bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for endogenous proteases, mRNA level and insulin as multiple mechanisms of N-cadherin down-regulation during retinal development
Authors: Roark Eileen F. , Paradies Nancy E. , Lagunowich Laura A. , Grunwald Gerald B.
Published: April 2021

Our previous studies of the role of cell adhesion in retinal development have focused on the expression and function of N-cadherin, the predominant calcium dependent intercellular adhesion protein of neural tissues. During the course of retinal development, N-cadherin expression undergoes significant qualitative and quantitative changes in its pattern of expression, most prominently a sharp down-regulation of expression throughout most of the retina. The present studies were directed at investigating the epigenetic mechanisms that could mediate this loss of N-cadherin from the retina. Using an in vitro intact retinal organ culture system, results were obtained which suggest that insulin enhances the down-regulation of N-cadherin expression in a protein-synthesis-dependent fashion. Furthermore, the metalloprotease inhibitor 1,10-phenanthroline inhibits the loss of N-cadherin from the retina. While N-cadherin is down-regulated in organ culture, other cell adhesion molecules, which are not down-regulated in vivo, are also not down-regulated in organ culture. The defined organ culture medium conditioned by the retina accumulates both a soluble 90 ×103Mr N-terminal fragment of N-cadherin as well as a number of secreted proteases. Both of these components are also shown to be present in vivo in the vitreous humor. Northern blot analysis indicates a single mRNA encoding N-cadherin in the retina and no evidence for a second message that could encode the 90 ×103Mr fragment. However, the amount of N-cadherin mRNA detectable on northern blots decreases during development. The results reported here suggest that the down-regulation of N-cadherin that occurs during retinal development is possibly mediated by multiple mechanisms, which include turnover at the cell surface mediated by endogenous proteolysis, reduced levels of N-cadherin mRNA and modulation by growth factors.
Cytoplasmic calcium and Fucus egg activation
Authors: Roberts S. K. , Gillot I. , Brownlee C.
Published: April 2021

Eggs of the marine brown alga, Fucus serratus, exhibit small transient elevations of cytosolic Ca2+ of variable magnitude, corresponding to the onset of the fertilization potential. Microinjection of Ca2+ buffers (BAPTA (1-2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid)) at concentrations sufficient to block any global fertilization-associated Ca2+cyt elevation did not inhibit egg activation (monitored as exocytosis of cell wall) or subsequent development. However, egg activation could be inhibited with higher buffer concentrations. Br2BAPTA (Kd = 1.6 μM) was a more effective inhibitor of egg activation than BAPTA (Kd = 0.17 μM). Localized microinjection of Ca2+ produced only localized cell wall exocytosis at the injection site. Eggs injected with Br2BAPTA at intracellular concentrations, which blocked egg activation, exhibited prolonged fertilization potentials. 45Ca2+ influx across the plasma membrane increased during fertilization. Our results show that a large transient global elevation of Ca2+cyt is not necessary for Fucus egg activation but rather a localized elevation to micromolar levels results, at least in part, from increased Ca2+ influx across the plasma membrane. This is needed for early fertilization events, including the generation of the fertilization potential and cell wall secretion.
Ecdysone receptor expression in the CNS correlates with stage-specific responses to ecdysteroids during Drosophila and Manduca development
Authors: Truman James W. , Talbot William S. , Fahrbach Susan E. , Hogness David S.
Published: April 2021

In insects, the ecdysteroids act to transform the CNS from its larval to its adult form. A key gene in this response is the ecdysone receptor (EcR), which has been shown in Drosophila to code for 3 protein isoforms. Two of these isoforms, EcR-A and EcR-B1, are prominently expressed in the CNS and we have used isoform-specific antibodies to examine their fluctuations through postembryonic life. EcR expression at the onset of metamorphosis is extremely diverse but specific patterns of EcR expression correlate with distinct patterns of steroid response. Most larval neurons show high levels of EcR-B1 at the start of meta-morphosis, a time when they lose larval features in response to ecdysteroids. Earlier, during the larval molts, the same cells have no detectable receptors and show no response to circulating ecdysteroids; later, during the pupal-adult transformation, they switch to EcR-A expression and respond by maturing to their adult form. During the latter period, a subset of the larval neurons hyperexpress EcR-A and these cells are fated to die after the emergence of the adult.

The stem cells for the imaginal neurons show prominent EcR-B1 expression during the last larval stage correlated with their main proliferative period. Most imaginal neurons, by contrast, express only EcR-A when they sub-sequently initiate maturation at the start of metamorphosis. The imaginal neurons of the mushroom bodies are unusual amongst imaginal neurons in expressing the B1 isoform at the start of metamorphosis but they also show regressive changes at this time as they lose their larval axons. Imaginal neurons of the optic lobe show a delayed expression of EcR-B1 through the period when cell-cell interactions are important for establishing connections within this region of the CNS.

