Evolutionary Genetics of the Cavefish Astyanax mexicanus.

Blind and depigmented fish belonging to the species Astyanax mexicanus are outstanding models for evolutionary genetics. During their evolution in the darkness of caves, they have undergone a number of changes at the morphological, physiological, and behavioral levels, but they can still breed with their river-dwelling conspecifics. The fertile hybrids between these two morphotypes allow forward genetic approaches, from the search of quantitative trait loci to the identification of the mutations underlying the evolution of troglomorphism. We review here the past 30years of evolutionary genetics on Astyanax: from the first crosses and the discovery of convergent evolution of different Astyanax cavefish populations to the most recent evolutionary transcriptomics and genomics studies that have provided researchers with potential candidate genes to be tested using functional genetic approaches. Although significant progress has been made and some genes have been identified, cavefish have not yet fully revealed the secret of their adaptation to the absence of light. In particular, the genetic determinism of their loss of eyes seems complex and still puzzles researchers. We also discuss future research directions, including searches for the origin of cave alleles and searches for selection genome-wide, as well as the necessary but missing information on the timing of cave colonization by surface fish.

Islet1 as a marker of subdivisions and cell types in the developing forebrain of Xenopus.

We provide a detailed and exhaustive description of forebrain regions, nuclei, and neuronal populations which express the LIM-homeodomain transcription factor Islet1 during Xenopus development. To this end, Isl1 immunofluorescence staining was combined with other regional or neuronal markers such as Nkx2.1, Pax6, GABA, nitric oxide synthase, tyrosine hydroxylase or calretinin, and with tract tracing experiments to differentiate projection neurons from interneurons. We interpret and discuss the results with regard to the developmental origin of some previously "ambiguous" nuclei of the septum and the amygdala. Thus, Isl1 appears like a prominently expressed determinant of striatal and striatal-derived regions of the subpallium, including the central amygdala, together with the dorsal septal nucleus and the lateral septum. In the diencephalon, Isl1 is a conspicuous marker of the prethalamus, the chiasmatic regions, and the preoptic region (including important dopaminergic populations). The tuberal and mammillary parts of the hypothalamus also strongly express Isl1. From a comparative point of view, a major difference with mammals is the scarce expression of Isl1 in the embryonic medial ganglionic eminence, which is notably devoid of Isl1 expression in mammals, and the important retrochiasmatic and mammillary Isl1 expression, both also devoid of Isl1 expression in mammals. Finally, we provide evidence for the existence in Xenopus of a "new" caudal medial telencephalic nucleus, the POC (for preoptic commissural area), which was recently described in mammals.

Phylogenomic analysis and expression patterns of large Maf genes in Xenopus tropicalis provide new insights into the functional evolution of the gene family in osteichthyans.

We have performed an exhaustive characterization of the large Maf family of basic leucine zipper transcription factors in vertebrates using the genome data available, and studied the embryonic expression patterns of the four paralogous genes thus identified in Xenopus tropicalis. Our phylogenetic analysis shows that, in osteichthyans, the large Maf family contains four orthology classes, MafA, MafB, c-Maf and Nrl, which have emerged in vertebrates prior to the split between actinopterygians and sarcopterygians. It leads to the unambiguous assignment of the Xenopus laevis XLmaf gene, previously considered a MafA orthologue, to the Nrl class, the identification of the amphibian MafA and c-Maf orthologues and the identification of the zebrafish Nrl gene. The four X. tropicalis paralogues display partially redundant but nevertheless distinct expression patterns in the somites, developing hindbrain, pronephros, ventral blood island and lens. Comparisons with the data available in the mouse, chick and zebrafish show that these large Maf expression territories are highly conserved among osteichthyans but also highlight a number of differences in the timing of large Maf gene expression, the precise extent of some labelled territories and the combinations of paralogues transcribed in some organs. In particular, the availability of robust phylogenies leads to a reinterpretation of previous expression pattern comparisons, suggesting an important part for function shuffling within the gene family in the developing lens. These data highlight the importance of exhaustive characterizations of gene families for comparative analyses of the genetic mechanisms, which control developmental processes in vertebrates.

The LIM-homeodomain gene family in the developing Xenopus brain: conservation and divergences with the mouse related to the evolution of the forebrain.

