Too much of a good thing: retinoic acid as an endogenous regulator of neural differentiation and exogenous teratogen.

Retinoic acid (RA) is essential for both embryonic and adult growth, activating gene transcription via specific nuclear receptors. It is generated, via a retinaldehyde intermediate, from retinol (vitamin A). RA levels require precise regulation by controlled synthesis and catabolism, and when RA concentrations deviate from normal, in either direction, abnormal growth and development occurs. This review describes: (i) how the pattern of RA metabolic enzymes controls the actions of RA; and (ii) the type of abnormalities that result when this pattern breaks down. Examples are given of RA control of the anterior/posterior axis of the hindbrain, the dorsal/ventral axis of the spinal cord, as well as certain sex-specific segments of the spinal cord, using varied animal models including mouse, quail and mosquitofish. These functions are highly sensitive to abnormal changes in RA concentration. In rodents, the control of neural patterning and differentiation are disrupted when RA concentrations are lowered, whereas inappropriately high concentrations of RA result in abnormal development of cerebellum and hindbrain nuclei. The latter parallels the malformations seen in the human embryo exposed to RA due to treatment of the mother with the acne drug Accutane (13-cis RA) and, in cases where the child survives beyond birth, a particular set of behavioural anomalies can be described. Even the adult brain may be susceptible to an imbalance of RA, particularly the hippocampus. This report shows how the properties of RA as a neural induction agent and organizer of segmentation can explain the consequences of RA depletion and overexpression.

Whole-mount procedures for simultaneous visualization of nerves, neurons, cartilage and bone.

Herein are described two techniques to selectively label and/or stain afferent and efferent fibers and their cell bodies and simultaneously stain bone and cartilage. One technique uses anti-acetylated alpha-tubulin immunohistochemistry to follow the course, peripheral branching, and origin of the ventral spinal nerve innervating the axial musculature and a second uses anterograde and retrograde transport of selectively applied 3 kDa biotin dextran amines to identify specific afferent and efferent projections and their cell bodies. Both procedures can be combined with an enzyme clearing and staining procedure for the simultaneous visualization of bone (alizarin red S) and cartilage (alcian blue) in whole-mount preparations. Myelinated and unmyelinated nerve fibers are stained dark brown with the 3,3' diaminobenzidine tetrahydrochloride (DAB) reaction product, whereas cartilage is stained blue (alcian blue) and bone is red (alizarin red S). The combination of these procedures provides a simultaneous three-dimensional understanding of the topography of afferent and efferent projections and their cell bodies and the axial and appendicular skeletal system, which is key to understanding the compartmental relationships of the spinal cord with respect to the axial and appendicular skeleton.

A mechanism for anterior transposition of the anal fin and its appendicular support in the western mosquitofish, gambusia affinis affinis

The interosseal and suspensory ligaments of the axial and appendicular skeleton of the Western Mosquitofish, Gambusia affinis affinis were investigated in whole-mounted late embryonic and adult female and male G. a. affinis stained with alcian blue and alizarin red S, cleared, and viewed using differential interference contrast. The interosseal and suspensory ligaments of late embryonic female G. a. affinis are reduced prior to sexual differentiation and continue reduced in adult females. However, in late embryonic male G. a. affinis the interosseal and suspensory ligaments are well developed prior to sexual differentiation and become robust in adult males. Treating late embryonic female G. a. affinis with either 30.0 or 40. 0 &mgr;g/ diet of 17alpha-methyltestosterone led to the premature calcification of the hemal spine of the 13th vertebra. Treating late embryonic female G. a. affinis with 17alpha-methyltestosterone did not lead to the remodeling of the hemal spines of the 14th-16th vertebrae. Thus, in all female G. a. affinis treated with 17alpha-methyltestosterone, no anterior transposition of the anal fin and its appendicular support was observed. However, treating late embryonic male G. a. affinis with 30.0 &mgr;g/g diet of 17alpha-methyltestosterone led to the premature calcification of the hemal spine of the 13th vertebra, blocking the anterior transposition in only 62.5% of the males treated, and treating late embryonic male G. a. affinis with 40.0 &mgr;g/g diet of 17alpha-methyltestosterone led to the premature calcification of the hemal spine of the 13th vertebra, blocking the anterior transposition in all (100.0%) males treated. Treatment with either 30.0 or 40.0 &mgr;g/g diet of 17alpha-methyltestosterone appeared not to completely effect the interosseal and suspensory ligaments of late embryonic male G.a. affinis as evident by the male-specific remodeling of the hemal spines of the 14th-16th vertebrae. The results of this study implicate the interosseal and suspensory ligaments in the male-specific remodeling of the axial and appendicular skeletal elements. Following the resorption of the 13th hemal spine, the interosseal and suspensory ligaments in late embryonic male G. a. affinis develop enough tension to cause directional growth, bending, and elongation of the 14th-16th hemal spines, thus providing a mechanism for the anterior transposition of the sexually dimorphic anal fin and its appendicular support. The data also support the need to revise the interosseal and suspensory ligament nomenclature by assigning the designation of 'female' and 'male' to these ligaments.