Overall, the appearance of the two receptor isoforms in cells correlates with different types of steroid responses: EcR-A predominates when cells are undergoing maturational responses whereas EcR-B1 predominates during proliferative activity or regressive responses. The heterogeneity of EcR expression at the start of metamorphosis presumably reflects the diverse origins and requirements of the neurons that nevertheless are all exposed to a common hormonal signal.
Localization of egg cytoplasm that promotes differentiation to epidermis in embryos of the ascidian Halocynthia roretzi
Authors: Nishida Hiroki
Published: April 2021

Embryogenesis in ascidians is of the mosaic type. This property suggests the presence of cytoplasmic factors in the egg that are responsible for specification of the developmental fates of early blastomeres. The epidermal cells that surround the entire tadpole larva originate exclusively from blastomeres of the animal hemisphere of early embryos. To obtain direct evidence for cytoplasmic determinants of epidermis fate, we carried out cytoplasmic transfer experiments by fusing blastomeres and anucleate cell fragments from various regions of eggs and embryos. Initially, presumptive non-epidermis blastomeres (blastomeres from the vegetal hemisphere) were fused to cytoplasmic fragments from various regions of blastomeres of 8-cell embryos of Halocynthia roretzi, and development of epidermal cells was monitored by following the expression of an epidermis-specific antigen, as well as by observations of morphology and the secretion of larval tunic materials. Formation of epidermis was observed when vegetal blastomeres were fused with cytoplasmic fragments from the presumptive epidermis blastomeres. The results suggested that cytoplasmic factors that promoted epidermis differentiation (epidermis determinants) were present in epidermis progenitors. Vegetal blastomeres only manifested this change in fate when fused with cytoplasmic fragments of roughly equal or larger size. Next, to examine the presence and localization of epidermis determinants in the uncleaved egg, cytoplasmic fragments from various regions of unfertilized and fertilized eggs were fused with the vegetal blastomeres. The results suggested that epidermis determinants were already present in unfertilized eggs and that they were segregated by movements of the ooplasm after fertilization. After the first phase of ooplasmic segregation, these determinants were widely distributed, with the highest activity being located in the equatorial region. There were no indications of regional differences in the activity within the equatorial region of eggs at this stage. After the second phase of ooplasmic segregation, prior to the first cleavage, the activity moved in the animal direction, namely, to the animal hemisphere, from which future epidermis-lineage blastomeres are normally formed.
TGFβ1 inhibits branching morphogenesis and N-myc expression in lung bud organ cultures
Authors: Serra Rosa , Pelton Ron W. , Moses Harold L.
Published: April 2021

Lung buds isolated from 11.5 days post coitum mouse embryos survive and undergo branching morphogenesis in culture. This organ culture system was used to examine the role of TGFβ1 and N-myc expression in lung branching morphogenesis. By 24 hours, TGFβ1 reversibly inhibited branching morphogenesis in a concentration-dependent manner. N-myc is known to be expressed during embryonic development in epithelial cells involved in branching mor-phogenesis and homozygous null N-myc mice have defects in lung development. In the present study, TGFβ1 was shown to inhibit the steady-state level of N-myc RNA 3to 4-fold at 14 and 48 hours of treatment as measured by northern blot and RNase protection analysis. Suppression of N-myc expression in epithelium was confirmed by in situ hybridization. Since inhibition of N-myc occurred prior to the observed changes in morphology and previous genetic studies have demonstrated and important role for N-myc in lung developoment, a model is proposed in which TGFβ1 inhibits tracheobronchial development by inhibiting expression of N-myc.
The kreisler mouse: a hindbrain segmentation mutant that lacks tworhombomeres
Authors: McKay Ian J. , Muchamore Ian , Krumlauf Robb , Maden Malcolm , Lumsden Andrew , Lewis Julian
Published: April 2021

kreisler is a recessive mutation resulting in gross malformation of the inner ear of homozygous mice. The defects in the inner ear are related to abnormalities in the hindbrain of the embryo, adjacent to the ear rudiments. At E9.5, the neural tube posterior to the boundary between the third and fourth rhombomeres, r3 and r4, appears unsegmented, and the region that would normally correspond to r4 is unusually thick-walled and contains many dying cells. The absence of morphological segmentation in the posterior hindbrain corresponds to an altered pattern of gene expression in that region, with major abnormalities posterior to the r4/5 boundary and minor abnormalities anterior to it. From the expression patterns at E9.5 of Krox-20, Hoxb-1 (Hox 2.9), Hoxb-2 (Hox 2.8), Hoxa-3 (Hox 1.5), Hoxd-4 (Hox 4.2) and cellular retinoic-acid binding protein I (CRABP I), it appears that the fundamental defect is a loss of r5 and r6. Correspondingly, the glossopharyngeal ganglion and nerve, associated with r6 are missing and the abducens nerve, which originates from r5 and r6, is also absent. Examination of Krox-20 expression at stages as early as E8.5 indicates that Krox-20 fails ever to be expressed in its r5 domain in the homozygous kreisler mutant. The abnormal amount of cell death is seen only later. An interpretation is that the cells that would normally become specified at an early stage as r5 and r6 adopt an r4 character instead, producing an excess of r4 cells that is disposed of subsequently by cell death.
igloo, a GAP-43-related gene expressed in the developing nervous system of Drosophila
Authors: Neel Victor A. , Young Michael W.
Published: April 2021

