermaphrodites (XX). DPY-30 is required for the sex-specific association of DPY-27 (a chromosome condensation protein homolog) with the hermaphrodite X chromosomes. Loss of dpy-30 activity results in XX-specific lethality. We demonstrate that dpy-30 encodes a novel nuclear protein of 123 amino acids that is present in both hermaphrodites and males (XO) throughout development. DPY-30 itself is not associated with the X chromosomes, nor is its pattern of expression perturbed by mutations in the gene hierarchy that controls dosage compensation. Therefore, DPY-30 is a ubiquitous factor that is likely to promote the hermaphrodite-specific association of DPY-27 with X by affecting the activity of a sex-specific dosage compensation gene. In XO animals, DPY-30 is required for developmental processes other than dosage compensation: coordinated movement, normal body size, correct tail morphology and mating behavior. We demonstrate that rescue of both the XX-specific lethality and the XO-specific morphological defects caused by dpy-30 mutations can be achieved by inducing dpy-30 transcripts either in the mother or in the embryo through the end of gastrulation. dpy-30 appears to be cotranscribed in an operon with a novel RNA-binding protein.
Overexpression of parathyroid hormone-related protein or parathyroid hormone in transgenic mice impairs branching morphogenesis during mammary gland development
Authors: Wysolmerski John J. , McCaughern-Carucci James F. , Daifotis Anastasia G. , Broadus Arthur E. , Philbrick William M.
Published: April 2021

Parathyroid hormone-related protein (PTHrP) was originally discovered as the tumor product that causes humoral hypercalcemia of malignancy. PTHrP is now known to be widely expressed in many normal fetal tissues where it may participate in the regulation of organogenesis. In this report, we document that overexpression of PTHrP in myoepithelial cells in the mammary glands of transgenic mice resulted in a form of breast hypoplasia characterized by a profound defect in branching morphogenesis of the developing mammary duct system. In addition, transgenic mice manifested a defect in lobuloalveolar development during pregnancy that seemed to be, in part, the consequence of an impaired ability to form terminal ducts in response to estrogen and progesterone stimulation. The effects of PTHrP on branching morphogenesis during breast development appeared to be the result of aminoterminal PTH-like sequences that signal through the PTH/PTHrP receptor, since overexpression of parathyroid hormone itself in the mammary glands of transgenic mice caused a similar developmental phenotype, and delivery of PTHrP (1–36) via locally implanted slow-release pellets impaired breast development in normal mice. These results suggest that PTHrP, which is a native product of mammary epithelial and myoepithelial cells may participate in normal breast development, perhaps as a locally secreted growth inhibitor.
Specificity of CNS and PNS regulatory subelements comprising pan-neural enhancers of the deadpan and scratch genes is achieved by repression
Authors: Emery John F. , Bier Ethan
Published: April 2021

The Drosophila pan-neural genes deadpan (dpn) and scratch (scrt) are expressed in most or all developing neural precursor cells of the central nervous system (CNS) and peripheral nervous system (PNS). We have identified a cisacting enhancer element driving full pan-neural expression of the dpn gene which is composed of independent CNSand PNS-specific subelements. We have also identified CNSand PNS-specific subelements of the scrt enhancer. Deletion analysis of the dpn and scrt PNS-specific subelements reveals that PNS specificity of these two evolutionarily unrelated enhancers is achieved in part by repression of CNS expression. We discuss the implications of the striking organizational similarities of the dpn, scrt, and sna pan-neural enhancers.
The division abnormally delayed (dally) gene: a putative integral membrane proteoglycan required for cell division patterning during postembryonic development of the nervous system in Drosophila
Authors: Nakato Hiroshi , Futch Tracy A. , Selleck Scott B.
Published: April 2021

We have devised a genetic screen to obtain mutants affecting cell division patterning in the developing central nervous system of Drosophila. The division abnormally delayed (dally) locus was identified using a combination of “enhancer trap” and behavioral screening methods. The ordered cell cycle progression of lamina precursor cells, which generate synaptic target neurons for photoreceptors, is disrupted in dally mutants. The first of two lamina precursor cell divisions shows a delayed entry into mitosis. The second division, one that is triggered by an intercellular signal from photoreceptor axons, fails to take place. Similar to lamina precursors, cells that generate the ommatidia of the adult eye show two synchronized divisions found along the morphogenetic furrow in the eye disc and the first division cycle in dally mutants displays a delayed progression into M phase like that found in the first lamina precursor cell division. dally mutations also affect viability and produce morphological defects in several adult tissues, including the eye, antenna, wing and genitalia. Sequencing of a dally cDNA reveals a potential open reading frame of 626 amino acids with homology to a family of Glypican-related integral membrane proteoglycans. These heparan sulfate-containing proteins are attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol linkage. Heparan sulfate proteoglycans may serve as co-receptors for a variety of secreted proteins including fibroblast growth factor, vascular endothelial growth factor, hepatocyte growth factor and members of the Wnt, TGF-β and Hedgehog families. The cell division defects found in dally mutants implicate the Glypican group of integral membrane proteoglycans in the control of cell division during development.
Translational control of oskar generates Short OSK, the isoform that induces pole plasm assembly
Authors: Markussen Finn-Hugo , Michon Anne-Marie , Breitwieser Wolfgang , Ephrussi Anne
Published: April 2021

