Jacques Picard

Early neurogenesis and teratogenesis in whole mouse embryo cultures. Histochemical, immunocytological and ultrastructural study of the premigratory neuronal-glial units in normal mouse embryo and in mouse embryos influenced by cocaine and retinoic acid.

Abstract. Yolk sacs of postimplantation mouse embryos were cultured in a mixture of human and rat sera. The central nervous system of these cultured normal embryos was studied from the stage of 5–9 somites (approximately 8.5 postcoital days) to 20–21 somites (approximately 9.5 postcoital days) and compared with in vivo embryos at the same stages. This developmental period covers most of the neural tube closure, the early premigratory differentiation of the neuroectodermal epithelium, and the glial commitment of a population of germinative cells. The neuronal and glial elements of the in vitro cultivated embryos were found to be identical to the corresponding neural tissue in in vivo embryos (light and electron microscopic comparisons); the morphological identity between the in vivo and in vitro embryos was confirmed by morphometry and by stainings revealing the differentiation of the glial elements and precursors. The study of the neuronal-glial units in this material revealed that the fascicular organization of the radial glial cells occurs before the stage of 20 somites. When submitted to a single low dose of retinoic acid at the 7-somite stage, the expression of the epitope recognized by radial cell 2(RC2), a glial marker, is delayed in the in vitro embryos 12–16 hours, but the glycogen and the other glial parameters mature in time. The in vitro embryos exposed to cocaine at the 7-somite stage displayed a prosencephalon remaining deprived of almost all glial cytological features during the entire culture period, although the other developmental parameters evolved normally. This in vitro whole embryo model seems to be a powerful tool for studying early neurogenesis and teratogenesis.

Loss of Function but No Gain of Function Caused by Amino Acid Substitutions in the Hexapeptide of Hoxa1 In Vivo

ABSTRACT Homeodomain containing transcription factors of the Hox family play critical roles in patterning the anteroposterior embryonic body axis, as well as in controlling several steps of organogenesis. Several Hox proteins have been shown to cooperate with members of the Pbx family for the recognition and activation of identified target enhancers. Hox proteins contact Pbx via a conserved hexapeptide motif. Previous biochemical studies provided evidence that critical amino acid substitutions in the hexapeptide sequence of Hoxa1 abolish its interaction with Pbx. As a result, these substitutions also abolish Hoxa1 activity on known target enhancers in cellular models, suggesting that Hoxa1 activity relies on its capacity to interact with Pbx. Here, we show that mice with mutations in the Hoxa1 hexapeptide display hindbrain, cranial nerve, and skeletal defects highly reminiscent of those reported for the Hoxa1 loss of function. Since similar hexapeptide mutations in the mouse Hoxb8 and the Drosophila AbdA proteins result in activity modulation and gain of function, our data demonstrate that the functional importance of the hexapeptide in vivo differs according to the Hox proteins.

Cell- and stage-specific expression of Mage genes during mouse spermatogenesis

The human MAGE-A1 and MAGE-A3 genes direct the expression of antigens recognized on melanoma cell lines by autologous cytolytic T lymphocytes (van der Bruggen et al. 1994a, 1994b). They belong to a family of related genes located in regions q28 (MAGEA genes; De Plaen et al. 1994), p21.3 (MAGE-B genes; Muscatelli et al. 1995), and q26 (MAGE-C1 gene; Lucas et al. 1998) of the X Chromosome (Chr). Several MAGE genes are expressed in tumors of various histological types, but none of them is expressed in healthy tissues except male germ cells and placenta (De Plaen et al. 1994; Dabovic et al. 1995; Takahashi et al. 1995; Lurquin et al. 1997; Lucas et al. 1998). By hybridizing mouse genomic libraries with a MAGE-A1 probe, we identified three nearly identical murine genes and named them Smage1to 3 (De Backer et al. 1995). Smage3is autosomal, whereas Smage1and2 are located on the X Chr, in a region syntenic to the human Xp21-22 region that contains the MAGE-B genes. These three murine genes were, therefore, renamedMage-bgenes. Further screening revealed the existence of 8 additional murine genes that were called Mage-a,although their equivalence with any group of human MAGE genes is not formally established yet (De Plaen et al. 1999). Expression of Mage-aand-b genes has been detected by RT-PCR in embryonic stem cells, in embryos, in tumor cell lines, and in testis where Mage-a3, -a4, -a5and the threeMage-bgenes are expressed (De Backer et al. 1995 and unpublished data; De Plaen et al. 1999). Using oligonucleotide in situ hybridization on testis sections, the Mage-b genes have been shown to be expressed in the round spermatids (Chomez et al. 1995). The function of MAGE proteins is unknown. As testis is the only healthy adult organ where MAGE and Magegenes are expressed, it has been suggested that these genes may have some role in spermatogenesis (Chomez et al. 1995). Spermatogenesis, the production of spermatozoa from spermatogonia, can be divided into three major steps: (1) the multiplication of spermatogonia by the process of mitosis; (2) meiosis, which reduces the chromosome number from diploid to haploid and is initiated by the entry of spermatogonia into the prophase of the first meiotic division. These cells, called primary spermatocytes, divide to form secondary spermatocytes, which divide again to form round spermatids; (3) the maturation of round spermatids to elongated spermatids and their subsequent transformation into the complex structure of the spermatozoon, a process called spermiogenesis. In mammals, spermatogenesis proceeds as a wave along the seminiferous tubule. In mice and rats, a transverse section in the tubule shows specific associations of cells characterizing successive tubular stages (Roosen-Runge and Giesel 1950). Thus, mouse testis provided us the opportunity to assess whether Mage genes display possible stage-specific expression during spermatogenesis. As a first step to identify possible Magegene function(s) in the testis, we compared the mRNA expression ofMage-a and Mage-b genes on healthy adult mouse testis sections. The cell types as well as the tubular stages where Mage-aand Mage-bgenes are expressed were assessed by radioactive in situ hybridization (ISH). Expression of both families of genes was detected inside the seminiferous tubules, no signal above background being detected in interstitial tissue containing Leydig cells.