GAP-43 (growth-associated protein, 43×103Mr) is an essential, membrane-associated, neuronal phosphoprotein in vertebrates. The protein is abundantly produced in the growth cones of developing and regenerating neurons, and it is phosphorylated upon induction of long-term potentiation (LTP). Prior work has identified GAP-43-like proteins only in chordates. In this paper, a nervous system-specific gene from Drosophila melanogaster is described that encodes two proteins sharing biochemical activities and sequence homology with GAP-43. The region of homology encompasses the calmodulin-binding domain and protein kinase C (PKC) phosphorylation site of GAP-43. The fly proteins are shown to bind Drosophila calmodulin (CaM), and are phosphorylated by purified PKC after a fashion predicted from prior work with vertebrate GAP-43. GAP-43 is modified by palmitoylation. An amino-terminal myris-toylation site is described for the Drosophila protein, which may play a similar role in membrane association in the fly. While a small family of GAP-43-related genes has been recognized in vertebrates, only a single gene appears to be present in the fly. As the Drosophila gene encodes two proteins, each with multiple calmodulin-binding domains and repeated sites for PKC phosphorylation, it may afford functions provided by the family of vertebrate genes.
Homeotic complex and teashirt genes co-operate to establish trunk segmental identities in Drosophila
Authors: Zulueta Pablo de , Alexandre Edith , Jacq Bernard , Kerridge Stephen
Published: April 2021

Homeotic genes determine the identities of metameres in Drosophila. We have examined functional aspects of the homeotic gene teashirt by ectopically expressing its product under the control of a heat-shock promoter during embryo-genesis. Our results confirm that the gene is critical for segmental identity of the larva. Under mild heat-shock con-ditions, the Teashirt protein induces an almost complete transformation of the labial to prothoracic segmental identity, when expressed before 8 hours of development. Positive autoregulation of the endogenous teashirt gene and the presence of Sex combs reduced protein in the labium explain this homeosis. Patterns in the maxillary and a more anterior head segment are partly replaced with trunk ones. Additional Teashirt protein has no effect on the identity of the trunk segments where the gene is normally expressed; teashirt function is overriden by some homeotic complex acting in the posterior trunk. Strong heat-shock regimes provoke novel defects: ectopic sense organs differentiate in posterior abdominal segments and trunk pattern elements differentiate in the ninth abdominal segment. Teashirt acts in a partially redundant way with certain homeotic complex proteins but co-operates with them for the estab-lishment of specific segment types. We suggest that Teashirt and HOM-C proteins regulate common sets of downstream target genes.
Regulation of gene expression in the mouse oocyte and early preimplantation embryo: developmental changes in Sp1 and TATA box-binding protein, TBP
Authors: Worrad Diane M. , Ram Prahlad T. , Schultz Richard M.
Published: April 2021

We previously demonstrated that an Sp1-dependent reporter gene is preferentially expressed in G2 of the 1-cell mouse embryo following microinjection of the male pronucleus when compared to microinjection of the female pronucleus (P. T. Ram and R. M. Schultz, 1993, Dev. Biol. 156, 552–556). We also noted that expression of the reporter gene is not observed following microinjection of the germinal vesicle of the fully grown oocyte. In the present study, we examined expression of this reporter gene during oocyte growth, as well as the nuclear concentration of two transcription factors, Sp1 and the TATA box-binding protein, TBP, during oocyte growth and the first cell cycle. The extent of reporter gene expression decreases during oocyte growth and this decrease correlates with the decrease in nuclear concentration of Sp1, as determined by confocal immunofluorescent microscopy. In addition, results of immunoblotting experiments also indicate a similar decrease in the total concentration of Sp1 during oocyte growth. The nuclear concentration of TBP also decreases during oocyte growth, as determined by confocal immunofluorescent microscopy. Following fertilization, the pronuclear concentration of these two transcription factors increases in a time-dependent fashion and the concentration of each is greater in the male pronucleus as compared to the female pronucleus. For each pronucleus and for each transcription factor, this increase in nuclear concentration is inhibited by aphidicolin, which inhibits DNA synthesis. Last, the increase in nuclear concentration of these two proteins observed between the 1-cell and 2-cell stages does not require transcription or cytokinesis.
A hierarchy of cross-regulation involving Notch, wingless, vestigial and cut organizes the dorsal/ventral axis of the Drosophila wing
Authors: Neumann Carl J. , Cohen Stephen M.
Published: April 2021

Short-range interaction between dorsal and ventral cells establishes an organizing center at the dorsal/ventral compartment boundary that controls growth and patterning of the wing. We report here that the dorsal/ventral organiser is built though a hierarchy of regulatory interactions involving the Notch and wingless signal transduction pathways and the vestigial gene. wingless and vestigial are activated in cells adjacent to the dorsal/ventral boundary by a Notch-dependent signal. vestigial is initially expressed under control of an early dorsal/ventral boundary enhancer that does not depend on wingless activity. Similarly, activation of wingless does not require vestigial function, showing that wingless and vestigial are parallel targets of the Notch pathway. Subsequently, vestigial is expressed in a broad domain that fills the wing pouch. This second phase of vestigial expression depends on Wingless function in cells at the dorsal/ventral boundary. In addition, the Notch and Wingless pathways act synergistically to regulate expression of cut in cells at the dorsal/ventral boundary. Thus Wingless can act locally, in combination with Notch, to specify cell fates, as well as at a distance to control vestigial expression. These results suggest that secreted Wingless protein mediates both longrange and short-range patterning activities of the dorsal/ventral boundary.
Genetic control of epithelial tube fusion during Drosophila tracheal development
Authors: Samakovlis Christos , Manning Gerard , Steneberg Pär , Hacohen Nir , Cantera Rafael , Krasnow Mark A.
Published: April 2021

