Axolotl Eggs Research Articles

THE MEXICAN AXOLOTL´S (Ambystoma mexicanum) EARLY DEVELOPMENT AND SURVIVAL IS AFFECTED BY COMMERCIAL GRADE MALATHION AND DICHLORVOS ORGANOPHOSPHORUS PESTICIDES

The wild population of the Mexican axolotl (Ambystoma mexicanum) is subject to a dramatic decline as a consequence of deteriorating conditions in its natural habitat, Xochimilco. The common use of organophosphorus pesticides (OPPs) in the region affects water quality and is partially responsible for the axolotl decline. This could be a consequence of the delicate nature of axolotls, which are struggling to survive under contaminated conditions at early stages of development. In this regard, we aimed to extend the knowledge of OPPs effect on the survival of axolotls in their early stage of development, within the context of detrimental environment conditions using commercial grade OPPs. Fertilized axolotl eggs were treated with malathion (MLT), dichlorvos (DDVP), and a depurated group in which the OPPs exposure was stopped earlier, as well as an untreated control group. Changes in hatching, survival, size, and morphology were monitored and analyzed in embryos and early larvae. Our results showed that MLT and DVPP accelerate egg hatching and mortality even after the early removal of OPPs from the medium. OPPs also caused a reduced size and morphological abnormalities. It is proposed that such abnormalities would jeopardize the survival of the wildlife axolotl. Our results suggest that OPPs can cause irreversible damage to the axolotl embryos, stopping their normal development, causing death and reducing their chances of survival in their natural environment.

Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).

Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR-Cas9 system. The protocol, which takes 7-8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs. The direct use of purified CAS9-NLS (CAS9 containing a C-terminal nuclear localization signal) protein allows the prompt formation of guide RNA (gRNA)-CAS9-NLS ribonucleoprotein (RNP) complexes, which accelerates the creation of double-strand breaks (DSBs) at targeted genomic loci in single-cell-stage axolotl eggs. With this protocol, a substantial number of F0 individuals harboring a homozygous-type frameshift mutation can be obtained, allowing phenotype analysis in this generation. In the presence of targeting constructs, insertions of exogenous genes into targeted axolotl genomic loci can be achieved at efficiencies of up to 15% in a non-homologous end joining (NHEJ) manner. Our protocol bypasses the long generation time of axolotls and allows direct functional analysis in F0 genetically manipulated axolotls. This protocol can be potentially applied to other animal models, especially to organisms with a well-characterized transcriptome but lacking a well-characterized genome.

Generation of Transgenic Axolotls (Ambystoma mexicanum): Figure 1.

INTRODUCTIONInjection of RNA, DNA, or morpholinos into amphibian and fish embryos is a standard technique used to test gene function during development or to make transgenic animals. The techniques used to inject axolotl (Ambystoma mexicanum) embryos are very similar to those used for Xenopus embryos, with a few modifications to account for the larger egg size and “softer” cytoplasm. For example, amphibian embryos must be “dejellied” prior to injection. Chemical treatments used to remove the jellycoat for Xenopus eggs are not reliable for axolotl eggs; thus, dejellying is performed manually. In addition, greater injection volumes (typically 50 nL) are used with axolotl embryos, with correspondingly larger total amounts of DNA and RNA, as compared with Xenopus. Injection of plasmid DNA into the axolotl egg results in varying frequencies of integration (depending on the plasmid DNA) within one of the first few cell divisions. The integration efficiency is significantly increased if the plasmid DNA contains SceI meganuclease sites and is coinjected with SceI meganuclease, as described in this protocol.

Axolotl (Ambystoma mexicanum) In Vitro Fertilization

INTRODUCTIONBreeding of axolotl (Ambystoma mexicanum) can be accomplished by natural or artificial methods. Although natural matings are a very easy way of obtaining axolotl embryos, in vitro fertilization can have other advantages. For example, through in vitro fertilization, it is possible to know the exact time of fertilization. This protocol describes preparation of a sperm suspension and in vitro fertilization of axolotl eggs after injection of females with follicle stimulating hormone (FSH).