At the posterior pole of the Drosophila oocyte, oskar induces a tightly localized assembly of pole plasm. This spatial restriction of oskar activity has been thought to be achieved by the localization of oskar mRNA, since mislocalization of the RNA to the anterior induces anterior pole plasm. However, ectopic pole plasm does not form in mutant ovaries where oskar mRNA is not localized, suggesting that the unlocalized mRNA is inactive. As a first step towards understanding how oskar activity is restricted to the posterior pole, we analyzed oskar translation in wild type and mutants. We show that the targeting of oskar activity to the posterior pole involves two steps of spatial restriction, cytoskeleton-dependent localization of the mRNA and localization-dependent translation. Furthermore, our experiments demonstrate that two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full oskar activity. Finally, we show that when oskar RNA is localized, accumulation of Oskar protein requires the functions of vasa and tudor, as well as oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by oskar.
Establishment of Drosophila imaginal precursor cells is controlled by the Arrowhead gene
Authors: Curtiss Jennifer , Heilig Joseph S.
Published: April 2021

Metamorphosis in Drosophila melanogaster requires synchronization of numerous developmental events that occur in isolated imaginal precursor tissues. The imaginal primordia are established during embryonic stages and are quiescent for much of larval life. The Arrowhead gene is necessary for establishment of proper numbers of cells within a subset of imaginal precursor tissues. Loss-offunction mutations in Arrowhead reduce the number of abdominal histoblasts and salivary gland imaginal ring cells before the proliferative stages of their development. The number of abdominal histoblasts in mutant animals is approximately half that of wild-type, as might result from failure of a single early division of these cells. A neomorphic Arrowhead allele results in the specific loss of the retinal precursors by the early third instar, before they have begun to differentiate. Since Arrowhead mutations affect only subsets of imaginal tissue, there must be distinctions in the developmental regulation of different imaginal precursors. Arrowhead may be part of a regulatory pathway responsible for establishing the proper number of abdominal histoblasts and salivary gland imaginal ring cells. The neomorphic Arrowhead allele, which may cause misexpression of the Arrowhead gene in the eye-antenna imaginal disc, interferes with the establishment or proliferation of retinal precursor cells.
Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro
Authors: Soudais Claire , Bielinska Malgorzata , Heikinheimo Markku , MacArthur Craig A. , Narita Naoko , Saffitz Jeffrey E. , Simon M. Celeste , Leiden Jeffrey M. , Wilson David B.
Published: April 2021

Transcription factor GATA-4 belongs to a family of zinc finger proteins involved in lineage determination. GATA-4 is first expressed in yolk sac endoderm of the developing mouse and later in cardiac tissue, gut epithelium and gonads. To delineate the role of this transcription factor in differentiation and early development, we studied embryoid bodies derived from mouse embryonic stem (ES) cells in which both copies of the Gata4 gene were disrupted. Light and electron microscopy demonstrated that embryoid bodies formed from wild-type and heterozygous deficient ES cells were covered with a layer of visceral yolk sac endoderm, whereas no yolk sac endoderm was evident on the surface of the homozygous deficient embryoid bodies. Independently selected homozygous deficient cell lines displayed this distinctive phenotype, suggesting that it was not an artifact of clonal variation. Biochemical markers of visceral endoderm formation, such as α-feto-protein, hepatocyte nuclear factor-4 and binding sites for Dolichos biflorus agglutinin, were absent from the homozygous deficient embryoid bodies. Examination of other differentiation markers in the mutant embryoid bodies, studies of ES cell-derived teratocarcinomas and chimeric mouse analysis demonstrated that GATA-4-deficient ES cells have the capacity to differentiate along other lineages. We conclude that, under in vitro conditions, disruption of the Gata4 gene results in a specific block in visceral endoderm formation. These homozygous deficient cells should yield insights into the regulation of yolk sac endoderm development and the factors expressed by visceral endoderm that influence differentiation of adjoining ectoderm/mesoderm.
A role for En-2 and other murine homologues of Drosophila segment polarity genes in regulating positional information in the developing cerebellum
Authors: Millen Kathleen J. , Hui Chi-Chung , Joyner Alexandra L.
Published: April 2021