During development of tubular networks such as the mammalian vascular system, the kidney and the Drosophila tracheal system, epithelial tubes must fuse to each other to form a continuous network. Little is known of the cellular mechanisms or molecular control of epithelial tube fusion. We describe the cellular dynamics of a tracheal fusion event in Drosophila and identify a gene regulatory hierarchy that controls this extraordinary process. A tracheal cell located at the developing fusion point expresses a sequence of specific markers as it grows out and contacts a similar cell from another tube; the two cells adhere and form an intercellular junction, and they become doughnut-shaped cells with the lumen passing through them. The early fusion marker Fusion-1 is identified as the escargot gene. It lies near the top of the regulatory hierarchy, activating the expression of later fusion markers and repressing genes that promote branching. Ectopic expression of escargot activates the fusion process and suppresses branching throughout the tracheal system, leading to ectopic tracheal connections that resemble certain arteriovenous malformations in humans. This establishes a simple genetic system to study fusion of epithelial tubes.
Overexpression of the tinman-related genes XNkx-2.5 and XNkx-2.3 in Xenopus embryos results in myocardial hyperplasia
Authors: Cleaver Ondine B. , Patterson Kristin D. , Krieg Paul A.
Published: April 2021

Drosophila tinman is an NK-class homeobox gene required for formation of the dorsal vessel, the insect equivalent of the vertebrate heart. Vertebrate sequences related to tinman, such as mouse Nkx-2.5, chicken cNkx-2.5, Xenopus XNkx-2.5 and XNkx-2.3 are expressed in cardiac precursors and in tissues involved in induction of cardiac mesoderm. Mice which lack a functional Nkx-2.5 gene die due to cardiac defects. To determine the role of tinman-related sequences in heart development, we have overexpressed both XNkx-2.3 and XNkx-2.5 in Xenopus laevis embryos. The resulting embryos are morphologically normal except that they have enlarged hearts. The enlarged heart phenotype is due to a thickening of the myocardium caused by an increase in the overall number of myocardial cells (hyperplasia). Neither ectopic nor precocious expression of cardiac differentiation markers is detectable in overexpressing embryos. These results suggest that both XNkx-2.3 and XNkx-2.5 are functional homologues of tinman, responsible for maintenance of the heart field.
Morphogenesis of the C. elegans hermaphrodite uterus
Authors: Newman Anna P. , White John G. , Sternberg Paul W.
Published: April 2021

We have undertaken electron micrographic reconstruction of the Caenorhabditis elegans hermaphrodite uterus and determined the correspondence between cells defined by their lineage history and differentiated cell types. In this organ, many cells do not move during morphogenesis and the cell lineage may function to put cells where they are needed. Differentiated uterine cell types include the toroidal ut cells that make structural epithelium, and specialized utse and uv cells that make the connection between the uterus and the vulva. A cell fate decision in which the anchor cell (AC) induces adjacent ventral uterine intermediate precursor cells to adopt the π fate, rather than the ground state ρ, has profound consequences for terminal differentiation: all π progeny are directly involved in making the uterine-vulval connection whereas all ρ progeny contribute to ut toroids or the uterine-spermathecal valve. In addition to specifying certain uterine cell fates, the AC also induces the vulva. Its multiple inductions thereby function to coordinate the connection of an internal to an external epithelium. The AC induces the π cells and ultimately fuses with a subset of their progeny. This is an example of reciprocal cell-cell interaction that can be studied at single cell resolution. The AC is thus a transitory cell type that plays a pivotal role in organizing the morphogenesis of the uterine-vulval connection.
Proteoglycans are required for maintenance of Wnt-11 expression in the ureter tips
Authors: Kispert Andreas , Vainio Seppo , Shen Liya , Rowitch David H. , McMahon Andrew P.
Published: April 2021

Development of the metanephric kidney requires the concerted interaction of two tissues, the epithelium of the ureteric duct and the metanephric mesenchyme. Signals from the ureter induce the metanephric mesenchyme to condense and proliferate around the ureter tip, reciprocal signals from the mesenchyme induce the ureter tip to grow and to branch. Wnt genes encode secreted glycoproteins, which are candidate mediators of these signaling events. We have identified three Wnt genes with specific, non-over-lapping expression patterns in the metanephric kidney, Wnt-4, Wnt-7b and Wnt-11. Wnt-4 is expressed in the condensing mesenchyme and the comma- and S-shaped bodies. Wnt-7b is expressed in the collecting duct epithelium from 13.5 days post coitum onward. Wnt-11 is first expressed in the nephric duct adjacent to the metanephric blastema prior to the outgrowth of the ureteric bud. Wnt-11 expression in Danforth’s short-tail mice suggests that signaling from the mesenchyme may regulate Wnt-11 activation. During metanephric development, Wnt-11 expression is confined to the tips of the branching ureter. Maintenance of this expression is independent of Wnt-4 signaling and mature mesenchymal elements in the kidney. Moreover, Wnt-11 expression is maintained in recombinants between ureter and lung mesenchyme suggesting that branching morphogenesis and maintenance of Wnt-11 expression are independent of metanephric mesenchyme-specific factors. Interference with proteoglycan synthesis leads to loss of Wnt-11 expression in the ureter tip. We suggest that Wnt-11 acts as an autocrine factor within the ureter epithelium and that its expression is regulated at least in part by proteoglycans.
Expression of constitutively active Notch arrests follicle cells at a precursor stage during Drosophila oogenesis and disrupts the anterior-posterior axis of the oocyte
Authors: Larkin Michele Keller , Holder Kristin , Yost Cynthia , Giniger Edward , Ruohola-Baker Hannele
Published: April 2021

