bluuebunny/crossref_metadata_2025 huggingface dataset:
Three maternal coordinate systems cooperate in the patterning of the Drosophila head
Authors: Grossniklaus U. , Cadigan K.M. , Gehring W.J.
Published: April 2021

In contrast to the segmentation of the embryonic trunk region which has been extensively studied, relatively little is known about the development and segmentation of the Drosophila head. Proper development of the cephalic region requires the informational input of three of the four maternal coordinate systems. Head-specific gene expression is set up in response to a complex interaction between the maternally provided gene products and zygotically expressed genes. Several zygotic genes involved in head development have recently been characterized. A genetic analysis suggests that the segmentation of the head may use a mechanism different from the one acting in the trunk. The two genes of the sloppy paired locus (slp1 and slp2) are also expressed in the embryonic head. slp1 plays a predominant role in head formation while slp2 is largely dispensible. A detailed analysis of the slp head phenotype suggests that slp is important for the development of the mandibular segment as well as two adjacent pregnathal segments (antennal and ocular). Our analysis of regulatory interactions of slp with maternal and zygotic genes suggests that it behaves like a gap gene. Thus, phenotype and regulation of slp support the view that slp acts as a head-specific gap gene in addition to its function as a pair-rule and segment polarity gene in the trunk. We show that all three maternal systems active in the cephalic region are required for proper slp expression and that the different systems cooperate in the patterning of the head. The terminal and anterior patterning system appear to be closely linked. This cooperation is likely to involve a direct interaction between the bcd morphogen and the terminal system. Low levels of terminal system activity seem to potentiate bcd as an activator of slp, whereas high levels down-regulate bcd rendering it inactive. Our analysis suggests that dorsal, the morphogen of the dorsoventral system, and the head-specific gap gene empty spiracles act as repressor and corepressor in the regulation of slp. We discuss how positional information established independently along two axes can act in concert to control gene regulation in two dimensions.
Cell cycles and clonal strings during formation of the zebrafish central nervous system
Authors: Kimmel Charles B. , Warga Rachel M. , Kane Donald A.
Published: April 2021

Cell lineage analysis of central nervous system progenitors during gastrulation and early segmentation in the zebrafish reveals consistent coupling of specific morphogenetic behaviors with particular cell cycles. Cells in single clones divide very synchronously. Cell divisions become progressively oriented, and act synergistically with oriented intercalations during the interphases of zygotic cell cycles 15 and 16 to extend a single lineage into a long, discontinuous string of cells aligned with the nascent embryonic axis. Dorsalwards convergence brings the string to the midline and, once there, cells enter division 16. This division, or sometimes the next one, and the following cell movement reorient to separate siblings across the midline. This change converts the single string into a bilateral pair of strings, one forming a part of each side of the neural tube. The stereotyped cellular behaviors appear to account for the previously reported clonal restriction in cell fate and to underlie morphogenesis of a midline organ of proper length and bilateral shape. Regulation of cellular morphogenesis could be cell-cycle dependent.
A maternal homeobox gene, Bombyx caudal, forms both mRNA and protein concentration gradients spanning anteroposterior axis during gastrulation
Authors: Xu Xin , Xu Pin-Xian , Suzuki Yoshiaki
Published: April 2021

We have isolated a caudal (cad) homologue from a cDNA library of Bombyx mori embryos. The Bombyx cad cDNA encodes a protein of 244 amino acids. The homology between Drosophila and Bombyx homeodomains is 80%. Similar to Drosophila cad, there is no YPWM peptide sequence along the upstream of homeodomain. Northern blot hybridization with a Bombyx cad probe revealed the presence of single maternal transcript of 2.3 kb. A stronger signal of the transcripts was detected in unfertilized eggs and in eggs up to 36 hours after deposition. The transcripts decreased rapidly by 2 days and a weak signal was maintained until hatching. To analyse its spatial expression pattern, we have established a novel frozen sectioning method for in situ hybridization and immunohistochemistry experiments. The results showed that Bombyx cad transcripts accumulated first in the nurse cells and transferred into the oocyte at a defined time during oogenesis. The maternal transcripts of Bombyx cad formed a concentration gradient spanning the anteroposterior axis during the gastrulation stage and were restricted to the anal pad, the most posterior domain, after 2 days of embryogenesis; the Drosophila cad mRNA revealed the corresponding expression profile during the syncytial blastoderm stage. The Bombyx cad protein was not detected in the ovary and the first 9 hours of eggs, but was first detected evenly during cellular blastoderm stage. During gastrulation, Bombyx cad protein concentration gradients shifted along the anteroposterior axis coinciding with the shifting of the mRNA concentration gradients. This is the first example of a concentration gradient along anteroposterior axis that may be involved in segmentation in insect embryos other than the long germ-band type. A comparison of the timing and conservation of mRNA as well as protein gradient formation in Drosophila and Bombyx might help to explain differences in the body plans and give some clues to elucidate the mechanism and function related to mRNA and protein concentration gradients.
bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS
Authors: Merry Diane E. , Veis Deborah J. , Hickey William F. , Korsmeyer Stanley J.
Published: April 2021

