scholarly journals Reproductive response of Calanus helgolandicus. II. In situ inhibition of embryonic development

1995 ◽  
Vol 129 ◽  
pp. 97-105 ◽  
Author(s):  
M Laabir ◽  
SA Poulet ◽  
A Ianora ◽  
A Miralto ◽  
A Cueff
Keyword(s):  
Embryonic Development ◽  
Reproductive Response ◽  
Calanus Helgolandicus

In situ hybridization reveals co-expression of embryonic and adult alpha globin genes in the earliest murine erythrocyte progenitors

Development ◽  
1992 ◽  
Vol 116 (4) ◽  
pp. 1041-1049 ◽  
Author(s):  
A. Leder ◽  
A. Kuo ◽  
M.M. Shen ◽  
P. Leder
Keyword(s):  
In Situ Hybridization ◽  
Embryonic Development ◽  
Fetal Liver ◽  
Globin Gene ◽  
Yolk Sac ◽  
Globin Genes ◽  
Globin Mrna ◽  
Mature Adult ◽  
Alpha Globin

Murine erythropoiesis begins with the formation of primitive red blood cells in the blood islands of the embryonic yolk sac on day 7.5 of gestation. By analogy to human erythropoiesis, it has been thought that there is a gradual switch from the exclusive expression of the embryonic alpha-like globin (zeta) to the mature adult form (alpha) in these early mouse cells. We have used in situ hybridization to assess expression of these two globin genes during embryonic development. In contrast to what might have been expected, we find that there is simultaneous expression of both zeta and alpha genes from the very onset of erythropoiesis in the yolk sac. At no time could we detect expression of embryonic zeta globin mRNA without concomitant expression of adult alpha globin mRNA. Indeed, adult alpha transcripts exceed those of embryonic zeta in the earliest red cell precursors. Moreover, the pattern of hybridization reveals co-expression of both genes within the same cells. Even in the fetal liver, which supersedes the yolk sac as the major site of murine fetal erythropoiesis, there is a brief co-expression of zeta and alpha genes followed by the exclusive expression of the adult alpha genes. These data indicate an important difference in hematopoietic ontogeny between mouse and that of human, where zeta expression precedes that of alpha. In addition to resolving the embryonic expression of these globin genes, our results suggest that the embryonic alpha-like globin gene zeta may be physiologically redundant, even during the earliest stages of embryonic development.

af9 Regulates gata2 Expression During Early Hemangioblast Specification and Vascular Pattern Formation In Zebrafish.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2600-2600
Author(s):  
Giuseppe Germano ◽  
Ilaria Guariento ◽  
Natascia Tiso ◽  
Blaine W. Robinson ◽  
Enrico Moro ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Pattern Formation ◽  
Embryonic Development ◽  
Blood Cells ◽  
Erythroid Cell ◽  
Fusion Partner ◽  
Vascular Pattern ◽  
Cell Stage ◽  
Whole Mount