During early development, there are numerous instances where a bipotent progenitor divides to give rise to two progeny cells with different fates. The Notch gene of Drosophila and its homologues in other metazoans have been implicated in many of these cell fate decisions. It has been argued that the role of Notch in such instances may be to maintain cells in a precursor state susceptible to specific differentiating signals. This has been difficult to prove, however, due to a lack of definitive markers for precursor identity. We here perform molecular and morphological analyses of the roles of Notch in ovarian follicle cells during Drosophila oogenesis. These studies show directly that constitutively active Notch arrests cells at a precursor stage, while the loss of Notch function eliminates this stage. Expression of moderate levels of activated Notch leads to partial transformation of cell fates, as found in other systems, and we show that this milder phenotype correlates with a prolonged, but still transient, precursor stage. We also find that expression of constitutively active Notch in follicle cells at later stages leads to a defect in the anterior-posterior axis of the oocyte.
Pointed, an ETS domain transcription factor, negatively regulates the EGF receptor pathway in Drosophila oogenesis
Authors: Morimoto Alyssa M. , Jordan Katherine C. , Tietze Kyria , Britton Jessica S. , O’Neill Elizabeth M. , Ruohola-Baker Hannele
Published: April 2021

Spatially regulated activation of the Drosophila epidermal growth factor (EGF) receptor by its ligand, Gurken, is required for establishment of the dorsal/ventral axis of the oocyte and embryo. During mid-oogenesis, Gurken is concentrated at the dorsal-anterior of the oocyte and is thought to activate the EGF receptor pathway in adjacent follicle cells. In response to this signal, dorsal follicle cell fate is determined. These cells further differentiate into either appendage-producing or midline cells, resulting in patterning in the dorsal follicle cell layer. We show here that Pointed, an ETS transcription factor, is required in dorsal follicle cells for this patterning. Loss of pointed results in the loss of midline cells and an excess of appendage-forming cells, a phenotype associated with overactivation of the EGF receptor pathway in the dorsal region. Overexpression of pointed leads to a phenotype similar to that generated by loss of the EGF receptor pathway. This suggests that Pointed normally down-regulates EGF receptor signaling in the midline to generate patterning in the dorsal region. Interestingly, pointed expression is induced by the EGF receptor pathway. These data indicate a novel antagonistic function for Pointed in oogenesis; in response to activation of the EGF receptor, pointed is expressed and negatively regulates the EGF receptor pathway, possibly by integrating information from a second pathway.
The caudal limit of Otx2 gene expression as a marker of the midbrain/hindbrain boundary: a study using in situ hybridisation and chick/quail homotopic grafts
Authors: Millet Sandrine , Bloch-Gallego Evelyne , Simeone Antonio , Alvarado-Mallart Rosa-Magda
Published: April 2021

Segmentation of the neural tube has been clearly shown in the forebrain and caudal hindbrain but has never been demonstrated within the midbrain/hindbrain domain. Since the homeobox-containing gene Otx2 has a caudal limit of expression in this region, we examined, mainly in chick embryos, the possibility that this limit could represent an interneuromeric boundary separating either two cerebellar domains or the mesencephalic and cerebellar primordia. In situ hybridisation with chick or mouse Otx2 probes showed the existence of a transient Otx2-negative area in the caudal mesencephalic vesicle, between stages HH10 and HH17/18 in chick, and at embryonic day 9.5 in mice. The first postmitotic neurons of the mesencephalon sensu stricto, as labelled with an antiβ-tubulin antibody, overlay the Otx2-positive neuroepithelium with a perfect match of the caudal limits of these two markers at all embryonic stages analysed (until stage HH20). Chick/quail homotopic grafts of various portions of the midbrain/hindbrain domain have shown that the progeny of the cells located in the caudal mesencephalic vesicle at stage HH10 are found within the rhombomere 1 as early as stage HH14. Furthermore, our results indicate that the cells forming the HH20 constriction (coinciding with the caudal Otx2 limit) are the progeny of those located at the caudal Otx2 limit at stage HH10 (within the mesencephalic vesicle). As a result, the Otx2-positive portion of the HH10 mesencephalic vesicle gives rise to the HH20 mesencephalon, while the Otx2-negative portion gives rise to the HH20 rostral rhombomere 1. Long-survival analysis allowing the recognition of the various grisea of the chimeric brains strongly supports the view that, as early as stage HH10, the caudal limit of Otx2 expression separates mesencephalic from isthmo/cerebellar territories. Finally, this study revealed unexpected rostrocaudal morphogenetic movements taking place between stages HH10 and HH16 in the mediodorsal part of the caudal Otx2-positive domain.
Evidence that Shh cooperates with a retinoic acid inducible co-factor to establish ZPA-like activity
Authors: Ogura Toshihiko , Alvarez Ignacio S. , Vogel Astrid , Rodríguez Concepción , Evans Ronald M. , Izpisúa Belmonte Juan Carlos
Published: April 2021