To gain insight into the molecular genetic basis of cerebellar patterning, the expression patterns of many vertebrate homologues of Drosophila segment polarity genes were examined during normal and abnormal cerebellar development, including members of the En, Wnt, Pax, Gli and Dvl gene families. Five of these genes were found to show transient, spatially restricted patterns of expression. Strikingly, expression of En-2, En-1, Wnt-7B and Pax-2 defined eleven similar sagittal domains at 17.5 dpc, reminiscent of the transient sagittal domains of expression of Purkinje cell markers which have been implicated in cerebellar afferent patterning. Postnatally, transient anterior/posterior differences in expression were observed for En-2, En-1, Gli and Wnt-7B dividing the cerebellum into anterior and posterior regions. The expression patterns of these genes were altered in cerebella of En-2 homozygous mutant mice, which show a cerebellar foliation patterning defect. Strikingly, four of the Wnt-7B expression domains that are adjacent to the En-2 domains are lost in En-2 mutant embryonic cerebella. These studies provide the first evidence of a potential network of regulatory genes that establish spatial cues in the developing cerebellum by dividing it into a grid of positional information required for patterning foliation and afferents. Taken together with previous gene expression studies, our data suggests that eleven sagittal domains and at least two anterior/posterior compartments are the basic elements of spatial information in the cerebellum.
Mouse embryos lacking RXRα are resistant to retinoic-acid-induced limb defects
Authors: Sucov Henry M. , Izpisúa-Belmonte Juan-Carlos , Gañan Yolanda , Evans Ronald M.
Published: April 2021

Embryonic exposure to the vitamin A metabolite retinoic acid (RA) causes malformations in numerous developing tissues, including the limbs, which serves as a model system of retinoic acid action. RA treatment of wild-type mouse embryos results in digit truncations and long bone reductions. These effects are mediated by products of the retinoic acid and retinoid X receptor genes (RARs and RXRs), members of the nuclear receptor family of liganddependent transcription factors. Mouse embryos homozygous for a mutation in the RXRα gene appear normal in limb development, although such embryos are phenotypically affected in other tissues. We now describe resistance to limb malformations normally induced by teratogenic RA exposure in the RXRα−/− background. RA treatments that cause limb defects in 100% of wild-type embryos fail to elicit malformations in RXRα homozygotes, implicating RXRα as a component in the teratogenic process in the limbs. Heterozygous embryos are intermediate in sensitivity to RA, suggesting the importance of RXRα gene dosage in limb teratogenesis. Expression of the RA-inducible gene RARβ2 was equivalent between wild-type and homozygous embryos after RA treatment. RA treatment also did not distinguish between wild-type and RXRα−/− embryos in the spatial expression of sonic hedgehog (Shh) and hoxd-12, two other genes implicated in limb development. However, the quantitative level of hoxd-12 expression was elevated in RXRα−/− embryos. These observations indicate that transcriptional processes which are inappropriately regulated in the mouse limb by exogenous RA require RXRα for their execution, and that specific teratogenic processes, as well as specific normal developmental processes under vitamin A control, occur through individual members of the RXR and RAR families.
Lineage restriction of the myogenic conversion factor myf-5 in the brain
Authors: Tajbakhsh Shahragim , Buckingham Margaret E.
Published: April 2021

myf-5 is one of four transcription factors belonging to the MyoD family that play key roles in skeletal muscle determination and differentiation. We have shown earlier by gene targeting nlacZ into the murine myf-5 locus that myf-5 expression in the developing mouse embryo is closely associated with the restriction of precursor muscle cells to the myogenic lineage. We now identify unexpected expression of this myogenic factor in subdomains of the brain. myf-5 expression begins to be detected at embryonic day 8 (E8) in the mesencephalon and coincides with the appearance of the first differentiated neurons; expression in the secondary prosencephalon initiates at E10 and is confined to the ventral domain of prosomere p4, later becoming restricted to the posterior hypothalamus. This expression is observed throughout embryogenesis. No other member of the MyoD family is detected in these regions, consistent with the lack of myogenic conversion. Furthermore, embryonic stem cells expressing the myf-5/nlacZ allele yield both skeletal muscle and neuronal cells when differentiated in vitro. These observations raise questions about the role of myf-5 in neurogenesis as well as myogenesis, and introduce a new lineage marker for the developing brain.
Motoneuron fate specification revealed by patterned LIM homeobox gene expression in embryonic zebrafish
Authors: Appel Bruce , Korzh Vladimir , Glasgow Eric , Thor Stefan , Edlund Thomas , Dawid Igor B. , Eisen Judith S.
Published: April 2021