Cell death is a common feature of neural development in all vertebrates. The bcl-2 proto-oncogene has been shown to protect a variety of cell types from programmed cell death. We have examined the distribution of bcl-2 protein in the developing and adult nervous systems. bcl-2 protein is widespread during embryonic development. Proliferating neuroepithelial cells of ventricular zones as well as the postmitotic cells of the cortical plate, cerebellum, hippocampus and spinal cord express bcl-2. Postnatally, bcl-2 is principally retained in the granule cells of the cerebellum and dentate gyrus of the hippocampus. bcl-2 expression in the CNS declines with aging. In the peripheral nervous system, neurons and supporting cells of sympathetic and sensory ganglia retain substantial bcl-2 protein throughout life. The widespread expression of bcl-2 in CNS and PNS neurons during embryonic development and its selective retention in the adult PNS is consistent with a role for bcl-2 in regulating neuronal survival. In addition, the expression of bcl-2 in some neuronal populations beyond the recognized period of cell death is suggestive of a role for bcl-2 beyond simply protecting neurons from developmental cell death.
Cell contact regulates neuroblast formation in the Caenorhabditis elegans lateral epidermis
Authors: Austin Judith , Kenyon Cynthia
Published: April 2021

A single line of epidermal seam cells lies along each side of the nematode C. elegans. During normal development, one of these cells, V5, produces a neuroblast that will give rise to a sensory structure, the postdeirid. If seam cells located either anterior or posterior to V5 are ablated however, this neuroblast formation is blocked. Because of this requirement for the presence of adjacent seam cells, we have asked whether V5’s ability to produce a neuroblast depends on direct contact with its seam cell neighbors. We find that direct contact between seam cells is required for commitment to neuroblast production. Seam cells lose and reform their contacts with each other as they go through rounds of cell division during larval development. Signaling required for neuroblast formation occurs when the seam cells make contact after their first round of division. If this contact is prevented, no neuroblast is made; when it is delayed, the time of signaling is also delayed. The characteristics of these signals suggest that a seam cell must be part of a continuous epithelium in order to develop normally and that signaling may occur via a cell recognition/cell adhesion pathway. The effect of seam cell ablations on neuroblast formation is altered in mab-5(–) animals, suggesting that this HOM-C gene is part of the pathway by which seam cell signaling controls the decision to make a postdeirid neuroblast.
Cell-extracellular matrix interactions under in vivo conditions during interstitial cell migration in Hydra vulgaris
Authors: Zhang Xiaoming , Sarras Michael P.
Published: April 2021

Interstitial cell (I-cell) migration in hydra is essential for establishment of the regional cell differentiation pattern in the organism. All previous in vivo studies have indicated that cell migration in hydra is a result of cell-cell interactions and chemotaxic gradients. Recently, in vitro cell adhesion studies indicated that isolated nematocytes could bind to substrata coated with isolated hydra mesoglea, fibronectin and type IV collagen. Under these conditions, nematocytes could be observed to migrate on some of these extracellular matrix components. By modifying previously described hydra grafting techniques, two procedures were developed to test specifically the role of extracellular matrix components during in vivo I-cell migration in hydra. In one approach, the extracellular matrix structure of the apical half of the hydra graft was perturbed using β-aminopropi-onitrile and β-xyloside. In the second approach, grafts were treated with fibronectin, RGDS synthetic peptide and antibody to fibronectin after grafting was performed. In both cases, I-cell migration from the basal half to the apical half of the grafts was quantitatively analyzed. Statistical analysis indicated that β-aminopropionitrile, fibronectin, RGDS synthetic peptide and antibody to fibronectin all were inhibitory to I-cell migration as compared to their respective controls.

β-xyloside treatment had no effect on interstitial cell migration. These results indicate the potential importance of cell-extracellular matrix interactions during in vivo I-cell migration in hydra.
An increase in intracellular pH during neural induction in Xenopus
Authors: Sater Amy K. , Alderton Janet M. , Steinhardt Richard A.
Published: April 2021

In this paper, we show that an intracellular alkalinization of the dorsal ectoderm cells is among the earliest responses to neural induction in Xenopus. Planar explants of the dorsal marginal zone were prepared from embryos that had been microinjected during cleavage stages with the fluorescent pH indicator bis-carboxyethyl-carboxyfluores-cein-dextran (BCECF-dextran), and intracellular pH (pHi) was monitored continuously by emission ratio microfluo-rimetry. During stage 10.5, the dorsal ectoderm cells undergo a sustained intracellular alkalinization of approximately 0.1 pH units in response to neural induction; in the absence of the inductive signal, the pH of the dorsal ectoderm cells decreases slightly. Ectoderm cells within planar explants of the ventral marginal zone show little change in pH during a similar period. This increase in intracellular pH is inhibited by 4, 4′-dihydrodiisothio-cyanatostilbene-2, 2′-disulfonate (H2DIDS) or a low Na+/high Cl− medium, treatments that presumably affect anion transport. Under these conditions, expression of the anterior neural-specific homeobox gene engrailed is not detected, while the notochord-specific epitope recognized by the Tor-70 antibody is expressed in the presence of H2DIDS. This characteristic alkalinization is not evoked by pharmacological agents that reportedly alter ectodermal developmental pathways in Xenopus embryos, such as NH4Cl, phorbol esters, or cAMP-dependent protein kinase agonists. Our results suggest that an ionic regulatory event may participate in the regulation of gene expression in response to neural induction.
Activin-mediated mesoderm induction requires FGF
Authors: Cornell Robert A. , Kimelman David
Published: April 2021