Patterning across the anteroposterior axis of the vertebrate limb bud involves a signal from the polarizing region, a small group of cells at the posterior margin of the bud. Retinoic acid (RA; Tickle, C., Alberts, B., Wolpert, L. and Lee, J. (1982) Nature 296, 554–566) and Sonic hedgehog (Shh; Riddle, R. D. Johnson, R.L., Laufer, E. and Tabin, C.J. (1993) Cell 25, 1401-1416; Chang, D. T., Lopez, A., von Kessler, D. P., Chiang, C., Simandl, B. K., Zhao, R., Seldin, M. F., Fallon, J. F. and Beachy, P. A. (1994) Development 120, 3339–3353) have been independently postulated as such signals because they can mimic the mirror image digit duplication obtained after grafting polarizing cells to the anterior of limb buds. Here we show that a embryonal carcinoma cell line, P19, transfected with a Shh expression vector shows low polarizing activity, but when cultured with retinoic acid, duplications like those induced by the polarizing region (ZPA) arise. Complete duplications are also obtained by cotransfecting P19 Shh cells with a constitutively active human retinoic acid receptor (VP16-hRARα). These data suggest that Shh and RA cooperate in generating ZPA activity and that Shh, while essential, may not act alone in this process.
A critical period for the specification of motor pools in the chick lumbosacral spinal cord
Authors: Matise Michael P. , Lance-Jones Cynthia
Published: April 2021

When 3-4 segments of the chick lumbosacral neural tube are reversed in the anterior-posterior axis at stage 15 (embryonic day 2.5), the spinal cord develops with a reversed organization of motoneurons projecting to individual muscles in the limb (C. Lance-Jones and L. Landmesser (1980) J. Physiol. 302, 581-602). This finding indicated that motoneuron precursors or components of their local environment were specified with respect to target by stage 15. To identify the timing of this event, we have now assessed motoneuron projections after equivalent neural tube reversals at earlier stages of development.

Lumbosacral neural tube segments 1-3 (± one segment cranial or caudal) were reversed in the anterior-posterior axis at stages 13 and 14 (embryonic day 2). The locations of motoneurons innervating two thigh muscles, the sartorius and femorotibialis, were mapped via retrograde horseradish peroxidase labeling at stages 35-36 (embryonic days 9-10). In a sample of embryos, counts were made of the total number of motoneurons in the lateral motor columns of reversed segments. The majority of motoneurons projecting to the sartorius and femorotibialis were in a normal position within the spinal cord. Segmental differences in motor column size were also similar to normal. These observations indicate that positional cues external to the LS neural tube can affect motoneuron commitment and number at stages 13-14. Since these observations stand in contrast to results following stage 15 reversals, we conclude that regional differences related to motoneuron target identity are normally specified or stabilized within the anterior LS neural tube between stages 14 and 15.

To examine the role of the notochord in this process, neural tube reversals were performed at stages 13-14 as described above, except that the underlying notochord was also reversed. Projections to the sartorius and femorotibialis muscles did not differ significantly from those in embryos with neural tube reversals alone, indicating that the notochord is not the source of cues for target identity at stages 13-14.
Procuste1 mutants identify two distinct genetic pathways controlling hypocotyl cell elongation, respectively in dark- and light-grown Arabidopsis seedlings
Authors: Desnos Thierry , Orbovic Vladimir , Bellini Catherine , Kronenberger Jocelyne , Caboche Michel , Höfte Jan Traas and Herman
Published: April 2021

Plant morphogenesis is dependent on a tight control of cell division and expansion. Cell elongation during post-embryonic hypocotyl growth is under the control of a light-regulated developmental switch. Light is generally believed to exert its effects on hypocotyl elongation through a phytochrome- and blue-light receptor-mediated inhibitory action on a so far unknown cell elongation mechanism.

We describe here a new class of allelic mutants in Arabidopsis, at the locus PROCUSTE1 (prc1-1 to -4), which have a hypocotyl elongation defect specifically associated with the dark-grown developmental program. Normal hypocotyl elongation is restored in plants grown in white, blue or red light. In agreement with this, the constitutive photomorphogenic mutation cop1-6, which induces a deetiolated phenotype in the dark, is epistatic to prc1-2 for the hypocotyl phenotype.

Epistasis analyses in red and blue light respectively, indicate that phytochrome B but not the blue light receptor HY4, is required for the switch from PRC1-dependent to PRC1-independent elongation.