In zebrafish, individual primary motoneurons can be uniquely identified by their characteristic cell body positions and axonal projection patterns. The fate of individual primary motoneurons remains plastic until just prior to axogenesis when they become committed to particular identities. We find that distinct primary motoneurons express particular combinations of LIM homeobox genes. Expression precedes axogenesis as well as commitment, suggesting that LIM homeobox genes may contribute to the specification of motoneuronal fates. By transplanting them to new spinal cord positions, we demonstrate that primary motoneurons can initiate a new program of LIM homeobox gene expression, as well as the morphological features appropriate for the new position. We conclude that the patterned distribution of different primary motoneuronal types within the zebrafish spinal cord follows the patterned expression of LIM homeobox genes, and that this reflects a highly resolved system of positional information controlling gene transcription.
Ommatidial polarity in the Drosophila eye is determined by the direction of furrow progression and local interactions
Authors: Strutt David I. , Mlodzik Marek
Published: April 2021

The adult eye of Drosophila is a highly ordered structure. It is composed of about 800 ommatidia, each displaying precise polarity. The ommatidia are arranged about an axis of mirror image symmetry, the equator, which lies along the dorsoventral midline of the eye. We use hedgehog pathway mutants to induce ectopic morphogenetic furrows and use these as a tool to investigate the establishment of ommatidial polarity. Our results show that ommatidial clusters are self- organising units whose polarity in one axis is determined by the direction of furrow progression, and which can independently define the position of an equator without reference to the global coordinates of the eye disc.
Two distinct pathways for the localization of RNAs at the vegetal cortex in Xenopus oocytes
Authors: Kloc Malgorzata , Etkin Laurence D.
Published: April 2021

We found that there are two major pathways by which RNAs are localized at the vegetal cortex during oogenesis of Xenopus laevis. One of these, through which Xlsirts, Xcat2 and Xwnt11 are localized, involves transport during stages 1 and 2 of oogenesis via a region of the mitochondr-ial cloud that we call the message transport organizer or METRO. This pathway involved three steps, transport of RNA from the GV to the mitochondrial cloud, sorting of the RNAs to specific regions of the METRO, and translo-cation to and anchoring at the vegetal cortex. These three RNAs exhibit a distinct pattern of spatial localization within the METRO when they approach the vegetal cortex. The other pathway is used by Vg1. We detected Vg1 throughout the oocyte cytoplasm during stages 1 and 2. During stage 3 it was translocated to the vegetal cortex and associated with the cortex overlapping the region at which the Xlsirt, Xcat2, and Xwnt11 RNAs are anchored. Our results also showed that anchoring of these RNAs was dependent in part on actin microfilaments, but was inde-pendent of microtubules. These results demonstrate a novel mechanism of translocation and RNA sorting used by RNAs several of which may be involved in the establish-ment of the embryonic body axis.
The somatic sex determines the requirement for ovarian tumor gene activity in the proliferation of the Drosophila germline
Authors: Nagoshi Rod N. , Patton J. Scott , Bae Eunkyung , Geyer Pamela K.
Published: April 2021

Gametogenesis in Drosophila requires sex-specific interactions between the soma and germline to control germ cell viability, proliferation, and differentiation. To determine what genetic components are involved in this interaction, we examined whether changes in the sexual identity of the soma affected the function of the ovarian tumor (otu) and ovo genes. These genes are required cell autonomously in the female germline for germ cell proliferation and differentiation. Mutations in otu and ovo cause a range of ovarian defects, including agametic ovaries and tumorous egg cysts, but do not affect spermatogenesis. We demonstrate that XY germ cells do not require otu when developing in testes, but become dependent on otu function for proliferation when placed in an ovary. This soma-induced requirement can be satisfied by the induced expression of the 98×103Mr OTU product, one of two isoforms produced by differential RNA splicing. These results indicate that the female somatic gonad can induce XY germ cells to become ‘female-like’ because they require an oogenesis-specific gene. In contrast, the requirement for ovo is dependent on a cell autonomous signal derived from the X:A ratio. We propose that differential regulation of the otu and ovo genes provides a mechanism for the female germline to incorporate both somatic and cell autonomous inputs required for oogenesis.
XlPOU 2, a noggin-inducible gene, has direct neuralizing activity
Authors: Witta Samir E. , Agarwal Veena R. , Sato Sheryl M.
Published: April 2021

XlPOU 2, a member of the class III POU domain family, is expressed initially in Spemann’s organizer, and later, in discrete regions of the developing nervous system in Xenopus laevis. XlPOU 2 may act downstream from initial neural induction events, since it is activated by the neural inducer, noggin. To determine if XlPOU 2 participates in the early events of neurogenesis, synthetic mRNA was microinjected into specific blastomeres of the 32-cell stage embryo. Misexpression of XlPOU 2 in the epidermis causes a direct switch in cell fate from an epidermal to a neuronal phenotype. In the absence of mesoderm induction, XlPOU 2 has the ability to induce a neuronal phenotype in uncommitted ectoderm. These data demonstrate the potential of XlPOU 2 to act as a master regulator of neurogenesis.
Soluble and cell-bound forms of steel factor activity play distinct roles in melanocyte precursor dispersal and survival on the lateral neural crest migration pathway
Authors: Wehrle-Haller Bernhard , Weston James A.
Published: April 2021

Trunk neural crest cells segregate from the neuroepithelium and enter a ‘migration staging area’ lateral to the embryonic neural tube. After some crest cells in the migration staging area have begun to migrate on a medial pathway, a subpopulation of crest-derived cells remaining in the migration staging area expresses mRNAs for the receptor tyrosine kinase, c-kit, and tyrosinase-related protein-2, both of which are characteristic of melanocyte precursors. These putative melanocyte precursors are subsequently observed on the lateral crest migration pathway between the dermatome and overlying epithelium, and then dispersed in nascent dermal mesenchyme.