The early patterning of mesoderm in the Xenopus embryo requires signals from several intercellular factors, including mesoderm-inducing agents that belong to the fibroblast growth factor (FGF) and TGF-β families. In animal hemisphere explants (animal caps), basic FGF and the TGF-β family member activin are capable of converting pre-ectodermal cells to a mesodermal fate, although activin is much more effective at inducing dorsal and anterior mesoderm than is basic FGF. Using a dominant-negative form of the Xenopus type 1 FGF receptor, we show that an FGF signal is required for the full induction of mesoderm by activin. Animal caps isolated from embryos that have been injected with the truncated FGF receptor and cultured with activin do not extend and the induction of some genes, including cardiac actin and Xbra, is greatly diminished, while the induction of other genes, including the head organizer-specific genes gsc and Xlim-1, is less sensitive. These results are consistent with the phenotype of the truncated FGF receptor-injected embryo and imply that the activin induction of mesoderm depends on FGF, with some genes requiring a higher level of FGF signaling than others.
The Drosophila maternal-effect mutation grapes causes a metaphase arrest at nuclear cycle 13
Authors: Fogarty Patrick , Kalpin Robert F. , Sullivan William
Published: April 2021

grapes (grp) is a second chromosome (36A-B) maternal-effect lethal mutation in Drosophila melanogaster. We demonstrate that the syncytial nuclear divisions of grp-derived embryos are normal through metaphase of nuclear cycle 12. However, as the embryos progress into telophase of cycle 12, the microtubule structures rapidly deteriorate and midbodies never form. Immediately following the failure of midbody formation, sister telophase products collide and form large tetraploid nuclei. These observations suggest that the function of the midbody in the syncytial embryo is to maintain separation of sister nuclei during telophase of the cortical divisions. After an abbreviated nuclear cycle 13 interphase, these polyploid nuclei progress through prophase and arrest in metaphase. The spindles associated with the arrested nuclei are stable for hours even though the microtubules are rapidly turning over. The nuclear cycle 13 anaphase separation of sister chromatids never occurs and the chromosomes, still encompassed by spindles, assume a telophase conformation. Eventually neighboring arrested spindles begin to associate and form large clusters of spindles and nuclei. To determine whether this arrest was the result of a disruption in normal developmental events that occur at this time, both grp-derived and wild-type embryos were exposed to X-irradiation. Syncytial wild-type embryos exhibit a high division error rate, but not a nuclear-cycle arrest after exposure to low doses of X-irradiation. In contrast, grp-derived embryos exhibit a metaphase arrest in response to equivalent doses of X-irradiation. This arrest can be induced even in the early syncytial divisions prior to nuclear migration. These results suggest that the nuclear cycle 13 metaphase arrest of unexposed grp-derived embryos is independent of the division-cycle transitions that also occur at this stage. Instead, it may be the result of a previously unidentified feedback mechanism.
The relationship between ovarian and embryonic dorsoventral patterning in Drosophila
Authors: Roth Siegfried , Schüpbach Trudi
Published: April 2021

In Drosophila, the dorsoventral asymmetry of the egg chamber depends on a dorsalizing signal that emanates from the oocyte. This signal is supplied by the TGFα-like gurken protein whose RNA is localized to the dorsal-anterior corner of the oocyte. gurken protein is the potential ligand of the Drosophila EGF receptor homolog (torpedo), which is expressed in the follicular epithelium surrounding the oocyte. Here, we describe how changes in the dorsalizing germ-line signal affect the embryonic dorsoventral pattern. A reduction in strength of the germ-line signal as produced by mutations in gurken or torpedo does not change the slope of the embryonic dorsoventral morphogen gradient, but causes a splitting of the gradient ventrally. This leads to embryos with two partial dorsoven-tral axes. A change in distribution of the germ-line signal as caused by fs(1)K10, squid and orb mutations leads to a shift in the orientation of the embryonic dorsoventral axis relative to the anterior-posterior axis. In extreme cases, this results in embryos with a dorsoventral axis almost parallel to the anterior-posterior axis. These results imply that gurken, unlike other localized cytoplasmic determinants, is not directly responsible for the establishment of cell fates along a body axis, but that it restricts and orients an active axis-forming process which occurs later in the follicular epithelium or in the early embryo.
New functions of the Drosophila rhomboid gene during embryonic and adult development are revealed by a novel genetic method, enhancer piracy
Authors: Noll Rebekka , Sturtevant Mark A. , Gollapudi Raghava R. , Bier Ethan
Published: April 2021

Localized expression of the Drosophila rhomboid (rho) gene has been proposed to hyperactivate EGF-Receptor signaling in specific cells during development of the embryo and adult. In this report we use a novel transposon based genetic method, enhancer piracy, to drive ectopic expression of a rho cDNA transgene by endogenous genomic enhancers. Many enhancer piracy transposon-rho insertions cause dominant phenotypes, over half of which cannot be duplicated by ubiquitous expression of rho. Genetic interactions between various dominant enhancer piracy alleles and mutations in the EGF-R/RAS signaling pathway indicate that many of these novel phenotypes result from ectopic activation of EGF-R signaling.