A comparative analysis of LIM-homeodomain (LIM-hd) expression patterns in the developing stage 32 Xenopus brain is presented. x-Lhx2, x-Lhx7, and x-Lhx9 were isolated and their expression, together with that of x-Lhx1 and x-Lhx5, was analyzed in terms of prosomeric brain development and LIM-hd combinatorial code and compared with mouse expression data. The results show an almost complete conservation of expression patterns in the diencephalon. The Lhx1/5 and Lhx2/9 subgroups label the pretectum/ventral thalamus/zona limitans versus the dorsal thalamus, respectively, in alternating stripes of expression in both species. Conversely, strong divergences in expression patterns are observed between the telencephalon of the two species for Lhx1/5 and Lhx2/9. Lhx7 exhibits particularly conservative patterns and is proposed as a medial ganglionic eminence marker. The conservation of diencephalic segments is proposed to mirror the conservative nature of diencephalic structures across vertebrates. In contrast, the telencephalic divergences are proposed to reflect the emergence of significant novelty in the telencephalon (connectivity changes) at the anamniote/amniote transition. Moreover, the data allow the new delineation of pallial and subpallial domains in the developing frog telencephalon, which are compared with mouse subdivisions. In the pallium, the mouse combinatorial expression of LIM-hd is notably richer than in the frog, again possibly reflecting evolutionary changes in cortical connectivity.

Lhx9 and Lhx9alpha LIM-homeodomain factors: genomic structure, expression patterns, chromosomal localization, and phylogenetic analysis.

Lhx9 is a LIM-homeodomain (LIM-hd) transcription factor expressed in the embryonic mouse brain. We report the isolation of Lhx9alpha, a cDNA encoding a truncated isoform of Lhx9 that lacks the recognition helix of the homeodomain and differs from Lhx9 cDNA in its 3'-coding and 3'-UTR sequences. Isolation of the Lhx9 gene showed that Lhx9 and Lhx9alpha are coded by six exons spanning 10 kb of genomic sequence and that Lhx9alpha is an isoform generated by alternative splicing of the fifth exon. Lhx9 was mapped to the subtelomeric region of chromosome 1. Further molecular analysis showed that Lhx9 is a new candidate gene for the unidentified dreher (dr) mutation in mouse. The comparison of genomic structure and molecular phylogenetic analysis led to the identification of six groups of LIM-hd proteins, a basis for further classification and knowledge of their evolutionary relationships. To investigate a possible role for Lhx9alpha, the expression patterns of Lhx9 and Lhx9alpha were compared during embryogenesis. Lhx9alpha was expressed at lower levels than Lhx9, with a similar but distinct pattern in the brain, especially in the neocortex. We suggest that Lhx9alpha could function as an endogenous dominant-negative form of Lhx9 during development, both to regulate in space and time the transcriptional effects of Lhx9 and to add a degree of refinement to the LIM-hd code.

Lhx9: a novel LIM-homeodomain gene expressed in the developing forebrain.

A novel LIM-homeodomain gene, Lhx9, was isolated by degenerate RT-PCR followed by mouse embryonic library screening. Lhx9 cDNA encodes a protein that is most closely related to Drosophila apterous and rodent Lhx2 proteins. The Lhx9 spatiotemporal pattern of expression during embryogenesis was similar but distinct from Lhx2. Highest expression levels were found in the diencephalon, telencephalic vesicles, and dorsal mesencephalon. Domains of expression respected the proposed neuromeric boundaries (). Lhx9 was also expressed in the spinal cord, forelimb and hindlimb mesenchyme, and urogenital system. Although Lhx9 expression was sustained in diencephalon and mesencephalon from embryonic day 10.5 (E10.5) to postnatal stages, it was transient in the future cerebral cortex, where it was turned off between E14.5 and E16.5. Lhx9 expression was highest if not exclusively located (depending on the region of interest) in the intermediate and mantle zones, as opposed to the mitotic ventricular zone. Lhx9 protein was tested for interaction with the recently discovered cofactors of LIM-homeodomain proteins and was found to interact strongly both with CLIM1 and CLIM2. The expression pattern and structural characteristics of Lhx9 suggest that it encodes a transcription factor that might be involved in the control of cell differentiation of several neural cell types. Furthermore, Lhx9 protein could act in a combinatorial manner with other LIM-homeodomain factors expressed in overlapping pattern.

Engrailed and retinotectal topography.

We examine the role of the Engrailed homeobox gene in establishment of local tectal topography. In the mesencephalon, a gradient of Engrailed appears early and defines the rostrocaudal axis of the tectum. Various experiments that cause ectopic Engrailed expression cause predictable readjustments of the retinotectal map. The newly discovered 'realisators' of the retinotopic map, such as receptor tyrosine kinase ligands ELF-1 and RAGS could be controlled directly by Engrailed. Indeed, recent results show that Engrailed regulates the expression of these ligands. The Engrailed gradient itself appears to be set up by signals including FGF8 and WNTI, allowing us to begin to trace the molecular cascade that is responsible for the correct wiring of the visual projection back into the early embryo.