Melanocyte precursors transiently require the c-kit ligand, Steel factor for survival. Although Steel factor mRNA is transiently expressed in the dorsal dermatome before the onset of trunk neural crest cell dispersal on the lateral pathway, it is no longer produced by dermatomal cells when melanocyte precursors have dispersed in the dermal mesenchyme. To assess the role of Steel factor in migration of melanocyte precursors on the lateral pathway, we analyzed melanocyte precursor dispersal and fate on the lateral pathway of two different Sl mutants, Sl, a null allele, and Sld, which lacks cell surface-associated Steel factor but produces a soluble form. No melanocyte precursors were detected in the dermatome of embryos homozygous for the Sl allele or in W mutants that lack functional c-kit. In contrast, in embryos homozygous for the Sld allele, melanocyte precursors appeared on the lateral pathway, but subsequently disappear from the dermis. These results suggest that soluble Steel factor is required for melanocyte precursor dispersal on the lateral pathway, or for their initial survival in the migration staging area. In contrast, membrane-bound Steel factor appears to promote melanocyte precursor survival in the dermis.
Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3
Authors: Pierce Sarah B. , Kimelman David
Published: April 2021

Dorsal axis formation in the Xenopus embryo can be induced by the ectopic expression of several Wnt family members. In Drosophila, the protein encoded by the Wnt family gene, wingless, signals through a pathway that antagonizes the effects of the serine/threonine kinase zeste-white 3/shaggy. We describe the isolation and characterization of a Xenopus homolog of zeste-white 3/shaggy, Xgsk-3. A kinase-dead mutant of Xgsk-3, Xgsk-3K→R, has a dominant negative effect and mimics the ability of Wnt to induce a secondary axis by induction of an ectopic Spemann organizer. Xgsk-3K→R, like Wnt, induces dorsal axis formation when expressed in the deep vegetal cells, which do not contribute to the axis. These results indicate that the dorsal fate is actively repressed by Xgsk-3, which must be inactivated for dorsal axis formation to occur. Furthermore, our work suggests that the effects of Xgsk-3K→R are mediated by an additional intercellular signal.
Proteinase expression in early mouse embryos is regulated by leukaemia inhibitory factor and epidermal growth factor
Authors: Harvey M. B. , Leco K. J. , Arcellana-Panlilio M. Y. , Zhang X. , Edwards D. R. , Schultz G. A.
Published: April 2021

Several proteinases from different multigene families have been implicated in the uterine invasion required for estab-lishment of pregnancy in some mammals. In this study, the expression of matrix metalloproteinase gelatinase B (MMP-9), urokinase-type plasminogen activator (uPA) and their inhibitors was investigated during early mouse embryo development. Transcripts for tissue inhibitors of metalloproteinases (TIMP-1,-2,-3) and uPA receptor were detected throughout pre- and peri-implantation develop-ment whilst MMP-9 and uPA mRNAs were first detected in peri-implantation blastocysts associated with the invasive phase of implantation. Through use of in situ hybridization, it was shown that MMP-9 transcripts were strongly expressed in the network of trophoblast giant cells at the periphery of implanting 7.5 day embryos and TIMP-3 transcripts were strongly expressed in the decidua imme-diately adjacent to the implanting embryo. uPA transcripts were preferentially expressed in the ectoplacental cone and its derivatives. Because these proteinases are regulated by growth factors and cytokines in other tissues, the effect of leukaemia inhibitory factor (LIF) and epidermal growth factor (EGF) on their activity was investigated. Both LIF and EGF, like the proteinases, have been implicated in peri-implantation development. Blastocysts collected on day 4 of pregnancy were cultured 2 days in TCM 199 + 10% fetal bovine serum to allow outgrowth followed by 24 hour culture in defined media containing either LIF or EGF. Conditioned media were assayed for uPA activity by a chromogenic assay and MMP activity by gelatin zymog-raphy. Both LIF and EGF stimulated uPA and MMP-9 activity in blastocyst outgrowths after 3 days of culture (day 7). Proteinase activity was assayed again at the 5th to 6th day of culture (day 9 to 10). EGF was found to have no effect whereas LIF decreased production of both pro-teinases. These results demonstrate that proteinase activity in early embryos can be regulated by growth factors and cytokines during the implantation process and, in particu-lar, they demonstrate the possible involvement of LIF in establishment of the correct temporal programme of pro-teinase expression.
Branching morphogenesis of embryonic mouse lung epithelium in mesenchyme-free culture
Authors: Nogawa Hiroyuki , Ito Takaaki
Published: April 2021