Patterned mis-expression of the rho cDNA transgene cor- relates in several cases with localized dominant enhancer piracy phenotypes. Enhancer piracy lines reveal an unan- ticipated role for rho in imaginal disc formation and provide the first evidence that mis-expression of rho is suf- ficient for converting entire intervein sectors into veins. Enhancer piracy may prove to be a general strategy for obtaining dominant alleles of a gene of interest in diverse insects, worms, plants, and potentially in vertebrates such as mice and fish.
Timing of neuronal death in trkA, trkB and trkC mutant embryos reveals developmental changes in sensory neuron dependence on Trk signalling
Authors: Piñón Luzia G. P. , Minichiello Liliana , Klein Rüdiger , Davies Alun M.
Published: April 2021

The sensory neurons of the embryonic mouse trigeminal ganglion are supported in culture by different neurotrophins at successive stages of development. Initially the neurons survive in response to BDNF and NT3 and later switch to becoming NGF-dependent (Buchman, V. I. and Davies, A. M. (1993), Development 118, 989-1001). To determine if this in vitro switch in neurotrophin responsiveness is physiologically relevant, we studied the timing of neuronal death in the trigeminal ganglia of embryos that are homozygous for null mutations in the trkA, trkB and trkC genes, which encode receptor tyrosine kinases for NGF, BDNF and NT3, respectively. In wild-type embryos, the number of pyknotic nuclei increased from E11 to peak between E13 and E14, and decreased gradually at later ages, becoming negligible by birth. Neuronal death in the trigeminal ganglia of trkA−/− embryos also peaked between E13 and E14, but was almost threefold greater than in wild-type embryos at this stage. Whereas there was no significant difference between the number of pyknotic nuclei in trkA−/− and wild-type embryos at E11 and E12, there was a substantial increase in the number of pyknotic nuclei in the trigeminal ganglia of trkB−/− at these earlier stages. Counts of the total number of neurons in E13 trigeminal ganglia revealed a marked decrease in trkB−/− but not trkA−/− or trkC−/− embryos. Consistent with the later onset of excessive neuronal death in trkA−/− embryos, there was a marked decrease in the neuronal complement of the trigeminal ganglia of trkA−/− embryos at E15. These results demonstrate that TrkB signalling is required for the in vivo survival of many trigeminal neurons during the early stages of target field innervation before they become NGF-dependent.
Distinct spatial and temporal functions for PS integrins during Drosophila wing morphogenesis
Authors: Brabant Marc C. , Fristrom Dianne , Bunch Thomas A. , Brower Danny L.
Published: April 2021

At the onset of pupariation in the Drosophila wing, the PS1 and PS2 integrins are expressed preferentially on the dorsal and ventral wing epithelia, respectively. Clonal analysis experiments have indicated that integrins are required to maintain the tight association of the wing surfaces. Surprisingly, we find that even in clones of cells lacking integrins the wing layers become apposed early in metamorphosis. However, following the normal period of wing separation, large integrin mutant clones do not become re-apposed in the pupa, and integrins are not organized in basal plaques in cells opposite a mutant clone. Paradoxically, our experiments indicate that at least one integrin function requires different integrins on the dorsal and ventral wing surfaces, however in some cases both αPS subunits can function to some degree on each wing surface. Finally, overexpression of an αPS subunit throughout the wing leads to a dominant wing blister phenotype, and the critical period for this phenotype is the beginning of pupariation. These data indicate that integrin requirements in wing morphogenesis can be separated into early (prepupal) and late (pupal) functions. The late function seems to reflect the traditional view of integrins as cell-matrix adhesion proteins. The early requirement, which probably requires dorsoventral segregation of PS1 and PS2, suggests functions for PS1 and PS2 in signaling events that regulate morphogenesis.
BMP signaling during bone pattern determination in the developing limb
Authors: Kawakami Yasuhiko , Ishikawa Tetsuya , Shimabara Masakiyo , Tanda Nobuya , Enomoto-Iwamoto Motomi , Iwamoto Masahiro , Kuwana Takashi , Ueki Ayako , Noji Sumihare , Nohno Tsutomu
Published: April 2021

To examine the role of BMP signaling during limb pattern formation, we isolated chicken cDNAs encoding type I (BRK-1 and BRK-2) and type II (BRK-3) receptors for bone morphogenetic proteins. BRK-2 and BRK-3, which constitute dual-affinity signaling receptor complexes for BMPs, are co-expressed in condensing precartilaginous cells, while BRK-1 is weakly expressed in the limb mesenchyme. BRK-3 is also expressed in the apical ectodermal ridge and interdigital limb mesenchyme. BRK-2 is intensely expressed in the posterior-distal region of the limb bud. During digit duplication by implanting Sonic hedgehog-producing cells, BRK-2 expression is induced anteriorly in the new digit forming region as observed for BMP-2 and BMP-7 expression in the limb bud. Dominant-negative effects on BMP signaling were obtained by over-expressing kinase domain-deficient forms of the receptors. Chondrogenesis of limb mesenchymal cells is markedly inhibited by dominant-negative BRK-2 and BRK-3, but not by BRK-1. Although the bone pattern was not disturbed by expressing individual dominant-negative BRK independently, preferential distal and posterior limb truncations resulted from co-expressing the dominant-negative forms of BRK-2 and BRK-3 in the whole limb bud, thus providing evidence that BMPs are essential morphogenetic signals for limb bone patterning.
Role of Bicaudal-D in patterning the Drosophila egg chamber in mid-oogenesis
Authors: Swan Andrew , Suter Beat
Published: April 2021