Post-transcriptional control of c -myc RNA during early development analyzed in vivo with a Xenopus-axolotl heterologous system

We have set up a heterologous in vivo system to study gene regulation at the post-transcriptional level during early development. This system uses two amphibian species, Xenopus laevis and Ambystoma mexicanum (axolotl), the development of which is three to four times slower than that of X. laevis. The stability of three different synthetic X. laevis c -myc transcripts was followed after injection into fertilized axolotl eggs. One transcript is 2.2 kilobases (kb) long (full-length). The second is 1.5-kb long with most of the 3′ untranslated region (3′UTR) removed, and the third corresponds to the 3′UTR (0.7-kb). The behavior of the endogenous axolotl c -myc RNA was compared with the exogenous injected c -myc transcripts. Our results show the existence of several developmental timers controlling degradation of the c -myc molecules. The first is activated at oocyte maturation and affects both the endogenous and exogenous (2.2- and 1.5-kb) transcripts containing the coding regions. A second timer could be linked to the number of cell divisions since fertilization (6th–7th cleavages) and involves the endogenous c -myc RNAs. Another timer could involve the c -myc mRNA molecule itself, because when injected into axolotl eggs, the half-life of the 2.2-kb X. laevis transcript appears to be independent of the axolotl context. After injection into axolotl fertilized eggs, the behavior of this X. laevis full-length c -myc molecule reveals an unexpected increase in the intensity of its autoradiographic signals. This increase occurs independently of events linked to mid-blastula transition and preliminary investigations are discussed.

Primary structure of neutral and acidic oligosaccharide-alditols derived from the jelly coat of the Mexican axolotl. Occurrence of oligosaccharides with fucosyl(alpha 1-3)fucosyl(alpha 1-4)-3-deoxy-D-glycero-D-galacto-nonulosonic acid and galactosyl(alpha 1-4)[fucosyl(alpha 1-2)]galactosyl(beta 1-4)-N-acetylglucosamine sequences.

Six major neutral and acidic oligosaccharide-alditols were prepared from the jelly coat of Mexican axolotl eggs. These compounds were demonstrated to contain 3-deoxy-D-glycero-D-galacto-nonulosonic acid (dNloA) and L-fucose (Fuc). The structures of the six major oligosaccharides were established as follows: [sequence: see text]

Ectopic expression of a genomic fragment containing a homeobox causes neural defects in the axolotl

An axolotl (Ambystoma mexicanum) genomic fragment containing the Ahox1 homeobox was placed under the control of the mouse hsp68 promoter, which seems to function constitutively in the axolotl. The resulting construct was injected into fertilized axolotl eggs to see if it would perturb development. Of the injected embryos, 20% showed severe reduction of the anterior neural plate. Later in development, these embryos had small heads, no eyes, and appeared to lack the normal regionalization of the brain. An additional 35% of the embryos were less severely affected, but had reduced or missing eyes. Control embryos, including ones injected with a construct missing the DNA recognition helix of the homeobox, developed normally.

Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.

The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the descendants of the injected blastomere. Cytological observations of the injected eggs show, in the arrested blastomeres, enlarged nuclei always surrounded by an intact nuclear envelope and containing uncondensed chromatin. The possible role of ras protein in meiosis and mitosis is discussed.

Heat-shock-induced grey crescent formation in axolotl eggs and oocytes: the role of gravity

ABSTRACT Axolotl eggs were heat shocked (36·8°C, 10min) inside their jelly layers. Heat shock (HS) was shown to induce the precocious appearance of a grey crescent (GC) in a number of eggs immediately after fertilization (Benford & Namenwirth, 1974). It was also demonstrated that this phenomenon occurs in fertilized or artificially activated eggs only when they are shocked within after spawning. The GC forms still later in heated unfertilized, nonactivated eggs. The role of the jelly layers is considered to be mechanical: a proportion of eggs is maintained in a tilted position until the egg is able to orient animal pole upwards under the influence of gravity as a late consequence of activation. The jelly layers are not essential if the eggs are artificially tilted or rotated during HS. GC formation can also be induced in in vitro maturing oocytes, provided they are tilted during HS. Gravity thus plays an essential role in the cytoplasmic rearrangements leading to HS-induced GC formation. Our results indicate a synergistic action between heat and gravity in this process. The cytological appearance of the GC formed in those experiments is that of a ‘Born’s crescent’ with a conspicuous ‘vitelline wall’ (Pasteels, 1964). When oocytes are enucleated before maturation, HS has no effect on GC formation. A nuclear factor is therefore essential, as has been demonstrated in early GC formation induced by inhibitors of protein synthesis. Finally, incorporation of amino acids into oocyte proteins appears to be rapidly inhibited by HS (from 5 min). However, we cannot conclude that GC formation is in fact triggered by inhibition of protein synthesis. It is also likely that HS disrupts cytoskeletal structure, hence facilitating cytoplasmic rearrangements. Nevertheless, these results are in agreement with the scheme we recently proposed for GC formation in the rotated axolotl oocyte (Gautier & Beet-schen, 1985).