Embryonic mouse lung epithelium was separated from its mesenchyme and cultured under mesenchyme-free conditions. When covered with Matrigel, the cultured epithelium underwent branching morphogenesis in medium containing acidic fibroblast growth factor (aFGF), in which the epithelial cells constructed a simple columnar cell layer forming a lumen, as seen in normal development. The epithelial growth and branching morphogenesis induced by aFGF was completely inhibited by an antibody against aFGF. Heparin caused extra epithelial growth in cooperation with aFGF, but its use resulted in luminal expansion instead of enhanced branching. Basic FGF induced abnormal morphogenesis of the epithelium, though the lumen formed was lined by a simple columnar cell layer. Epidermal growth factor could not maintain epithelial cell growth, and the epithelium became a smaller and smoother ball than that at the start of cultivation. When covered with a collagen gel instead of Matrigel, the epithelium remained in its initial form, neither newly branching nor becoming a smooth ball, in the presence of aFGF. These results show that the epithelium of lung rudiments was able to branch under mesenchyme-free culture conditions in which a basement membrane matrix and aFGF were substitutes for the mesenchyme.
Regulation of regional differences in the differentiation of cerebral cortical neurons by EGF family-matrix interactions
Authors: Ferri Raymond T. , Levitt Pat
Published: April 2021

Both lineage-based and epigenetic regulation have been postulated as mechanisms to control the formation of discrete areas in the cerebral cortex, but specific genes or signaling pathways that may be involved have yet to be defined. In this paper, we examine whether progenitors, isolated from the cerebral wall prior to neurogenesis, can respond to exogenous cues by adopting a region-specific phenotype. The expression of the limbic system-associated membrane protein (LAMP), a neuron-specific marker of limbic cortical areas, was monitored in cultured neurons arising from precursors harvested from presumptive perirhinal (limbic) and sensorimotor (nonlimbic) zones at embryonic day 12 in the rat. Neuronal phenotype in all cultures was identified by expression of microtubule-associated protein-2 (MAP2). On a substrate of poly-lysine, over 80% of the precursors from the limbic area that differentiate into neurons express a LAMP+ phenotype. Approximately 20% of the neurons generated from precursors of the sensorimotor region become LAMP+. However, modification of the microenvironment had a significant effect on the differentiation of the sensorimotor precursors. When the nonlimbic precursors are grown on Matrigel, there is a 2-fold increase in the number of MAP2+/LAMP+ double-labeled neurons. Dissection of the Matrigel components reveals that in combination with growth factor-deficient Matrigel or collagen type IV, epidermal growth factor and transforming growth factor-α increase LAMP expression in the sensorimotor population. Delaying the addition of growth factor until after most cell division had ceased failed to increase the number of LAMP+ neurons. Another growth factor in Matrigel, platelet-derived growth factor, does not produce the same effect. Our results indicate that local signals can regulate the differentiation of cortical progenitors, providing a potential mechanism for establishing an early commitment to specific regional phenotypes in the developing cerebral wall that relate to future functional domains in the cortex.
Conditional root expansion mutants of Arabidopsis
Authors: Hauser Marie-Theres , Morikami Atsushi , Benfey Philip N.
Published: April 2021

Regulation of cell expansion is essential to the formation of plant organs. We have characterized 21 mutations, representing six loci, that cause abnormal cell expansion in the root of Arabidopsis thaliana. The phenotype of these mutants is conditional upon the rate of root growth. Calculation of cell volumes indicated that the mutations resulted in defects in either the orientation or the extent of expansion or in both. Analysis of cortical microtubules in the mutants suggested that a shift in the orientation of cell expansion may not be dependent on a change in the orientation of the microtubules. Double mutant combinations resulted in loss of the conditional phenotype suggesting that the genes may act in a similar pathway or encode partially redundant functions.
Patterning of the mesoderm in Xenopus: dose-dependent and synergistic effects of Brachyury and Pintallavis
Authors: O’Reilly M.-A. J. , Smith J. C. , Cunliffe V.
Published: April 2021

Widespread expression of the DNA-binding protein Brachyury in Xenopus animal caps causes ectopic mesoderm formation. In this paper, we first show that two types of mesoderm are induced by different concentrations of Brachyury. Animal pole explants from embryos injected with low doses of Xbra RNA differentiate into vesicles containing mesothelial smooth muscle and mesenchyme. At higher concentrations somitic muscle is formed. The transition from smooth muscle formation to that of somitic muscle occurs over a two-fold increase in Brachyury concentration.