Sex differences in endogenous retinoid release in the post-embryonic spinal cord of the western mosquitofish, Gambusia affinis affinis.

In this study we have shown sex differences in endogenous retinoic acid synthesis and retinaldehyde dehydrogenase activity in the post-embryonic spinal cords of immature female and male G. a. affinis. The F9 reporter cell assay and the zymography bioassay showed that the endogenous retinoic acid levels correlated with the levels of the endogenous enzyme(s) responsible for retinoic acid synthesis. These data also showed that both the endogenous retinoic acid levels and the enzyme(s) were higher in spinal cord segments 8-16 of immature males than in immature females. We have also shown that exogenous treatment of 17 alpha-methyltestosterone results in the masculinization of the immature female's anal fin and its appendicular support elements as well as the endogenous synthesis of retinoic acid and retinaldehyde dehydrogenase activity. The F9 reporter cell assay showed that the endogenous retinoic acid levels were relatively unchanged in spinal cord segments 8-16 of immature males treated with 17 alpha-methyltestosterone. However, the F9 reporter cell assay showed that the endogenous retinoic acid levels in spinal cord segments 8-16 of immature females treated with 17 alpha-methyltestosterone were markedly higher than levels observed in the immature males. The data also showed that the activity of the enzyme(s) responsible for the synthesis of endogenous retinoic acid was higher in spinal cord segments 8-16 of immature females treated with 17 alpha-methyltestosterone than in immature males treated with 17 alpha-methyltestosterone. Currently under investigation is the question of what role the endogenous enzyme(s) responsible for the synthesis of retinoic acid plays either alone or in concert with androgen in organizing hormone-dependent sexually dimorphic areas in the teleost body plan.

Organizational-activational concept revisited: sexual differentiation in an atherinomorph teleost.

Because of its numerous sexually dimorphic characters, the Western mosquitofish Gambusia affinis affinis is an excellent vertebrate system for addressing questions concerning sexual differentiation. In this review, the actions and limits of gonadal sex steroids, specifically testosterone, on the development of the sexually dimorphic anal fin and its axial and appendicular support are described. Data from our laboratory show that the critical period in this species for the anterior transposition of the male anal fin and its appendicular support occurs during the late embryonic period and that this differentiation of the male phenotypic genital areaduring the critical period is regulated by androgen.

Development of the anal fin appendicular support in the western mosquitofish, Gambusia affinis affinis (Baird and Girard, 1854): a reinvestigation and reinterpretation.

Development of the sexually dimorphic anal fin appendicular support of an internal fertilizing bony fish Gambusia affinis affinis was investigated by staining whole-mounted embryos, immature, and adult female and male G. a. affinis with alizarin red S and alcian blue. The tissue was examined histologically to assess development of the amphicelous centrum and to verify specificity of the stains. Our data confirm earlier claims about the development of the male and female characteristics in this species, and we provide for the first time direct embryonic evidence suggesting that development of the sexually dimorphic anal fin appendicular support is biphasic: (1) anteriorization of the most anterior caudal segments, and (2) growth and elongation of hemal arches of vertebrae 14-16. The first process involves a sequential homeotic transformation of hemal arches of vertebrae 11-13 through resorption of mineralized connective tissue, thus forming parapophyses that bear pleural ribs. This process begins in undifferentiated embryos and proceeds similarly in postnatal males and females. During the same period, the second process, likely induced by male gonadal hormones, causes the addition of mineralized connective tissue at the hemal arches of vertebrae 14-16. This second process, which occurs only in males, elongates the hemal arches of vertebrae 14-16 anteriorly. This elongation apparently translocates the anal fin appendicular support (including parts of the hemal spine of the hemal arch of vertebra 13) to the level of vertebra 11. It appears that the developmental programs of both female and male G. a. affinis create an area of 6 vertebrae which are markedly different from any vertebrae anterior to 11 and posterior to 16. We propose to term the area including these vertebrae and the associated anal fin, the genital area. We also propose that the first process, homeotic transformation of caudal into precaudal segments, is regulated by differential expression of control genes, such as homeobox genes, whereas the second process is regulated by gene expression under the control of male gonadal hormones. Conflicting data in the literature can be resolved with this model. Appropriate tests of the model are proposed.