The Bicaudal-D (Bic-D) gene is required early in Drosophila oogenesis for the differentiation of an oocyte from one of a cluster of 16 interconnected germarial cells. To analyze the role of Bic-D later in oogenesis, we have con-structed Drosophila lines in which Bic-D expression is under the control of the hsp70 promoter. In these flies, Bic-D activity can be induced early in oogenesis, allowing an oocyte to be made. Then, by shifting females to non-inducing conditions, Bic-D levels are depleted for the remainder of oogenesis. Using this system, we find that Bic-D is indeed required in the later stages of oogenesis. In ovaries from mutant females, oocyte growth is reduced, apparently due to defects in nurse-cell-to-oocyte transport. Smaller oocyte size results in the misalignment of follicle cells and the underlying germ line, leading to ventralization of dorsal follicle cells and to defects in centripetal cell migration. In addition, we show that Bic-D is required for the localization of specific mRNAs at both the anterior and posterior of the oocyte.
The role of bone morphogenetic proteins in vertebral development
Authors: Monsoro-Burq Anne-Hélène , Duprez Delphine , Watanabe Yuji , Bontoux Martine , Vincent Christine , Brickell Paul , Douarin Nicole Le
Published: April 2021

This study first shows a striking parallel between the expression patterns of the Bmp4, Msx1 and Msx2 genes in the lateral ridges of the neural plate before neural tube closure and later on, in the dorsal neural tube and superficial midline ectoderm. We have previously shown that the spinous process of the vertebra is formed from Msx1- and 2-expressing mesenchyme and that the dorsal neural tube can induce the differentiation of subcutaneous cartilage from the somitic mesenchyme. We show here that mouse BMP4- or human BMP2-producing cells grafted dorsally to the neural tube at E2 or E3 increase considerably the amount of Msx-expressing mesenchymal cells which are normally recruited from the somite to form the spinous process of the vertebra. Later on, the dorsal part of the vertebra is enlarged, resulting in vertebral fusion and, in some cases (e.g. grafts made at E3), in the formation of a ‘giant’ spinous process-like structure dorsally. In strong contrast, BMP-producing cells grafted laterally to the neural tube at E2 exerted a negative effect on the expression of Pax1 and Pax3 genes in the somitic mesenchyme, which then turned on Msx genes. Moreover, scle-rotomal cell growth and differentiation into cartilage were then inhibited. Dorsalization of the neural tube, manifested by expression of Msx and Pax3 genes in the basal plate contacting the BMP-producing cells, was also observed.

In conclusion, this study demonstrates that differentiation of the ventrolateral and dorsal parts of the vertebral cartilage is controlled by different molecular mechanisms. The former develops under the influence of signals arising from the floor plate-notochord complex. These signals inhibit the development of dorsal subcutaneous cartilage forming the spinous process, which requires the influence of BMP4 to differentiate.
Extracellular Ca2+ entry and Ca2+ release from inositol 1,4,5-trisphosphate-sensitive stores function at fertilization in oocytes of the marine bivalve Mytilus edulis
Authors: Deguchi Ryusaku , Osanai Kenzi , Morisawa Masaaki
Published: April 2021

An oocyte of the marine bivalve Mytilus edulis, which is arrested at metaphase I, reinitiates meiosis at fertilization. The fertilized oocyte shows increases in intracellular Ca2+ ([Ca2+]i) comprising three different phases: an initial large [Ca2+]i transient, a subsequent low but sustained [Ca2+]i elevation, and repetitive small [Ca2+]i transients. In this study, we have investigated the sources and mechanisms of the sperm-induced [Ca2+]i increases. Application of methoxyverapamil (D-600), an inhibitor of voltage-dependent Ca2+ influx, suppressed the initial [Ca2+]i transient but did not affect the following two phases of [Ca2+]i changes. Injection of heparin, an antagonist of the inositol 1,4,5-trisphosphate (IP3) receptor, inhibited the later two phases without much affecting the initial transient. Combined application of D-600 and heparin almost completely abolished the three phases of the sperm-induced [Ca2+]i changes. Furthermore, Ca2+ influx caused by seawater containing excess K+ was blocked by D-600 but not by heparin, and IP3-induced Ca2+ release caused by photolysis of injected ‘caged’ derivatives of IP3 was blocked by heparin but not by D-600. These results strongly suggest that two types of Ca2+ mobilization systems, the extracellular Ca2+ entry responsible for an initial [Ca2+]i transient and the IP3 receptor-mediated Ca2+ release responsible for the following two phases of [Ca2+]i changes, function at fertilization of Mytilus oocytes.
VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development
Authors: Kukk Eola , Lymboussaki Athina , Taira Suvi , Kaipainen Arja , Jeltsch Michael , Joukov Vladimir , Alitalo Kari
Published: April 2021

The vascular endothelial growth factor family has recently been expanded by the isolation of two new VEGF-related factors, VEGF-B and VEGF-C. The physiological functions of these factors are largely unknown. Here we report the cloning and characterization of mouse VEGF-C, which is produced as a disulfide-linked dimer of 415 amino acid residue polypeptides, sharing an 85% identity with the human VEGF-C amino acid sequence. The recombinant mouse VEGF-C protein was secreted from transfected cells as VEGFR-3 (Flt4) binding polypeptides of 30-32×103Mr and 22-23×103Mr which preferentially stimulated the autophosphorylation of VEGFR-3 in comparison with VEGFR-2 (KDR). In in situ hybridization, mouse VEGF-C mRNA expression was detected in mesenchymal cells of postimplantation mouse embryos, particularly in the regions where the lymphatic vessels undergo sprouting from embryonic veins, such as the perimetanephric, axillary and jugular regions. In addition, the developing mesenterium, which is rich in lymphatic vessels, showed strong VEGF-C expression. VEGF-C was also highly expressed in adult mouse lung, heart and kidney, where VEGFR-3 was also prominent. The pattern of expression of VEGF-C in relation to its major receptor VEGFR-3 during the sprouting of the lymphatic endothelium in embryos suggests a paracrine mode of action and that one of the functions of VEGF-C may be in the regulation of angiogenesis of the lymphatic vasculature.
Segmental expression of Hoxa-2 in the hindbrain is directly regulated by Krox-20
Authors: Nonchev Stefan , Vesque Christine , Maconochie Mark , Seitanidou Tania , Ariza-McNaughton Linda , Frain Monique , Marshall Heather , Sham Mai Har , Krumlauf Robb , Charnay Patrick
Published: April 2021