Alterations of mouse embryonic branchial nerves and ganglia induced by ethanol

An immunostaining technique using monoclonal antibodies to a neurofilament protein has allowed us to visualize defects in the development of cranial nerves and ganglia of 10 to 10.5 days mouse embryos following exposure to ethanol in whole embryo culture. Reference patterns for development of cranial nerves and ganglia of control mouse embryos explanted and examined when they had 25 to 34 pairs of somites were established. Additionally, control mouse embryos were grown in whole embryo culture for 48 h, with culture being initiated in embryos having 6 to 7 somite pairs. At the end of the culture period, only minor differences were observed between the control groups. An experimental group of embryos was cultured in the presence of increasing doses (1.6, 3.2, 4, and 4.8 g/l) of ethanol. Defects were observed in the development of the glossopharyngeal and vagus nerves. These abnormalities included absence of the dorsal root (superior ganglion) of IX, star-like shape of inferior ganglion IX, disorganization of the rootlets of nerve X and abnormal fibers between the two nerves and ganglia. These results suggest that the migration and patterning of neural crest cells derived from r6 and r7 may be particularly affected by ethanol. The results also demonstrate the usefulness of this approach in evaluating the susceptibility of the developing cranial nerves to toxicant exposure.

Cranial nerves and ganglia are altered after in vitro treatment of mouse embryos with valproic acid (VPA) and 4-en-VPA

Prenatal valproic acid (VPA) exposure results in neural tube defects and in the fetal valproate syndrome (FVS), associated with developmental delay. In the present study we investigate the alterations induced by VPA and one of its metabolite, 4-en-VPA, on specific neural structures: branchial nerves and ganglia. This study was performed on 8-9 pairs of somites mouse embryos exposed in vitro for 24 h to 0.75 mM of VPA or 1 mM of 4-en-VPA. After an additional culture period of 20 h without drug, the embryos were processed for whole mount immunostaining using the monoclonal antibody 2H3, directed against the 155 kDa neurofilament protein. This technique makes it possible to visualise the branchial nerves/ganglia. VPA and 4-en-VPA induced a delay in the development of the trigeminal (V), glossopharyngeal (IX) and vagus (X) nerves/ganglia. The development of the facial (VII) nerve was delayed to a lesser extend. These treatments also induced defects in the four ganglia. The main abnormalities were a reduced dorsal component of ganglion V, the absence of the dorsal root of ganglion IX, a disorganised dorsal part of ganglion X and diffuse ventral fibres in nerves VII-VIII. In addition, scattered fibres were observed around and between ganglia. In conclusion, VPA and 4-en-VPA deeply altered the differentiation of branchial nerves/ganglia. The dorsal part of the ganglia, arising from the rhombencephalic neural crest, was particularly sensitive. The disorganisation of fibres could possibly be explained by alteration of the extracellular matrix.

In vitro neuroteratogenicity of valproic acid and 4-en-VPA.