Expression of DNA ligase genes by ram spermatid nuclei and RNA in amphibian eggs.

During animal development and gametogenesis two DNA ligases are found and successively expressed. In this study the two DNA ligases present in the axolotl egg and the two ligases present during ram sperm cell maturation were distinguished by biochemical and immunological methods. The expression of the genes for the heavy and light ram DNA ligases has been studied using transplantation of spermatid and sperm nuclei in axolotl eggs. We found that ram DNA ligases were expressed in axolotl egg cytoplasm. The exclusion phenomenon between the heavy and light form of DNA ligase is species-specific and involves a cytoplasmic mediator. In the transplanted ram germ cell nuclei the heavy ram DNA ligase expression was found to be sensitive to inhibitors of transcription while the light one was not. When mRNA was used, no exclusion process was observed and both the heavy and light enzyme expression were sensitive to cycloheximide and not to aamanitin. These results are discussed in terms of the possible stability of the gene-regulated state following nuclear transfer.

Molecular duality of DNA ligase in axolotl corresponds to distinctive transcriptional information

Based upon the use of specific antibodies and sucrose gradient sedimentation analysis, the present work describes the use of the post-transcriptional equipment of the urodele egg to compare the information contained in two RNA samples extracted from respectively liver and activated axolotl eggs. It is shown that besides the normal DNA ligase activity present in the host Pleurodeles eggs, RNA can translate for the specific carried information revealing a difference between the two samples. Moreover, unlike in nuclear transplantation, the homologous DNA ligases are not mutually exclusive. These observations give a new convincing support of the genetic basis of the molecular duality of DNA ligases.

Specific genes in development

ABSTRACT A heavy form of DNA ligase (8S) replaces the light form (6S) of enzyme when Axolotl egg is fertilized or artificially activated at 18 °C. When eggs either fertilized or activated are maintained at 10 °C the heavy form of enzyme does not appear and the light DNA ligase remains undiminished.

DNA ligase in axolotl egg: A model for study of gene activity control

Replacement of the light form of DNA ligase (6 S) by the heavy form (8 S) in activated egg of Axolotl has been studied as a model for change in genetic activity exerted by the female pronucleus. Nuclear transplantation shows that a blastula nucleus is able to govern the replacement of the light ligase by the heavy one. The result is not the same if the grafted nucleus is taken from an androgenetic embryo, devoid of the heavy enzyme. Therefore the change in the properties of the female pronucleus appears stable and autoreproducible. Gamma irradiations delivered at different times after activation establish that the replacement of the ligase forms depends on an intact nucleus up to 3 hr 30 min after activation, and thereafter is achieved independently of any nuclear damage. Inhibitors of DNA replication impede the change of enzymatic form in reversible process, suggesting new chromatin synthesis as prerequisite for expression of the new genetic activity. The quantitative level of DNA ligase activity does not show any dose effect when one or many nuclei are present in the same cytoplasm. However, a change in nucleotide concentration results in a change in DNA ligase activity, indicating cytoplasmic control of enzymatic regulation.

Isolation of the messenger RNA for 8S DNA ligase in early developing axolotl egg and its cell free translation.

A new DNA ligase activity is expressed when the Axolotl eggs enter cleavage. The messenger RNA can be labelled by [3H] uridine thereby indicating its de novo synthesis. This new genetic expression is occurring just before cleavage and is the earliest found during Amphibian development. The newly synthesized [3H] mRNA can be translated in vitro in the rabbit reticulocyte lysate system. The resulting product is a 160 K protein specifically immunoprecipitated with the antiserum directed against 8S DNA ligase. This in vitro translated polypeptide exhibits 8S DNA ligase activity specific of activated or fertilized eggs but does not display 6S DNA ligase activity of non activated eggs.