The hindbrain is a segmented structure divided into repeating metameric units termed rhombomeres (r). The Hox family, vertebrate homologs of the Drosophila HOM-C homeotic selector genes, are expressed in rhombomere-restricted patterns and are believed to participate in regulating segmental identities. Krox-20, a zinc finger gene, has a highly conserved pattern of expression in r3 and r5 and is functionally required for their maintenance in mouse embryos. Krox-20 has been shown to directly regulate the Hoxb-2 gene and we wanted to determine if it was involved in regulating multiple Hox genes as a part of its functional role. Hoxa-2 is the only known paralog of Hoxb-2, and we examined the patterns of expression of the mouse Hoxa-2 gene with particular focus on r3 and r5 in wild type and Krox-20−/− mutant embryos. There was a clear loss of expression in r3, which indicated that Hoxa-2 was downstream of Krox-20. Using transgenic analysis with E. coli lacZ reporter genes we have identified and mapped an r3/r5 enhancer in the 5′ flanking region of the Hoxa-2 gene. Deletion analysis narrowed this region to an 809 bp BglII fragment, and in vitro binding and competition assays with bacterially expressed Krox-20 protein identified two sites within the enhancer. Mutation of these Krox-20 sites in the regulatory region specifically abolished r3/r5 activity, but did not affect neural crest and mesodermal components. This indicated that the two Krox-20 sites are required in vivo for enhancer function. Furthermore, ectopic expression of Krox-20 in r4 was able to transactivate the Hoxa-2/lacZ reporter in this rhombomere. Together our findings suggest that Krox-20 directly participates in the transcriptional regulation of Hoxa-2 during hindbrain segmentation, and is responsible for the upregulation of the r3 and r5 domains of expression of both vertebrate group 2 Hox paralogs. Therefore, the segmental phenotypes in the Krox-20 mutants are likely to reflect the role of Krox-20 in directly regulating multiple Hox genes.
REGA-1 is a GPI-linked member of the immunoglobulin superfamily present on restricted regions of sheath cell processes in grasshopper
Authors: Seaver Elaine C. , Carpenter Ellen M. , Bastiani Michael J.
Published: April 2021

REGA-1 is a glycoprotein localized to sheath cell processes in the developing CNS when NBs are producing progeny and neurons are maturing and extending processes. It is also present on a subset of muscles and on the lumenal surface of the ectoderm in the embryonic appendages when pioneer neurons are growing into the CNS. REGA-1 is associated with the extracellular side of the cell membrane by a glycosyl-phosphatidylinositol linkage. We have identified a cDNA clone encoding REGA-1 using a sequence from purified protein. Sequence analysis defines REGA-1 as a novel member of the immunoglobulin superfamily containing three immunoglobulin domains and one fibronectin type III repeat. Each Ig domain has distinct sequence characteristics that suggest discrete functions. REGA-1 is similar to other Ig superfamily members involved in cell adhesion events and neurite outgrowth.
Autonomous and nonautonomous Notch functions for embryonic muscle and epidermis development in Drosophila
Authors: Baker Rob , Schubiger Gerold
Published: April 2021

The Notch (N) gene encodes a cell signaling protein that mediates neuronal and epidermal determination in Drosophila embryos. N also regulates several aspects of myogenic development; embryos lacking N function have too many muscle founder cells and fail to properly differentiate somatic muscle. To identify cell-autonomous requirements for Notch function during muscle development, we expressed a Notch minigene in the mesoderm, but not in the ectoderm, of amorphic N− embryos. In these embryos, muscle founder hypertrophy is rescued, indicating that Notch is autonomously required by mesoderm cells to regulate the proper number of muscle founders. However, somatic muscle differentiation is only partially normalized, suggesting that Notch is also required in the ectoderm for proper muscle development.

Additionally, mesodermal expression of Notch partially rescues epidermal development in overlying neurogenic ectoderm. This is unexpected, since previous studies suggest that Notch is autonomously required by proneural ectoderm cells for epidermal development. Mesodermal expression of a truncated Notch protein lacking the extracellular domain does not rescue ventral epidermis, suggesting that the extracellular domain of Notch can non-autonomously rescue epidermal development across germ layers.
An extracellular matrix molecule that is selectively expressed during development is important for gastrulation in the sea urchin embryo
Authors: Berg Linnea K. , Chen Siming W. , Wessel Gary M.
Published: April 2021