Brachyury is required for differentiation of notochord in mouse and fish embryos, but even the highest concentrations of Brachyury do not induce this tissue in Xenopus animal caps. Co-expression of Brachyury with the secreted glycoprotein noggin does cause notochord formation, but it is difficult to understand the molecular basis of this phenomenon without knowing more about the noggin signal transduction pathway. To overcome this difficulty, we have now tested mesoderm-specific transcription factors for the ability to synergize with Brachyury. We find that coexpression of Pintallavis, but not goosecoid, with Brachyury causes formation of dorsal mesoderm, including notochord. Furthermore, the effect of Pintallavis, like that of Brachyury, is dose-dependent: a two-fold increase in Pintallavis RNA causes a transition from ventral mesoderm formation to that of muscle, and a further two-fold increase induces notochord and neural tissue. These results suggest that Pintallavis cooperates with Brachyury to pattern the mesoderm in Xenopus.
Role of MAP kinase in mesoderm induction and axial patterning during Xenopus development
Authors: LaBonne Carole , Burke Brenda , Whitman Malcolm
Published: April 2021

We have examined the role of MAP kinase during mesoderm induction and axial patterning in Xenopus embryos. MAP Kinase Phosphatase (MKP-1) was used to inactivate endogenous MAP kinase and was found to prevent the induction of early and late mesodermal markers by both FGF and activin. In whole embryos, MKP-1 was found to disrupt posterior axial patterning, generating a phenotype similar to that obtained with a dominant inhibitory FGF receptor. Overexpression of either constitutively active MAP kinase or constitutively active MAP kinase (MEK) was sufficient to induce Xbra expression, while only constitutively active MEK was able to significantly induce expression of muscle actin. When MAP kinase phosphorylation was used as a sensitive marker of FGF receptor activity in vivo, this activity was found to persist at a low and relatively uniform level throughout blastula stage embryos. The finding that a low level of MAP kinase phosphorylation exists in unstimulated animal caps and is absent in caps overexpressing a dominant inhibitory FGF receptor provides a basis for our previous observation that overexpression of this receptor inhibits activin induction. These results indicate that FGFdependent MAP kinase activity plays a critical role in establishing the responsiveness of embryonic tissues to mesoderm inducers.
Effect of activin and lithium on isolated Xenopus animal blastomeres and response alteration at the midblastula transition
Authors: Kinoshita Kei , Asashima Makoto
Published: April 2021

Dorsoventral mesoderm patterning in the amphibian embryo involves a series of interactions mediated by several peptide growth factors. Animal blastomeres isolated at the 8-cell stage are useful for studying mesoderm patterning, since they contain the prospective (uninduced) mesoderm region and allow examination of the default state of animal cells. When activin is applied to these dorsal and ventral animal half explants, a competence prepattern for responding to activin is observed. In order to investigate the characteristics of prepatterning, we treated animal blastomeres with the embryo dorsalizing agent LiCl. Treatment with lithium alone did not induce normal trunk mesoderm in either blastomere. Lithium did, however, alter the competence of animal blastomeres to activin. Dorsal mesoderm was formed in the ventral blastomeres, as well as in the dorsal blastomeres. This result reveals that the early dorsoventral polarity in the animal hemisphere is not fixed.

Using goosecoid(gsc) and Xwnt-8 genes as dorsal and ventral mesoderm markers, it was verified that lithium modifies the competence to activin. Unexpectedly, lithium treatment on its own resulted in gsc expression in the animal half explants. This suggests that embryo goosecoid expression may be induced by the effect of dorsal determination activity, but not by mesoderm induction. However, lithium induced also the expression of brachyury (Xbra) gene at very low levels. This would indicate the formation of dorsal-anterior mesoderm, which was not identified by the tissue observations.

Expression of Xwnt-8, a ventral mesoderm marker usually induced in blastula animal caps by activin, was hardly induced in the blastomere explants. We isolated whole animal half explants at the 8-cell stage and exposed to activin at different stages. It was found that the same concentration of activin induces gsc before the midblastula stage, and induces Xwnt-8 at later stages. This suggests that the response of animal blastomeres alters depending on the stage of activin signaling.
A 25.7×103Mr hydra metalloproteinase (HMP1), a member of the astacin family, localizes to the extracellular matrix of Hydra vulgaris in a head-specific manner and has a developmental function
Authors: Yan Li , Pollock Gail H. , Nagase Hideaki , Sarras Michael P.
Published: April 2021

Hydra extracellular matrix (ECM) is composed of a number of components seen in vertebrate ECM such as laminin, type IV collagen, fibronectin, and heparan sulfate proteoglycan. A number of functional studies have shown that hydra ECM plays an important role in pattern formation and morphogenesis of this simple metazoan. The present study was designed to identify matrix degrading proteinases in hydra and determine their potential function in hydra morphogenesis. Using SDS-PAGE gelatin-zymography, five gelatinolytic bands were identified with relative molecular masses of 67×103, 51-58×102 (a triplet) and 25-29×103, respectively. Inhibition studies indicated that all of these gelatinases were metalloproteinases. Gelatin-zymography indicated that there was a differential distribution of these gelatinases along the longitudinal axis of hydra, with the 67×103Mr gelatinase being concentrated in the body column, while the 51-58×103Mr gelatinase triplet and the 25-29×103Mr gelatinase concentrated in the head region. Purification procedures were successfully developed for the 25-29×103Mr metalloproteinase which has been termed hydra metalloproteinase 1 (HMP1) and which appeared as a single band with a SDS-PAGE mobility of 25.7×103Mr.