Abstract Abstract 2600 Background AF9 is a transcription factor that plays an essential role in hematopoiesis and embryonic development. The alteration of AF9 is principally associated in acute myeloid leukemia as fusion partner of human MLL (mixed-lineage leukemia) gene rearrangements. Zebrafish is an excellent model organism to study embryonic development and hematopoiesis. We have previously shown that zebrafish af9 is expressed within the intermediate cell mass (ICM), a site of primitive hematopoiesis in zebrafish. Here we study the loss of af9 in zebrafish development to further understand how af9 modulates early hematopoietic and embryonic development. Methods and results Two morpholino antisense oligos (MOs), designed to block translation and inhibit pre-mRNA splicing of af9, were co-injected in embryos at 1–2 cell stage. To control for off-target effects, two morpholino mismatch oligos were designed and co-injected. Efficacy of MOs was demonstrated by Western blot analysis and RT-PCR in controls and MO-injected embryos (morphants). In vivo monitoring of both morphants and control embryos was carried out by microscopy. Effects of af9 depletion on vasculature and erythropoiesis were evaluated in Tg(fli1:eGFP) and Tg(gata1:DsRed) transgenic lines, respectively. Whole-mount in situ hybridization of known hematopoietic markers was used to decipher the developmental time-points in which af9 regulates blood development. Following injection of two MOs at 1–2 cell stage, we compared the morphological features of the morphants with control embryos at about 24 hours post-fertilization (hpf). The af9 morphants showed small head and eyes, disruption of tail development and pronounced swelling in the posterior ICM. Circulating blood cells were reduced from 26 hpf to later stages of development. At 48 hpf the heart was enlarged, showed a paucity of blood-cells and pericardial edema. Decreased number of blood cells in morphant embryos was further confirmed by o-dianisidine staining at 48 hpf and 72 hpf and in living af9-knockdown gata1:DsRed transgenic animals, suggesting that the differentiation of erythroblasts remains insufficient or impaired. Concordant with this observation, we examined the expression of specific markers for early hematopoiesis (scl, lmo2 and gata2) and primitive erythropoiesis (gata1, hbbe, and band3) using whole-mount in situ hybridization (WISH). At the 5-somite stage, the early hematopoietic precursor marker gata2 was markedly increased while scl and lmo2 remained unaffected in af9 morphants. Interestingly, by 24 hpf gata2 was found to be specifically over-expressed in ICM while no change was observed for scl and lmo2 markers. Besides, the erythroid progenitors and mature erythrocyte markers gata1, band3 and hbbe displayed nearly normal expression. To further confirm the role af9 in early hematopoiesis, we examined its expression in moonshine, a mutant zebrafish with defects in erythroid maturation due to deficiency of tif1γ, a key regulator of hematopoietic gene expression. WISH analysis in moonshine showed loss of af9 expression in the ICM at 24 hpf, suggesting that af9 functions genetically downstream of tif1γ in normal erythroid cell development. To determine the effect of af9 on endothelial and vascular development, we performed knockdown of af9 in fli1:eGFP transgenic line. By 24 hpf, these morphants showed significant increase of fluorescence intensity in the posterior ICM and a clear perturbation in the inter-segmental vessels (ISV) of the trunk at 30 hpf, indicating that af9 is required for early steps in hemangioblast specification and vascular pattern formation in zebrafish. Conclusion af9 regulates gata2 expression during early hemangioblast specification and vascular pattern formation in zebrafish. af9 may also be involved in caudal segment morphogenesis. Taken together, these data provide the initial framework of a pathway that can be used to further integrate the molecular events regulating hemangioblast differentiation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Switchable genome editing via genetic code expansion

2018 ◽  
Author(s):  
Toru Suzuki ◽  
Maki Asami ◽  
Sanjay G. Patel ◽  
Louis Y. P. Luk ◽  
Yu-Hsuan Tsai ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Genetic Code ◽  
Genome Editing ◽  
Active Form ◽  
Mammalian Genome ◽  
Gene Drive ◽  
Genetic Code Expansion ◽  
Or Gene ◽  
Gene Drives

AbstractMultiple applications of genome editing by CRISPR-Cas9 necessitate stringent regulation and Cas9 variants have accordingly been generated whose activity responds to small ligands, temperature or light. However, these approaches are often impracticable, for example in clinical therapeutic genome editing in situ or gene drives in which environmentally-compatible control is paramount. With this in mind, we have developed heritable Cas9-mediated mammalian genome editing that is acutely controlled by the cheap lysine derivative, Lys(Boc) (BOC). Genetic code expansion permitted non-physiological BOC incorporation such that Cas9 (Cas9BOC) was expressed in a full-length, active form in cultured somatic cells only after BOC exposure. Stringently BOC-dependent, heritable editing of transgenic and native genomic loci occurred when Cas9BOC was expressed at the onset of mouse embryonic development from cRNA or Cas9BOC transgenic females. The tightly controlled Cas9 editing system reported here promises to have broad applications and is a first step towards purposed, spatiotemporal gene drive regulation over large geographical ranges.

scholarly journals How to distinguish between different cell lineages sharing common markers using combinations of double in-situ-hybridization and immunostaining in avian embryos: CXCR4-positive mesodermal and neural crest-derived cells

2020 ◽  
Vol 155 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Imadeldin Yahya ◽  
Marion Böing ◽  
Beate Brand-Saberi ◽  
Gabriela Morosan-Puopolo
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Embryonic Development ◽  
Neural Crest ◽  
Expression Patterns ◽  
Simultaneous Detection ◽  
Neural Crest Cells ◽  
Cell Lineages ◽  
Wide Range