The conditional hypocotyl growth defect is associated with a deformation of the hypocotyl surface due to an uncontrolled swelling of epidermal, cortical or endodermal cells, suggesting a defect in the structure of the expanding cell wall. A similar phenotype was observed in elongating roots, which was however, independent of the light conditions. The aerial part of mature mutant plants grown in the light was indistinguishable from the wild type.

prc1 mutants provide a means of distinguishing, for the first time, two genetic pathways regulating hypocotyl cell elongation respectively in dark- and light-grown seedlings, whereby light not only inhibits hypocotyl growth, but also activates a PRC1-independent cell elongation program.
The expression of trkB and p75 and the role of BDNF in the developing neuromuscular system of the chick embryo
Authors: McKay S. E. , Garner A. , Caldero J. , Tucker R. P. , Large T. , Oppenheim R. W.
Published: April 2021

The neurotrophin, brain-derived neurotrophic factor, prevents motoneuron cell death during the normal development of the chick embryo. Brain-derived neurotrophic factor is a ligand for the low-affinity NGF receptor, p75, and for the high-affinity neurotrophin receptor, trkB. If motoneurons respond directly to brain-derived neurotrophic factor then they must possess at least one, and possibly both, of these receptors during the period of naturally occurring cell death. Histological sections from the lumbar region of chick embryos were probed for the presence of trkB and p75 mRNA using digoxigenin-labeled anti-sense RNA probes. p75 mRNA was present in spinal cord motoneurons at stages of development that correlate with motoneuron cell death. Immunohistochemical localization also revealed that p75 protein was present in motoneurons, primarily along the ventral roots and developing intramuscular nerves. In contrast, trkB mRNA was not present in chick motoneurons until after the process of cell death was underway. The timing of trkB expression suggested that some motoneurons, i.e., those that die prior to the onset of trkB expression, may be insensitive to brainderived neurotrophic factor. This was confirmed by comparing the number of surviving motoneurons following different in vivo treatment paradigms. The evidence indicates that motoneurons undergo a temporal shift in sensitivity to brain-derived neurotrophic factor.
A Dictyostelium mutant with defective aggregate size determination
Authors: Brock Debra A. , Buczynski Greg , Spann Timothy P. , Wood Salli A. , Cardelli James , Gomer Richard H.
Published: April 2021

Starved Dictyostelium cells aggregate into groups of roughly 105 cells. We have identified a gene which, when repressed by antisense transformation or homologous recombination, causes starved cells to form large numbers of small aggregates. We call the gene smlA for small aggregates. A roughly 1.0 kb smlA mRNA is expressed in vegetative and early developing cells, and the mRNA level then decreases at about 10 hours of development. The sequence of the cDNA and the derived amino acid sequence of the SmlA protein show no significant similarity to any known sequence. There are no obvious motifs in the protein or large regions of hydrophobicity or charge. Immunofluorescence and staining of Western blots of cell fractions indicates that SmlA is a 35×103Mr cytosolic protein present in all vegetative and developing cells and is absent from smlA cells. The absence of SmlA does not affect the growth rate, cell cycle, motility, differentiation, or developmental speed of cells. Synergy experiments indicate that mixing 5% smlA cells with wild-type cells will cause the wild-type cells to form smaller fruiting bodies and aggregates. Although there is no detectable SmlA protein secreted from cells, starvation medium conditioned by smlA cells will cause wild-type cells to form large numbers of small aggregates. The component in the smlA-conditioned media that affects aggregate size is a molecule with a molecular mass greater than 100×103Mr that is not conditioned media factor, phosphodiesterase or the phosphodiesterase inhibitor. The data thus suggest that the cytosolic protein SmlA regulates the secretion or processing of a secreted factor that regulates aggregate size.
Function of posterior HoxD genes in the morphogenesis of the anal sphincter
Authors: Kondo Takashi , Dollé Pascal , Zákány Jozsef , Duboule Denis
Published: April 2021

Vertebrate 5′-located HoxD genes are expressed in the most caudal part of the digestive tract and their potential functions during gut development have been assessed by gene disruptions. We have inserted reporter lacZ sequences within the Hoxd-12 gene and analysed the morphology of the gut in these mice as well as in Hoxd-13 mutant animals. When homozygous, both mutations induce an important disorganization of the anorectal region. In particular, severe alterations of the smooth muscle layers of the rectum led to defective morphogenesis of the internal anal sphincter. Similarly, Hoxd-12 and Hoxd-13 functionally overlap during digit development. The function of these genes in the morphogenesis of the digestive system as well as their functional evolution are discussed.
CSF-1 deficiency in mice results in abnormal brain development
Authors: Michaelson M. Dror , Bieri Phyllis L. , Mehler Mark F. , Xu Hong , Arezzo Joseph C. , Pollard Jeffrey W. , Kessler John A.
Published: April 2021

Colony stimulating factor-1 (CSF-1) was initially identified as a growth factor for mononuclear phagocytes. This study examines the role of CSF-1 in the development of the central nervous system (CNS). CSF-1 treatment of neurons cultured from embryonic brain promoted survival and process outgrowth in a dose-dependent manner. By contrast, CSF-1 treatment of neurons cultured from the osteopetrotic (op/op) mouse, a null mutant for CSF-1, promoted significantly less process outgrowth, suggesting that there are neural abnormalities in op/op animals. Nuclease protection assays were used to determine whether CSF-1 and its receptor are expressed at times appropriate to regulate neural development. Both CSF-1 and its receptor are expressed in developing mouse brain, with a unique pattern of CSF-1 mRNA splice variant expression encoding secreted, and not membrane-bound, growth factor. To determine whether brain function is altered by null mutation of CSF-1, op/op mice were examined using electrophysiologic assays. Brainstem auditory and visual evoked potentials were both abnormal in op/op mice. Further, intracortical recordings revealed aberrant neuronal function within visual cortex and alterations in the cortical circuitry that balances excitation and inhibition. Daily CSF-1 injection of postnatal op/op mice largely rescued the abnormal neural phenotype, confirming that the absence of CSF-1 during development is responsible for the abnormalities. The effects of CSF-1 on cultured embryonic neural cells, the developmentally appropriate expression of CSF-1 and its receptor, and the neurological abnormalities in op/op mice suggest a role for CSF-1 in brain development.
Mechanisms of reduced fertility in Hoxa-10 mutant mice: uterine homeosis and loss of maternal Hoxa-10 expression
Authors: Benson Gail V. , Lim Hyunjung , Paria B. C. , Satokata Ichiro , Dey Sudhansu K. , Maas Richard L.
Published: April 2021