Engrailed, retinotectal targeting, and axonal patterning in the midbrain during Xenopus development: an antisense study.

Axonal tracts in the vertebrate brain seem to respect domains of homeobox gene expression. To test the role of engrailed in tract formation in the midbrain, we inhibited its expression using antisense (AS) oligonucleotides. Phosphorothioate-modified AS oligos caused navigational errors in both the optic projection (OP) and the intertectal commissure (ITC). These oligos, however, also inhibited bFGF binding to the brain. To determine whether these tract phenotypes were due to inhibition of bFGF function or engrailed expression, we used partially phosphorothioate-modified (pp) oligos, which inhibit engrailed expression but do not affect bFGF binding. These ppAS oligos caused the ITC phenotype but had no effect on the OP. Thus, interference with bFGF function correlates with the OP phenotype, while the ITC phenotype is directly related to engrailed expression.

Role of dopamine in the plasticity of glutamic acid decarboxylase messenger RNA in the rat frontal cortex and the nucleus accumbens.

The modulatory role of dopamine (DA) on the expression of mRNA encoding the large isoform of glutamic acid decarboxylase (GAD67), the biosynthesis enzyme of gamma aminobutyric acid (GABA), was examined in GABA neurons of two structures innervated by DA neurons originating from the ventral tegmental area (VTA): the medial frontal cortex (MFC) and the nucleus accumbens (NAcc). A bilateral electrolytic lesion of VTA was performed in rats to produce a DA denervation of both the MFC and NAcc. The efficacy of VTA lesions was verified by measurement of locomotor activity and by immunohistochemical detection of tyrosine hydroxylase in the mesencephalon. GAD67 mRNA was detected by in situ hybridization histochemistry using a 35S-labelled cDNA probe. Densitometric analysis of GAD67 mRNA hybridization signals revealed in VTA-lesioned rats a significant decrease (-24%) in GAD67 mRNA levels in the prelimbic area of the MFC and no significant effect in the anterior cingulate area or the frontoparietal cortex. Single cell analyses by computer-assisted grain counting showed that the decrease in GAD67 mRNA levels in prelimbic MFC was due to a change in GAD67 mRNA expression in a subpopulation of GABA interneurons located in the deep cortical layers (V-VI). By contrast, in the NAcc of VTA-lesioned rats, GAD67 mRNA levels were significantly increased in the anterior part and in the core but were unchanged in the shell part. These results suggest that in two target structures of VTA DA neurons, GAD67 mRNA expression is, in normal conditions, under a tonic stimulatory and a tonic inhibitory DA control in the MFC and the NAcc respectively. A schematic diagram is proposed for functional interactions between these structures.

Stimulation by nicotine of the spontaneous release of [3H]gamma-aminobutyric acid in the substantia nigra and in the globus pallidus of the rat.

The effect of (-)-nicotine on the spontaneous release of [3H]gamma-aminobutyric acid ([3H]GABA) was studied in vitro in rat substantia nigra (SN) and globus pallidus (GP) slices. In both structures, nicotine (10(-4) M) elicited a transient increase of [3H]GABA release lasting no more than 2.5 min. At the peak of the effect, a 18.5% and 25% increase of [3H]GABA was observed in GP and SN slices, respectively. At lower concentration (10(-5) M), nicotine produced a small but significant transient increase (+8%) in GP slices whereas this concentration was ineffective in SN slices. Pempidine (10(-5) M) totally antagonized the 10(-4) M nicotine-induced effect in SN and GP. The increase of [3H]GABA release elicited by 10(-4) M nicotine was abolished when Ca2+ concentration in the superfusion medium was lowered from 2.4 to 0.4 mM. To investigate a possible dopaminergic (DA) link in the response, we examined the sensitivity of the nicotine-induced effect to DA D1 (SCH23390) and D2 (sulpiride) receptor antagonists. In SN, SCH23390 (10(-6) M) abolished the 10(-4) M nicotine-induced effect. In GP, sulpiride (10(-5) M) failed to modify the response. Moreover, SCH23390 partially reversed the nicotine-induced effect (-37%) in GP. Taken together these results indicate that nicotine differentially modulate the [3H]GABA release in SN and GP. In SN, the nicotine-induced [3H]GABA release appears to be mediated by DA neurons. In GP, only a part of the nicotinic response involved a DA link. A possible direct stimulation of nicotinic receptors localized on striato-pallidal terminals is discussed.