Enzymes involved in DNA replication in the axolotl: I. Analysis of the forms and activities of DNA polymerase and DNA ligase during development

DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.

Evidence for a DNA ligase change related to early cleavage in axolotl egg

A definite change in the forms of DNA ligase appears when the axolotl egg enters cleavage. Sucrose gradient and phosphocellulose chromatography show that the a 6S form of DNA ligase exists before division, i.e. in unfertilised and fertilised egg, and a 8.2S form is present at the first division. N-ethylmaleimide sensitivity and heat stability are different for the two forms. The possible significance of this early change is discussed.

Induction of triploid and gynogenetic diploid axolotls (Ambystoma mexicanum) by hydrostatic pressure

AbstractHydrostatic pressure of 10,000 psi, applied 60 minutes from oviposition, for eight minutes, was used to suppress the formation of the second polar body in fertilized axolotl eggs. When this procedure was used on naturally fertilized eggs, triploid axolotls were produced with 97% efficiency. Artificial insemination with ultraviolet irradiation inactivated sperm in combination with this procedure yielded gynogenetic diploids with 92% efficiency. Survival was variable in both sets of experiments and ranged from 64% to 72%.

Tubulin and Microtubules in the Early Development of the Axolotl and Other Amphibia

SYNOPSIS. Unfertilized eggs of the axolotl, Ambystoma mexicanum , contain a pool of soluble tubulin accumulated during oogenesis. After initiation of cleavage the tubulin pool decreases somewhat and then remains constant through early development. Some properties of tubulin alter during development, but at least some of these changes are not due to changes in tubulin perse. However, the tubulin in axolotl oocytes, eggs, and embryos differs in some electrophoretic properties from tubulin in adult axolotl brain and testis. Equivalent differences were observed in Necturus maculosus tubulins. Heterogeneity of axolotl tubulins was confirmed by peptide mapping: Different patterns of peptides were formed by specific limited proteolysis of soluble tubulin from eggs and testis. The heterogeneity was more marked in the a than in the β subunit. Mobilization of soluble tubulin into the mitotic apparatus depends on the functioning of microtubule organizing centers after activation of the egg at fertilization. In eggs of the nc mutant axolotl there is a lesion in some step of activation, one effect of which is that even though the eggs contain an essentially normal pool of tubulin, microtubules fail to assemble, no mitotic apparatus forms, and embryonic development does not begin. These eggs can be partially corrected by injection of heterologous microtubule fragments, which elicit the mobilization of nc tubulin into arrays of microtubules, followed by initiation of cleavage and development to a partial blastula stage. The results of these experiments are discussed in comparison with other reports in the literature about the function of microtubule organizing centers during amphibian egg development.

Microtubule proteins in axolotl eggs and developing embryos

Tubulin from eggs and embryos of the Mexican axolotl was characterized by electrophoresis and colchicine binding. In urea-polyacrylamide gel electrophoresis, soluble axolotl egg tubulin migrated as two bands, identical to tubulins from sea urchin sperm and Drosophila eggs. However, in SDS-containing gels, on which the α and β subunits of standard tubulins were well resolved, axolotl egg tubulin migrated as a single band with an apparent molecular weight of 53,500. The method of disruption of the eggs affected both yield of tubulin from vinblastine sulfate precipitates and stability of the colchicine binding activity. The colchicine binding activity of soluble tubulin from gently disrupted eggs was specific and of high affinity, with properties similar to those reported for other tubulins. The tubulin pool in unfertilized eggs was determined to be approximately 2 μg/egg; the level decreased 20% after initiation of cleavage and then remained constant through development to postneurula stages. The colchicine binding activity of soluble tubulin from embryos was much less stable than that of unfertilized eggs and decreased further during development. No differences were found in properties of tubulin from eggs of several strains of normally pigmented axolotls; however, tubulin from albino eggs showed slightly different properties in both electrophoresis and colchicine binding. The colchicine binding activity of soluble tubulin accounts for only half the total activity in axolotl eggs; they possess, in addition, a particulate nontubulin colchicine binding activity.

The cleavage pattern of the axolotl egg studied by cinematography and cell counting

The temporal pattern of cleavage in the egg of the axolotl,Ambystoma mexicanum, was studied 1. by time-lapse microcinematography, and 2. by counting the total number of blastomeres dissociated at successive stages.