Andrés E. Carrasco

Cloning of an X. laevis gene expressed during early embryogenesis coding for a peptide region homologous to Drosophila homeotic genes.

A Xenopus laevis gene was cloned on the basis of cross-homology to a region conserved between several Drosophila homeotic genes. It was shown to be expressed during early development, starting at late gastrulation and producing three transcripts under specific temporal control. The frog gene contains a region coding for 60 amino acids, which is extremely conserved when compared to Drosophila homeotic genes in the region of the homeo domain. Southern blot analysis indicates that there are at least five such sequences in the Xenopus genome. The conserved region is very rich in arginine. This gene could perhaps represent the first development-controlling gene identified in vertebrates.

Glyphosate-Based Herbicides Produce Teratogenic Effects on Vertebrates by Impairing Retinoic Acid Signaling

The broad spectrum herbicide glyphosate is widely used in agriculture worldwide. There has been ongoing controversy regarding the possible adverse effects of glyphosate on the environment and on human health. Reports of neural defects and craniofacial malformations from regions where glyphosate-based herbicides (GBH) are used led us to undertake an embryological approach to explore the effects of low doses of glyphosate in development. Xenopus laevis embryos were incubated with 1/5000 dilutions of a commercial GBH. The treated embryos were highly abnormal with marked alterations in cephalic and neural crest development and shortening of the anterior-posterior (A-P) axis. Alterations on neural crest markers were later correlated with deformities in the cranial cartilages at tadpole stages. Embryos injected with pure glyphosate showed very similar phenotypes. Moreover, GBH produced similar effects in chicken embryos, showing a gradual loss of rhombomere domains, reduction of the optic vesicles, and microcephaly. This suggests that glyphosate itself was responsible for the phenotypes observed, rather than a surfactant or other component of the commercial formulation. A reporter gene assay revealed that GBH treatment increased endogenous retinoic acid (RA) activity in Xenopus embryos and cotreatment with a RA antagonist rescued the teratogenic effects of the GBH. Therefore, we conclude that the phenotypes produced by GBH are mainly a consequence of the increase of endogenous retinoid activity. This is consistent with the decrease of Sonic hedgehog (Shh) signaling from the embryonic dorsal midline, with the inhibition of otx2 expression and with the disruption of cephalic neural crest development. The direct effect of glyphosate on early mechanisms of morphogenesis in vertebrate embryos opens concerns about the clinical findings from human offspring in populations exposed to GBH in agricultural fields.

A homeo-box-containing gene expressed during oogenesis in xenopus

We report the isolation of a second Xenopus gene containing a homeo domain homologous to those of Drosophila. Out of 60 amino acids, 59 are conserved with Antennapedia, and 54 with a previously isolated frog gene. The predicted carboxy-terminus of this homeotic-like protein is 21 amino acids downstream from the conserved domain, ending with six glutamic acids in a row. The main interest of this gene lies in the fact that it is abundantly transcribed in large Xenopus oocytes, where it might be translated into proteins potentially involved in the early determination of embryonic cell types.

Notch activates sonic hedgehog and both are involved in the specification of dorsal midline cell-fates in Xenopus

We analysed the role of Notch signalling during the specification of the dorsal midline in Xenopus embryos. By activating or blocking the pathway we found that Notch expands the floor plate domain of sonic hedgehog and pintallavis and represses the notochordal markers chordin and brachyury, with a concomitant reduction of the notochord size. We propose that within a population of the early organiser with equivalent potential to develop either as notochord or floor plate, Notch activation favours floor plate development at the expense of the notochord, preferentially before mid gastrula. We present evidence that sonic hedgehog down-regulates chordin, suggesting that secreted Sonic hedgehog may be involved or reinforcing the cell-fate switch executed by Notch. We also show that Notch signalling requires Presenilin to modulate this switch.

Retinoic acid induces changes in the localization of homeobox proteins in the antero-posterior axis of Xenopus laevis embryos