GABA interneurons in the rat medial frontal cortex: characterization by quantitative in situ hybridization of the glutamic acid decarboxylase (GAD67) mRNA.

In situ hybridization of mRNA encoding one isoform of glutamic acid decarboxylase (GAD67) was performed in the rat medial frontal cortex (MFC) to characterize GABA interneurons. Qualitatively, the labelling obtained with a [35S]cDNA probe was in register with neurons and was never associated with glial cells. No obvious differences in the density of labelled cells were observed between the different areas of the MFC examined (infralimbic, prelimbic, anterior cingulate and precentral medial) and between the various cortical layers. Grain counting was performed on single cells in the various layers of the prelimbic and the anterior cingulate area, two main areas of the MFC. According to their grain density, neurons were arbitrarily classified as low, high and very high GAD67 mRNA content. The neurons with the high GAD67 mRNA content corresponded to around 50% of the labelled cells in all the layers and in both areas. In the prelimbic area, the neuronal population with a low GAD67 mRNA content varied from 50% in layers I and II-III to 40% in layers V-VI whereas the very high GAD67 mRNA content neurons corresponded to around 5% of the labelled neurons in all layers. In the anterior cingulate area the neuronal population showing low GAD67 mRNA content varied from 35% in layers I and II-III to 20% in layers V-VI. In this area, neurons with a very high GAD67 mRNA content were more numerous than in the prelimbic area: they varied from 15% in layers I and II-III to 30% in layers V-VI. Parallel to the presence of very highly labelled cells, GAD enzymatic activity measured both in the presence and in the absence of pyridoxal 5'-phosphate was higher in the anterior cingulate area than in the prelimbic area. The heterogeneity of GAD67 mRNA content at the cellular level might underlie the existence of subpopulations of GABA interneurons in the MFC and suggests a higher GABAergic inhibitory control in the anterior cingulate area than in the prelimbic area.

Expression of GAD mRNA in GABA interneurons of the rat medial frontal cortex.

The distribution of glutamic acid decarboxylase (GAD) mRNA containing cells was studied in the rat medial frontal cortex (MFC). The neurons labelled by the 35S-labelled cDNA probe were distributed uniformly throughout all the layers and represented 16% of the total neuronal population. It was possible to distinguish two cell populations expressing high and low levels of GAD mRNA corresponding to 63% and 27% of labelled cells, respectively. Concerning the laminar distribution of these two populations of GAD mRNA containing neurons, no marked difference was observed between the various areas of the MFC.

Synergism between D1 and D2 dopamine receptors in the inhibition of the evoked release of [3H]GABA in the rat prefrontal cortex.

In order to examine a possible interaction between D1 and D2 receptors in the dopaminergic control of the electrically-evoked release of [3H]GABA in the rat prefrontal cortex, the effects of D1 and D2 dopamine agonists were studied in vitro on cortical slices. The D1 agonist SKF38393 (10(-5) M) inhibited the electrically-evoked release of [3H]GABA. This effect was totally reversed by both the D1 antagonist SCH23390 (10(-7) M) and the D2 antagonist sulpiride (10(-5) M). We previously observed that maximal D2-mediated inhibition of the electrically-evoked release of [3H]GABA was obtained with 10(-7) M RU24926 and 10(-8) M LY171555. Here we showed that the inhibition produced by these two D2 agonists is also abolished by 10(-7) M SCH23390. In dopamine-depleted slices from reserpine-treated animals, it was not possible to detect an effect of either RU24926 (10(-7) M) or SKF38393 (10(-5) M), suggesting a permissive role of endogenous dopamine in the effect of either D2 or D1 agonist. Finally, SKF38393 used at a subliminar concentration (10(-6) M) was able to potentiate the effect of a liminar concentration of RU24926 (1.5 x 10(-8) M). Taken together these results strongly suggest that in the rat prefrontal cortex a D1-D2 receptor synergism is involved in the dopaminergic control of the electrically-evoked release of [3H]GABA.

Opposing effects of dopamine D2 receptor stimulation on the spontaneous and the electrically evoked release of [3H]GABA on rat prefrontal cortex slices.