Mouse embryos displaying 8 to 9 pairs of somites were cultured during 26 h in presence of 0.75 mM of VPA, or of 1 mM of 4-en-VPA. These concentrations induced approximately 50% of dysmorphogenic embryos. Irregular suture of caudal neural tube, abnormal head shape, cranial neural tube defects, and deformed optic vesicles were the most common defects observed with both compounds. The main differences in the types of dysmorphogeneses detected between the two compounds concerned the suture of the caudal neural tube and the telencephalic region. Other macroscopic effects induced by the two compounds were similar. Several of the observed abnormalities can be correlated with defects reported after in vivo exposure. The major alteration of the histological structure of the neural tube concerned a specific area in the hindbrain : VPA and 4-en-VPA induced an abnormal and irregular budding of the neuroepithelium at this level. Immunohistology with an antibody specific for radial glial fibers (RC-2) as well as SEM analysis showed a moderate effect on glial development, mainly after exposure to VPA.

Retinoic acid induces a tissue-specific deletion in the expression domain of Otx2.

The expression domain of Otx2, a gene essential for the development of the fore- and midbrain, has previously been shown to be affected by exposure to all-trans-retinoic acid (AT-RA). However, morphological abnormalities of the fore- and midbrain induced by exposure of early somite-stage embryos to AT-RA were not associated with abnormal Otx2 expression. To identify abnormal expression of developmental genes induced by exposure at early somite-stages, we performed a fine analysis of the expression domains of Otx2, Otx1, Emx2, and Pax-6 by combining in situ hybridization (ISH) with computer-assisted superpositions and three-dimensional reconstructions of these expression domains. No alteration in the relative location of the caudal boundaries of the expression domains of these genes was observed. The only abnormality was a deletion of the most cranial portion of the neural folds (NF).

Whole embryo culture of presomitic mouse embryos

Rat embryos explanted at the presomite stage and cultured through limb bud stages develop to well formed embryos and exhibit growth and differentiation which mimic those observed in vivo at corresponding stages. In contrast, the culture of presomite stage mouse embryos has proven to be much less successful. In the present study presomitic and 3-4 somite stage mouse embryos were cultured for 48 hr. Four stages of presomitic mouse embryos corresponding to late primitive streak, early neural plate, mid neural plate and late neural plate were cultured for 24 hr in a mixture of mouse serum (12.5%), rat serum (25%) and human serum (62.5%) and during a further period of 24 hr in a mixture of human (80%) and rat (20%) serum. Two explantation procedures were used. Embryos of 3-4 somites, cultured as a reference, developed in quite a similar way to embryos in vivo and in this group very few dysmorphogeneses were observed. In contrast, the development of earlier embryos was not reproducible and the two explantation procedures led to similar results.

Embryotoxicity of human sera from patients treated with isotretinoin

Sera of 20 patients treated with 20-40 mg isotretinoin/day were tested for embryotoxicity potential. For each patient, the first sample was taken before treatment (control sample) and the second was taken 2 months after the start of treatment (treated sample). Six embryos displaying six or seven pairs of somites were cultured for 26 hr in each serum sample, when sufficient serum was available. No deaths were observed in the control sample, whereas dead embryos (6%) were observed in the treated sample. The rates of malformed embryos were 13 and 81% in the control and in the treated sample, respectively. The most frequent abnormalities affected the cephalic neural tube, the branchial bars, the yolk sac circulation and the caudal neural tube. Growth and differentiation were significantly decreased in the treated sample. The concentrations of isotretinoin and of two metabolites (trans-retinoic acid and 4-oxo-isotretinoin) were measured in 12 sera. A correlation between embryotoxicity and concentration was established for two of the chemicals. Modulation of the embryotoxicity by drug-induced changes in the serum cannot be excluded.

All-trans-retinoic acid upregulates the expression of COUP-TFI in early-somite mouse embryos cultured in vitro

Exposure of embryos to an excess of retinoic acid (RA) modifies the spatio-temporal pattern of expression of developmental genes. RA regulates the expression of target genes through binding of the retinoid nuclear receptors (RARs and RXRs), as heterodimers, to regulatory cis-acting elements. COUP-TF factors, which are able to dimerize with the RXRs and to compete with the retinoid receptors for their DNA binding sites, are suspected to modulate the retinoid signal transduction pathway. Therefore, COUP-TF factors may be involved in the regulation of the expression of developmental genes and/or in the modifications induced by an excess of RA in the expression of these genes. The aim of this work is to assess whether RA-induced modifications in the expression of Krox-20 and Hox genes correlate with alterations of the expression of COUP-TF genes. In addition to spatial modifications in the expression patterns of Krox-20 and Hox genes, we report here an upregulation of the expression level of COUP-TFI after RA exposure. However, this abnormality did not spatially overlap with the modifications observed in the expression of Krox-20 and Hox genes. These data suggest an involvement of COUP-TFI in the generation of RA-induced abnormalities, but do not support the hypothesis of an involvement of this factor in the regulation of the expression of Hox or Krox-20 genes.