We have studied the localization of the proteins of Xeb1 and Xeb2, two homeobox (hbx)-containing genes that are expressed during the early development of Xenopus laevis. Both proteins are expressed in juxtaposed and partially overlapping domains along the antero-posterior axis of Xenopus laevis embryos, with clearly defined anterior boundaries. Xeb2 is predominantly expressed in the caudal region of the hindbrain, whereas the Xeb1 protein is located in the most rostral region of the spinal cord. Furthermore, both proteins are expressed in single cells dispersed in the lateral flanks of the embryo in positions that correlate with the expression domains in the neural tube. We suggest that these cells are migratory neural crest cells that have acquired positional information in the neural tube prior to migration. The Xeb2 protein was also detected in the most posterior branchial arches and the pronephros. In stage 45 embryos, nuclei of the IX-X cranial ganglia, the lung buds and cells spreading into the forelimb rudiment express the Xeb2 antigen. The Xeb1 protein was also detected in the lung buds and the forelimb rudiment. To examine the effect of retinoic acid on expression, gastrula embryos were treated with all-trans retinoic acid (RA). Increasing concentrations of RA caused progressive truncation of anterior structures. The most severely affected embryos lacked eyes, nasal pits, forebrain, midbrain and otic vesicles, and the anterior boundary of the hindbrain seemed to be displaced rostrally. This alteration correlates with a progressive displacement of the anterior boundary of the expression domain of Xeb2. On the other hand, 10(-6) M RA induces an ectopic site of Xeb1 expression at the anterior end of the central nervous system, located just anterior to the extended domain of Xeb2 whereas expression in the spinal cord remains unaffected.

Localization of Xenopus homoeo-box gene transcripts during embryogenesis and in the adult nervous system

Tissue distribution and localization of RNAs from the Xeb1 homoeo-box-containing gene were monitored with Northern blots and in situ hybridization. Xeb1 transcript distribution in larval stage embryos was established by blotting RNAs extracted from microdissected embryos. Those transcripts are restricted to a limited number of embryonic regions such as the dorsal trunk. The tissue/cell localization of Xeb1 transcripts was then monitored at several embryonic stages with in situ hybridization methods using [35S]RNA probes. These homoeo-box transcripts accumulated in a progressive and dynamic fashion. First localized in late gastrulae, they are distributed along the neural tube and in caudal mesoderm at later stages. By the swimming tadpole stage the spatial distribution of the homoeo-box transcripts is limited to specific regions of the central nervous system. Adult spinal cord shows the signal in specific neurons in the ventrolateral field of the gray matter.

Chapter Two – Pesticides Used in South American GMO-Based Agriculture: A Review of Their Effects on Humans and Animal Models

In South America, the incorporation of genetically modified organisms (GMO) engineered to be resistant to pesticides changed the agricultural model into one dependent on the massive use of agrochemicals. Different pesticides are used in response to the demands of the global consuming market to control weeds, herbivorous arthropods, and crop diseases. Here, we review their effects on humans and animal models, in terms of genotoxicity, teratogenicity, and cell damage. We also stress the importance of biomarkers for medical surveillance of populations at risk and propose the use of biosensors as sensitive resources to detect undesirable effects of new molecules and environmental pollutants. The compatibility of glyphosate, the most intensively used herbicide associated to GMO crops, with an integrated pest management for soybean crops, is also discussed.

The Notch-target gene hairy2a impedes the involution of notochordal cells by promoting floor plate fates in Xenopus embryos

We have previously shown that the early Xenopus organiser contains cells equally potent to give rise to notochord or floor plate, and that Notch signalling triggers a binary decision, favouring the floor plate fate at the expense of the notochord. Now, we present evidence that Delta1 is the ligand that triggers the binary switch, which is executed through the Notch-mediated activation of hairy2a in the surrounding cells within the organiser, impeding their involution through the blastopore and promoting their incorporation into the hairy2a+ notoplate precursors (future floor-plate cells) in the dorsal non-involuting marginal zone.

The Alzheimer-related gene presenilin-1 facilitates sonic hedgehog expression in Xenopus primary neurogenesis

We analyzed the influence of presenilins on the genetic cascades that control neuronal differentiation in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduced the number of N-tubulin+ primary neurons and modulated Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increased shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduced shh expression. Both shh and presenilin promoted cell proliferation and apoptosis, but the effects of shh were widely distributed, while those resulting from presenilin injection coincided with the range of shh signaling. We suggest that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling.

Whole‐Mount In Situ Hybridization and Detection of RNAs in Vertebrate Embryos and Isolated Organs

Nonisotopic in situ hybridization using intact embryos or organs is an important method for determining the spatial distribution of RNAs. Because it allows the analysis of large numbers of samples, it is amenable to temporal expression studies and comparison between different genotypes. It offers sensitivity and reproducibility. In addition, histological details are not lost during the staining process. The protocols in this unit can be used for whole-mount in situ hybridization in Xenopus, mouse, and chicken embryos, as well as dissected organs from mouse and chicken. Preparation of digoxigenin-labeled riboprobes is also described.