The extracellular matrix is important in the regulation of many cellular events of early development including migration, shape change, proliferation and gene expression. In the sea urchin embryo, disruption of the extracellular matrix results in selective defects in each of these events during gastrulation. Here we describe a new molecule of the extracellular matrix in Lytechinus variegatus, referred to as ECM 18, that has several important features. First, antibody interference of ECM 18 results in a profound but reversible inhibition of primary mesenchyme cell organization and endoderm morphogenesis during gastrulation. Second, during gastrulation, ECM 18 mRNA accumulates to highest levels in the invaginating endoderm and the ECM 18 protein is deposited in the basal lamina surrounding the archenteron as well as in other areas of the blastocoel wall. Immunolocalization by fluorescence and electron microscopy demonstrates the selective accumulation of ECM 18 in the extracellular matrix. Third, although the mRNA encoding ECM 18 is present throughout development, the protein accumulates only during gastrulation. ECM 18 protein is not detected in eggs or early embryos and analysis of polysome-associated mRNA suggests that at least part of the translational regulation of ECM 18 is at the level of ECM 18 mRNA-polysome formation. Finally, sequence analysis of ECM 18 shows that the protein contains a repeat sequence with a conserved cysteine motif, suggestive of involvement in proteinprotein interactions. Thus, ECM 18 appears to be important in mediating select morphogenetic changes during gastrulation and the pattern of its expression in the embryo is unique among the extracellular matrix molecules known in this embryo.
The Caenorhabditis elegans LIN-26 protein is required to specify and/or maintain all non-neuronal ectodermal cell fates
Authors: Labouesse Michel , Hartwieg Erika , Horvitz H. Robert
Published: April 2021

The C. elegans gene lin-26, which encodes a presumptive zinc-finger transcription factor, is required for hypodermal cells to acquire their proper fates. Here we show that lin26 is expressed not only in all hypodermal cells but also in all glial-like cells. During asymmetric cell divisions that generate a neuronal cell and a non-neuronal cell, LIN-26 protein is symmetrically segregated and then lost from the neuronal cell. Expression in glial-like cells (socket and sheath cells) is biologically important, as some of these neuronal support cells die or seem sometimes to be transformed to neuron-like cells in embryos homozygous for strong loss-of-function mutations. In addition, most of these glial-like cells are structurally and functionally defective in animals carrying the weak loss-of-function mutation lin-26(n156). lin-26 mutant phenotypes and expression patterns together suggest that lin-26 is required to specify and/or maintain the fates not only of hypodermal cells but also of all other non-neuronal ectodermal cells in C. elegans. We speculate that lin-26 acts by repressing the expression of neuronal-specific genes in non-neuronal cells.
Genetic analysis of Laminin A in Drosophila: extracellular matrix containing laminin A is required for ocellar axon pathfinding
Authors: García-Alonso Luis , Fetter Richard D. , Goodman Corey S.
Published: April 2021

Genetic analysis of the Laminin A (LamA) gene in Drosophila reveals that distinct classes of sensory axons have different requirements for extracellular matrix (ECM) containing laminin A versus epithelial cell surfaces. In the eye-antenna imaginal disc, the nerve from the three simple eyes (ocelli) to the brain is pioneered by a population of transient ocellar neurons whose axons extend on an ECM that covers and connects the disc epithelium and brain. Axons from neighboring mechanosensory (bristle) neurons extend under the ECM in direct contact with the surface of the disc cells, and pioneer a different axon pathway that enters the brain in a different location. In LamA mutants, the ocellar pioneer axons display striking pathfinding defects, while neighboring bristle axons appear normal; the ocellar pioneers usually extend in the proper direction, adhering to the epithelium and sometimes fasciculating with mechanosensory axons, but they invariably fail to reach the brain.
Evidence in the mouse for self-renewing stem cells in the formation of a segmented longitudinal structure, the myotome
Authors: Nicolas J. F. , Mathis L. , Bonnerot C.
Published: April 2021

A novel method of clonal analysis has been used in the mouse to define the cellular events that lead to the formation of a segmented longitudinal structure, the myotome. Progenitor cells of the myotome were randomly marked during development by intragenic homologous recombination in transgenic mice expressing a reporter laacZ gene. 153 clones corresponding to 7829 cells, that is 20% of the myotomal population of one embryo, were obtained from 3000 E11.5 embryos. Their analysis leads to the hypothesis that, at E11.5, the 41 segments of the myotome have been mainly produced from a unique, spatially organised pool of self-renewing stem cells that accompanies the formation of the anterior-posterior axis.
Switching the in vivo specificity of a minimal Hox-responsive element
Authors: Chan Siu-Kwong , Ryoo Hyung-Don , Gould Alex , Krumlauf Robb , Mann Richard S.
Published: April 2021

The homeodomain proteins encoded by the Hox complex genes do not bind DNA with high specificity. In vitro, Hox specificity can be increased by binding to DNA cooperatively with the homeodomain protein extradenticle or its vertebrate homologs, the pbx proteins (together, the PBC family). Here we show that a two basepair change in a Hox-PBC binding site switches the Hox-dependent expression pattern generated in vivo, from labial to Deformed. The change in vivo correlates with an altered Hox binding specificity in vitro. Further, we identify similar Deformed-PBC binding sites in the Deformed and Hoxb-4 genes and show that they generate Deformed or Hoxb-4 expression patterns in Drosophila and mouse embryos, respectively. These results suggest a model in which Hox-PBC binding sites play an instructive role in Hox specificity by promoting the formation of different Hox-PBC heterodimers in vivo. Thus, the choice of Hox partner, and therefore Hox target genes, depends on subtle differences between Hox-PBC binding sites.
The C. elegans gene pag-3 is homologous to the zinc finger proto-oncogene gfi-1
Authors: Jia Yiwen , Xie Guofeng , McDermott Joan B. , Aamodt Eric
Published: April 2021