The establishment of a receptive uterine environment is critical for embryonic survival and implantation. One gene that is expressed in the uterus during the peri-implantation period in mice and is required for female fertility is the homeobox gene Hoxa-10. Here we characterize the periimplantation defects in Hoxa-10 mutant females and investigate functions of Hoxa-10 in the uterine anlage during morphogenesis and in the adult uterus during pregnancy. Examination of pregnancy in Hoxa-10 mutant females has revealed failure of implantation as well as resorption of embryos in the early postimplantation period. Morphologic analysis of the mutant uterus has demonstrated homeotic transformation of the proximal 25% into oviduct. Histology and molecular markers confirm this anterior transformation. Furthermore, in situ hybridization shows that this region coincides with the anterior limit of embryonic Hoxa-10 expression in the urogenital ducts and a parallel transformation is observed in Hoxa-10 mutant males at the junction of the epididymis and ductus deferens. Female fertility could be compromised by either the homeotic transformation or the absence of Hoxa-10 function in the adult during pregnancy. To distinguish between these two potential mechanisms of infertility, wildtype blastocysts were transferred into mutant uteri distal to the transformed region on day 2.5 of pseudopregnancy. This procedure did not rescue the phenotype, suggesting that adult uterine expression of Hoxa-10 is required during pregnancy. Moreover, when implantation was experimentally delayed, homozygous uteri were able to support survival of blastocysts comparable to wild-type controls, indicating that the requirement for Hoxa-10 is intrinsic to implantation. While expression of LIF and HB-EGF appears unaffected in the mutant uteri, a decrease is observed in the intensity and number of blue dye reactions, an indicator of increased vascular permeability in response to implantation. In addition, mutant uteri exhibited decreased decidualization in response to artificial stimuli. These results show that Hoxa-10 is required during morphogenesis for proper patterning of the reproductive tract and in the adult uterus for peri-implantation events.
Positive and negative signals modulate formation of the Xenopus cement gland
Authors: Bradley Leila , Wainstock Daniel , Sive Hazel
Published: April 2021

The cement gland is a simple secretory organ that marks the anterior-most dorsal ectoderm in Xenopus embryos. In this study, we examine the timing of cement gland induction and the cell interactions that contribute to cement gland formation. Firstly, we show that the outer ectodermal layer, from which the cement gland arises, becomes specified as cement gland by mid-gastrula. Curiously, at early gastrula, the inner layer of the dorsal ectoderm, which does not contribute to the mature cement gland, is strongly and transiently specified as cement gland. Secondly, we show that the mid-gastrula dorsoanterior yolky endoderm, which comes to underlie the cement gland primordium, is a potent inducer of cement gland formation and patterning. The cement gland itself has an anteroposterior pattern, with the gene XA expressed only posteriorly. Dorsoanterior yolky endoderm greatly enhances formation of large, patterned cement glands in partially induced anterodorsal ectoderm, but is unable to induce cement gland in naive animal caps. Neural tissue is induced less frequently than cement gland by the dorsoanterior yolky endoderm, suggesting that the endoderm induces cement gland directly. Thirdly, we demonstrate that the ventral ectoderm adjacent to the cement gland attenuates cement gland differentiation late during gastrulation. The more distant ventral mesendoderm is also a potent inhibitor of cement gland formation. These are the first data showing that normal ventral tissues can inhibit cement gland differentiation and suggest that cement gland size and position may be partly regulated by negative signals. Previous work has shown that cement gland can be induced by neural plate and by dorsal mesoderm. Together, these data suggest that cement gland induction is a complex process regulated by multiple positive and negative cell interactions.
derailed is required for muscle attachment site selection in Drosophila
Authors: Callahan Christopher A. , Bonkovsky Joshua L. , Scully Audra L. , Thomas John B.
Published: April 2021

During development, muscles must form and attach at highly stereotyped positions to allow for coordinated movements. In Drosophila, muscles grow towards and attach to specifically positioned cells within the epidermis. At the molecular level, very little is known about how muscles recognize these attachment sites. The derailed gene encodes a receptor tyrosine kinase family member that is essential for the pathfinding ability of expressing neurons. Here we show that the Drl RTK is also expressed by a small subset of developing embryonic muscles and neighboring epidermal cells during muscle attachment site selection. In drl mutants, these muscles often fail to attach at appropriate locations although their epidermal attachment cells appear unaffected. These results show that, similar to its role in neuronal pathway recognition, the Drl RTK participates in a mechanism required for muscle attachment site selection. The data suggest that both neurons and muscles use common mechanisms to recognize their paths or targets, and that Drl plays an analogous role in both developing systems.