AbstractCell migration plays a crucial role in early embryonic development. The chemokine receptor CXCR4 has been reported to guide migration of neural crest cells (NCCs) to form the dorsal root ganglia (DRG) and sympathetic ganglia (SG). CXCR4 also plays an important part during the formation of limb and cloacal muscles. NCCs migration and muscle formation during embryonic development are usually considered separately, although both cell lineages migrate in close neighbourhood and have markers in common. In this study, we present a new method for the simultaneous detection of CXCR4, mesodermal markers and NCCs markers during chicken embryo developmental stages HH18–HH25 by combining double whole-mount in situ hybridization (ISH) and immunostaining on floating vibratome sections. The simultaneous detection of CXCR4 and markers for the mesodermal and neural crest cells in multiple labelling allowed us to compare complex gene expression patterns and it could be easily used for a wide range of gene expression pattern analyses of other chicken embryonic tissues. All steps of the procedure, including the preparation of probes and embryos, prehybridization, hybridization, visualization of the double labelled transcripts and immunostaining, are described in detail.

scholarly journals POSTERS. An immunocytochemical and in situ hybridisation study of embryonic development of the human diencephalon

2001 ◽  
Vol 199 (1) ◽  
pp. 217-227
Author(s):  
G. CLOWRY ◽  
S. LISGO ◽  
D.-M. HAGAN ◽  
S. ROBSON ◽  
T. SRACHAN ◽  
...  
Keyword(s):  
Embryonic Development ◽  
In Situ Hybridisation ◽  
Hybridisation Study

Oligodendrocyte and astrocyte development in rodents: An in situ and immunohistological analysis during embryonic development

Glia ◽  
2002 ◽  
Vol 40 (1) ◽  
pp. 25-43 ◽  
Author(s):  
Ying Liu ◽  
Yuanyuan Wu ◽  
Jeffrey C. Lee ◽  
Haipeng Xue ◽  
Larysa H. Pevny ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Immunohistological Analysis

scholarly journals Generation of monoclonal antibodies detecting specific epitopes in locust antennae

1993 ◽  
Vol 175 (1) ◽  
pp. 45-57
Author(s):  
J. Strotmann ◽  
I. Boekhoff ◽  
S. Goggerle ◽  
H. Breer
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Protein Synthesis ◽  
Monoclonal Antibodies ◽  
Embryonic Development ◽  
High Molecular Weight ◽  
Time Course ◽  
Antigen Expression

1. Following a tissue-specific screening paradigm, monoclonal antibodies have been generated that interact with distinct subpopulations of cells in locust antennae. 2. Antigens were identified as high molecular weight components. 3. Immunoreactivity was not detectable during embryonic development, but rapidly appeared within a few hours of hatching. 4. The time course of antigen expression in antennal cells could be followed in situ as well as in vitro. 5. Expression of monoclonal antibody B14/6D2-like immunoreactivity was prevented by blocking protein synthesis with cycloheximide.

The Zebrafish Homologue of the Murine Ecotropic Viral Integration Site-1 (. Evi-1) gene Regulates Zebrafish Embryonic Blood Development.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1461-1461
Author(s):  
Martina Konantz ◽  
Martijn H. Brugman ◽  
In-Hyun Park ◽  
George Q. Daley ◽  
Christiane Nuesslein-Volhard ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Development ◽  
Integration Site ◽  
Myeloid Cells ◽  
Ips Cells ◽  
Viral Integration ◽  
Lateral Plate Mesoderm ◽  
Lateral Plate ◽  
Hematopoietic Development