Mutations in the Caenorhabditis elegans gene pag-3 result in misexpression of touch receptor-specific genes in the BDU interneurons and in motility defects. We cloned pag-3 and found that the gene encodes a C2H2-type zinc finger protein related to the mammalian GFI-1 protein. Sequencing of the three pag-3 alleles showed that two apparent null alleles encode a nonsense mutation before the zinc fingers and a missense mutation in the fourth zinc finger that changes a coordinating histidine to a tyrosine. The third allele contains a nonsense mutation in the N-terminal region but is not a null allele. Northern analysis showed that a single pag-3 transcript of about 1.6 kb is present in embryos and L1, L2 and L3 larvae. pag-3 message levels were about twofold higher in pag-3 mutants than in wildtype animals, which suggested that pag-3 may negatively regulate its own expression. pag-3lacZ fusion genes were expressed in the BDU interneurons, the touch neurons, 11 VA and 11 VB ventral cord motor neurons, two AVF interneurons and in unidentified neurons of the retrovesicular ganglion. The BDU neurons and the ALM touch neurons are lineal sister cells in the AB.a lineage and the VA and VB motor neurons are lineal sister cells in the AB.p lineage. The VA motor neurons are required for backward movement and the VB motor neurons are required for forward movement. Mosaic analysis showed that the wildtype pag-3 gene is required in the AB.p lineage for coordinated movement and in the AB.a lineage to suppress touch neuron gene expression in the BDU neurons. Because pag-3 is expressed in both the BDU neurons and in the touch neurons, another protein(s) not expressed in the touch neurons may interact with pag-3 to repress touch neuron gene expression in the BDU neurons. Alternatively, another protein in the touch receptor cells may inactivate PAG-3 and allow expression of the touch receptor program. These results show that pag-3 is a temporally regulated gene that is expressed early in development and functions in multiple types of neurons. They also strongly suggest that the PAG-3 protein is a DNA-binding protein with properties similar to the mammalian proto-oncogene product GFI-1.
Xmsx-1 modifies mesodermal tissue pattern along dorsoventral axis in Xenopus laevis embryo
Authors: Maeda Ryu , Kobayashi Ako , Sekine Ryo , Lin Jih-Jing , Kung Hsiang-fu , Maéno Mitsugu
Published: April 2021

This study analyzes the expression and the function of Xenopus msx-1(Xmsx-1) in embryos, in relation to the ventralizing activity of bone morphogenetic protein-4 (BMP-4). Expression of Xmsx-1was increased in UV-treated ventralized embryos and decreased in LiCl-treated dorsalized embryos at the neurula stage (stage 14). Whole-mount in situ hybridization analysis showed that Xmsx-1is expressed in marginal zone and animal pole areas, laterally and ventrally, but not dorsally, at mid-gastrula (stage 11) and late-gastrula (stage 13) stages. Injection of BMP-4 RNA, but not activin RNA, induced Xmsx-1 expression in the dorsal marginal zone at the early gastrula stage (stage 10+), and introduction of a dominant negative form of BMP-4 receptor RNA suppressed Xmsx-1expression in animal cap and ventral marginal zone explants at stage 14. Thus, Xmsx-1 is a target gene specifically regulated by BMP-4 signaling. Embryos injected with Xmsx-1 RNA in dorsal blastomeres at the 4-cell stage exhibited a ventralized phenotype, with microcephaly and swollen abdomen. Histological observation and immunostaining revealed that these embryos had a large block of muscle tissue in the dorsal mesodermal area instead of notochord. On the basis of molecular marker analysis, however, the injection of Xmsx-1RNA did not induce the expression of α-globin, nor reduce cardiac α-actin in dorsal marginal zone explants. Furthermore, a significant amount of α-actin was induced and α-globin was turned off in the ventral marginal zone explants injected with Xmsx-1. These results indicated that Xmsx-1 is a target gene of BMP-4 signaling, but possesses a distinct activity on dorsal-ventral patterning of mesodermal tissues.
A role for Siamois in Spemann organizer formation
Authors: Fan Melinda J. , Sokol Sergei Y.
Published: April 2021

The vertebrate body plan is specified in the early embryo through the inductive influence of the organizer, a special region that forms on the dorsalmost side of the embryo at the beginning of gastrulation. In Xenopus, the homeobox gene Siamois is activated prior to gastrulation in the area of organizer activity and is capable of inducing a secondary body axis when ectopically expressed. To elucidate the function of endogeneous Siamois in dorsoventral axis formation, we made a dominant repressor construct (SE) in which the Siamois homeodomain was fused to an active repression domain of Drosophila engrailed. Overexpression of 1-5 pg of this chimeric mRNA in the early embryo blocks axis development and inhibits activation of dorsal, but not ventrolateral, marginal zone markers. At similar expression levels, SE proteins with altered DNA-binding specificity do not have the same effect. Coexpression of mRNA encoding wild-type Siamois, but not a mutated Siamois, restores dorsal development to SE embryos. Furthermore, SE strongly blocks axis formation triggered by β-catenin but not by the organizer product noggin. These results suggest that Siamois function is essential for β-catenin-mediated formation of the Spemann organizer, and that Siamois acts prior to noggin in specifying dorsal development.