The spontaneous and the electrically evoked release of [3H]GABA were studied in vitro on slices of rat medial prefrontal cortex. The slices were preincubated with [3H]GABA and then superfused with a Krebs' solution. The superfusion with a Ca(2+)-free medium progressively increased the spontaneous [3H]GABA release and strongly decreased the electrically evoked release of [3H]GABA (-65%). The effects of three dopaminergic D2 receptor agonists (RU24926, lisuride and LY171555) were studied on both the spontaneous and the electrically evoked [3H]GABA release. The spontaneous release of [3H]GABA was increased by exposure to each of these three D2 agonists. RU24926 produced a dose-dependent increase from 10(-9) to 3 x 10(-8) M and the maximal effect was totally abolished by the dopaminergic D2 receptor antagonist sulpiride (10(-5) M). With lisuride a progressive increase of [3H]GABA release was observed and a plateau value was reached with concentrations between 10(-7) and 10(-6) M. These effects were totally reversed by 10(-5) M sulpiride. The dose-response relation for LY171555 was bell-shaped, with a maximal effect being obtained with 10(-9) M) LY171555. This effect decreased with a higher concentration (10(-8) M) and finally was no longer observed for 10(-7) M LY171555. The maximal increase induced by LY171555 was totally abolished by 10(-5) M sulpiride. In contrast, the electrically evoked release of [3H]GABA was inhibited by these three D2 agonists. The IC50 value of the inhibition was 4.1 x 10(-8) M for RU24926 and 2 x 10(-7) M for lisuride. Sulpiride (10(-5) M) totally abolished the effect of 10(-7) M RU24926. In the concentration range of lisuride examined, a 50% reduction of the lisuride inhibition was obtained in the presence of sulpiride (10(-5) M). The dose-response curve obtained with LY171555 had a U-shape, with a maximal inhibition reached with 10(-8) M, whereas no effect was observed with 10(-6) M. The inhibition induced by 10(-8) M LY171555 was completely antagonized by 10(-5) M sulpiride. The D2 agonist-induced inhibition of the electrically evoked release of [3H]GABA was mimicked by dopamine endogenously released by 10(-5) M amphetamine. This effect was reversed by 10(-5) M sulpiride. Our data provide further evidence for a dopaminergic control of GABA interneurons in the prefrontal cortex. This regulation implies the activation of D2 dopaminergic receptors. The possible mechanisms underlying the opposite effects of D2 agonists on the spontaneous and the electrically evoked release of [3H]GABA are discussed.

[D2 dopaminergic receptor activation enhances the spontaneous release of 3H-GABA in the prefrontal cortex of rats, in vitro. The facilitating role of D1 dopaminergic receptors]. / L'activation des récepteurs D2 dopaminergiques stimule la libération spontanée de 3H-GABA dans le cortex préfrontal du rat, in vitro. Rôle permissif des récepteurs D1 dopaminergiques.

The effects of three D2 dopaminergic agonists on the spontaneous release of 3H-GABA have been studied on rat prefrontal cortical slices. LY171555 (10(-9) M), RU24926 (3 x 10(-8) M) and lisuride (10(-7) M) respectively enhanced the spontaneous release by 25, 20.5 and 23%. These effects were totally reversed by the D2 antagonist sulpiride (10(-5) M). Furthermore, subliminar concentration of RU24926 (10(-9) M) and of the D1 agonist SKF38393 (10(-6) M) induced a clear enhancement of the spontaneous release of 3H-GABA when they were superfused simultaneously. Our results suggest that in the prefrontal cortex, the spontaneous release of 3H-GABA is under an activatory D2 dopaminergic control. The activation of D1 receptors seems to have an enabling effect on this regulation.

[Effects of the stimulation of D1 and D2 dopaminergic receptors on the electrically induced release of gamma-(3H)-aminobutyric acid in the prefrontal cortex of the rat]. / Effets de la stimulation des récepteurs D1 et D2 dopaminergiques sur la libération d'acide gamma-[3H] aminobutyrique induite électriquement dans le cortex préfrontal du rat.

The effects of D1 and D2 dopaminergic agonists and antagonists on the electrically-evoked release of gamma-[3H] aminobutyric acid (3H-GABA) have been studied on rat prefrontal cortex slices. The major part of the electrically-evoked release of 3H-GABA appeared to be Ca++ dependent since a 62% decrease was observed when calcium was removed from the superfusion medium. Two specific D2 dopaminergic agonists, RU 24926 (10(-7) M) and lisuride (10(-6) M), respectively induced a 32% and a 50% inhibition of the electrically-evoked release of 3H-GABA. The selective D2 dopaminergic antagonists sulpiride (10(-5) M) totally abolished the effect of RU 24926 and partially abolished the effect of lisuride. The selective D1 agonist SKF 38393 (10(-5) M) did not affect 3H-GABA release. These results suggest that in the rat prefrontal cortex in vitro, the dopaminergic modulation of 3H-GABA release is mediated through D2 but not D1 receptors. The activation of D2 dopaminergic receptors induces an inhibition of the electrically-evoked release of 3H-GABA.