Abstract Abstract 1461 Poster Board I-484 The ecotropic viral integration site-1 (Evi-1) locus was originally identified as a common site of retroviral integration in murine myeloid tumors and was later shown to be one of the most potent oncogenenes associated with murine and human myeloid leukemia. More recent data suggest involvement of Evi-1 in embryonic hematopoiesis (Goyama et al, Cell Stem Cell 2008; Yuasa et al, EMBO J, 2005), yet the precise role and molecular regulation of Evi-1 during blood development remains poorly understood. The zebrafish model offers powerful tools for genetic and embryonic studies. Here, we study zebrafish embryonic development and human pluripotent stem cells to understand how evi-1 modulates early hematopoietic development. Loss-of-function studies were performed in vivo by injecting Morpholino oligonucleotides in zebrafish zygotes to inhibit evi-1 pre-mRNA splicing. To control for off-target effects, two separate morpholinos were designed and injected. N=100 zebrafish were analysed pro experiment in each group. Inhibition of evi-1 was confirmed by quantitative PCR comparison in morpholino-injected and control embryos. Hematopoietic development was followed in both morphants and wild-type embryos by simple microscopy and in situ hybridizations using known hematopoeitic markers in order to investigate the developmental time-point in which evi-1 regulates blood development. evi-1 morpholino injected zebrafisch embryo showed severely reduced numbers of circulating blood cells, consistent with the phenotype observed in Evi-1−/− mice. Additionally, hemorrhages were observed, suggesting concomittant defects of the endothelial lineage in evi-1 deficient fish. In situ hybridization analysis on 11-12 somite stage embryos revealed strong reduction of myeloid embryonic hematopoiesis (measured by pu.1 expression in the anterior lateral plate mesoderm), while no change was observed in primitive erythroid progenitor cells (monitored by gata1 expression) or overall in blood and endothelial precursors in the posterior lateral plate mesoderm (as monitored by scl expression). Taken together, our studies demonstrate a strong impact of evi-1 on zebrafish blood development, confirming the results from Evi-1−/− mice. As gata1 expression and therefore erythroid precursor cells in the posterior blood islands are unaffected in evi-1 morphants, our results support the hypothesis that the reduction of primitive yolk-sac erythrocytes in mutant mice was caused from hemorrhages from pericardial effusions. Since erythroid and myeloid cells derive from a common precursor, but gata1 expression was unaffected in knock-down embryos, we anticipate that evi-1 plays a specific role in the myeloid lineage, as shown by abolished pu.1 expression in the anterior LPM. evi-1 therefore probably affects differentiation, survival or proliferation of myeloid cells. Previous reports in adult hematopoietic cells show that evi-1 can interact with both gata1 and pu.1. However, our data suggest that this is not the case during embryonic development, since gata1 expression remained unaltered in morpholino-injected embryos. Furthermore, data in mice suggest that Evi-1 may modulate embryonic hematopoiesis by affecting hematopoietic stem cell proliferation through regulation of Gata2. Currently ongoing experiments in our laboratories focus on characterization of genetic interactions between evi-1, gata2 and pu.1 during zebrafish blood development. Amongst other, gata2 and respectively pu.1 mRNA are co-injected in evi-1 morphants to analyse whether they can rescue the blood phenotype. Moreover, selected findings in zebrafish embryonic development will be verified in the human using using in vitro differentiating human induced pluripotent stem (iPS) cells. First expression data generated by real-time PCR analysis showed differential expression of EVI-1 in embryoid bodies generated from human iPS cells, confirming our hypothesis that EVI-1 has specific effects during human blood development. Disclosures No relevant conflicts of interest to declare.

scholarly journals FERTILIDAD Y FECUNDIDAD DE MONTASTRAEA ANNULARIS EN UN ARRECIFE DEGRADADO

2016 ◽  
Vol 38 (2) ◽  
Author(s):  
Elvira Alvarado Ch. ◽  
Alberto Acosta
Keyword(s):  
Reproductive Effort ◽  
Total Mortality ◽  
Recruitment Rate ◽  
Medium Size ◽  
Partial Mortality ◽  
Structure Characteristics ◽  
Male Gametes ◽  
Reproductive Response ◽  
Montastraea Annularis

This study determined the gametogenic cycle, fecundity and fertility of Montastraea annularis, in the Isla Grande coral reef, Archipelago of Nuestra Señora del Rosario, one of the most degraded reefs in the Colombian Caribbean. The surveyed population is currently characterized by medium size colonies (ca. 3379 cm2), with 27 % of the ramets showing partial mortality and 10 % with total mortality. Colonies present high number of small sized ramets formed by !ssion (76 %, smaller than 50 cm2). Based on the above mentioned population structure characteristics, the aim of this research was to explain the low recruitment rate reported for this reef through the reproductive response of a population dominated by small ramets. Between September 2003-2004, tissue fragments were collected and histological slides were made to detail the development of oocytes and spermaries and to estimate fecundity. Additionally during September of 2003 and 2004, in situ spawned eggs were collected to quantify fecundity. Results showed that oogenesis occurred between May and September and the spermatogenesis between July and September. Fertility was low (35 % and 40 %, in situ and histology, respectively) as well as fecundity (20 and 11 eggs/cm2/year-1, in situ and histology, respectively). Of the 33 % of ramets that showed spermaries, only 3 % of the mesenteries presented male gametes and in these, the presence of sperm cysts was also scarce (4 espermaries/gonad). Although the size of the ramets did not show a statistical relationship with fertility or fecundity, ramets between 50 and 100 cm2 were found more fecund than smaller (<50 cm2) or larger ramets (>200 cm2). The poor reproductive effort found can, in part, explain the low number of small colonies in the studied population, which in turn is expected in degraded sites and in populations with high !ssion rates. The results suggest that the viability of the population of M. annularis at Isla Grande is de!ned by the low number of fertile ramets and the low fecundity, but especially by low production of male gametes.

Sign in / Sign up

Export Citation Format

Share Document