The N-terminal sequence of purified HMP1 indicated that it has structural homology with metalloproteinases that belong to the astacin family. Subsequent cloning and sequencing of cDNA clones confirmed the identification of HMP1 as an astacin-like metalloproteinase. Immunocytochemical studies with antibodies generated against the purified enzyme and to a synthetic peptide indicated that HMP1 was localized to the ECM of tentacles. Functional studies were performed in which purified HMP1, anti-HMP1 IgG, or suspected substrates of HMP1 (e.g. growth factors such as TGF-β1) were introduced into the inter-epithelial compartment of hydra using a ‘DMSO loading’ procedure. These studies indicated that HMP1 has a functional role during a number of developmental processes such as head regeneration and cell differentiation/trans-differentiation of tentacle battery cells.
Transcriptional control of tektin A mRNA correlates with cilia development and length determination during sea urchin embryogenesis
Authors: Norrander J. M. , Linck R. W. , Stephens R. E.
Published: April 2021

Previous studies have shown that tektin A, one of three integral filamentous protein components of outer doublet microtubules, is synthesized in sea urchins in an amount correlating to the length of embryonic cilia initially assembled or experimentally regenerated. To investigate further the molecular mechanism for the regulation of tektin synthesis, tektin cDNA clones were used to assess mRNA levels during ciliogenesis, zinc-induced animalization, deciliation-induced regeneration and theophylline-induced elongation. Possibly involved in centriole replication, low, near-constant levels of mRNA for all three tektins are present in the unfertilized egg and during cleavage stages. Preceded by new synthesis of tektin B and C mRNAs, tektin A mRNA is up-regulated during ciliogenesis, but only tektin A mRNA levels correlate directly with ciliary length in animalized embryos; the others augment larger, non-limiting pools of tektins B and C. Tektin mRNAs decrease to steady-state levels after ciliogenesis, but are up-regulated again when the embryos are deciliated, correlating with the length of cilia to be deployed. In a species where a 3-fold ciliary length increase can be induced by theophylline treatment of zinc-arrested embryos, the mRNAs accumulate to proportionately higher levels during arrest but are not translated until induction, whereupon they decrease inversely with ciliary elongation. This suggests transcriptional control with respect to mRNA amounts but post-transcriptional control with respect to the expression of this phenotype. These data are consistent with a model in which (1) tektin filaments serve as linear determinants of microtubule doublet structure, and (2) the fixed amount of tektin A mRNA and protein synthesis consequently limit the length of doublets that can be co-assembled from larger pools of tektins B and C, tubulin, and other components.
TGFα can act as a chemoattractant to perioptic mesenchymal cells in developing mouse eyes
Authors: Reneker Lixing W. , Silversides David W. , Patel Kavita , Overbeek Paul A.
Published: April 2021

Growth factors are believed to play an important role in regulating cell fate and cell behavior during embryonic development. Transforming growth factor α (TGFα), a member of the epidermal growth factor (EGF) superfamily, is a small polypeptide growth factor. Upon binding to its receptor, the EGF receptor (EGFR), TGFα can exert diverse biological activities, such as induction of cell proliferation or differentiation. To explore the possibility that TGFα might regulate cell fate during murine eye development, we generated transgenic mice that express human TGFα in the lens under the control of the mouse αA-crystallin promoter. The transgenic mice displayed multiple eye defects, including corneal opacities, cataracts and microphthalmia. At early embryonic stages TGFα induced the perioptic mesenchymal cells to migrate abnormally into the eye and accumulate around the lens. In situ hybridization revealed that the EGFR mRNA is highly expressed in the perioptic mesenchyme, suggesting that the migratory response is mediated by receptor activation. In order to test this model, the TGFα transgenic mice were bred to EGFR mutant waved-2 (wa-2) mice. We found that the eye defects of the TGFα transgenic mice are significantly abated in the wa-2 homozygote background. Because the EGFR mutation in the wa-2 mice is located in the receptor kinase domain, this result indicates that the receptor tyrosine kinase activity is critical for signaling the migratory response. Taken together, our studies demonstrate that TGFα is capable of altering the migratory decisions and behavior of perioptic mesenchyme during eye development.