Relationship between hindbrain segmentation, neural crest cell migration and branchial arch abnormalities in rat embryos exposed to fluconazole and retinoic acid in vitro

Abstract

Fluconazole (FLUCO) and retinoic acid (RA) can perturb morphogenesis of the branchial apparatus in rodent embryos exposed in vitro. The aim of the present study was to compare the effects induced by in vitro exposure to FLUCO or to RA on rhombomere organisation, neural crest cell (NCC) migration and cranial nerve differentiation using specific antibodies. For this purpose 9.5 d.p.c. rat embryos were exposed to teratogenic concentrations of FLUCO or RA; another group was exposed to no-effect concentrations of both agents. Expression of Hox-b1 and Krox20 (markers of specific rhombomeres) was altered after FLUCO and RA exposure. Furthermore, FLUCO and RA showed a synergistic effect. These results suggest that the observed branchial abnormalities are due to anomalous NCC migration related to incorrect organisation of specific rhombomeres.

Introduction

The clinical antifungal agent fluconazole (FLUCO, a bis-triazole derivative) has teratogenic activity toward cultured rodent embryos [1], [2], [3]. Triazole derivatives in general are potent antifungal agents that act by inhibiting the cytochrome P-450 mediated conversion of lanosterol to ergosterol [4]. The inhibitory potency of these compounds is not restricted to fung, but also occurs with a number of mammalian cytochrome P-450 dependent activities. For example, the inhibitory activity of triazole derivatives on CYP26, a P-450 enzyme that degrades retinoic acid (RA), has been demonstrated in vitro and in vivo in normal rats, as well as patients with acute promyelocytic leukaemia [5], [6]. RA, a Vitamin A derivative, is endogenously synthesized in the Vertebrata and is essential for normal embryonic development [7] and the maintenance of differentiation in some adult tissues [8]. It is well known that the excess or deficiency of retinoids induces abnormalities in mammalian embryos [9] and that the developing hindbrain and branchial region are particularly sensitive target tissues based on ample experimental and clinical evidence [10], [11], [12], [13], [14].

Triazole derivatives are able to induce branchial malformations similar to RA [2], [3], [15]. In particular, FLUCO induces specific branchial arch abnormalities in postimplantation mouse [1] or rat embryos [2], [3] that were exposed in vitro to concentrations of 150–250 μM and 125–500 μM, respectively. The observed abnormalities were similar to what were described following RA exposure in vitro [16], [17] and included hypoplasia, agenesis, fusion of first and second branchial arches. This raises the possibility that FLUCO and RA act by a similar mechanism of pathogenesis.

During embryogenesis, the branchial arch mesenchyme is composed of densely packed paraxial mesoderm surrounded by a peculiar derivative of cranial neural crest cells (NCC) sometimes referred to as ectomesenchyme. NCC are a migratory population of cells that originate at the dorsal lip of the neural folds [18], [19]. Crest cells that contribute to the morphogenesis of branchial arches and cranial nerve ganglia arise from the rhomboencephalic neural crest in an axial-level specific pattern [20], [21]. Development of branchial arch derivatives (as craniofacial structures), of specific branchiomotor nerves and cranial sensory ganglia, and the fate of NCC are in most cases correlated with their rhomboencephalic origin. This correlation has been described in terms of anatomical position and the expression of a specific combination of “pattern forming” genes that include Hox genes [20], [22]. Regional diversity in the hindbrain is achieved through the generation of seven to eight neuroepithelial compartments (rhombomeres). This segmentation, controlled by the Hox code and regulated by a number of signals including RA [23], is required to define the periodic organisation of hindbrain neurons and the migration pathways of NCC that colonise the branchial arches [24]. The most anterior rhombomeres (r1, r2, r3) and associated NCC do not express Hox genes, whereas NCC originating in more posterior regions and the related rhombomeres (r4–r8) generally express the same Hox code [24]. Similarly, two Hox transcription factors (Krox20 and Kreisler) are critically important for the development of rhombomeres 3/5 [25] and 5/6 [26], respectively.

Recently, we showed that FLUCO produces its teratogenic effects by interfering with NCC migration and without altering the extracellular matrix and the reciprocal induction between paraxial mesoderm and ectomesenchyme [3]. In particular, NCC seemed able to migrate but with altered migration pathways. A diffused NCC mass was detected near the rhomboencephalon in FLUCO exposed embryos, whereas controls showed three distinct migration streams from the rhomboencephalon to the branchial arches. Our hypothesis was that FLUCO exposure in vitro interferes with the regular hindbrain segmentation and rhombomere patterning and consequently alters the NCC distribution into branchial arch mesenchyme.

On this basis, the present study was undertaken to compare the effects induced by exposure to FLUCO or RA on branchial arch morphology, cranial nerve differentiation (using immunostaining with anti-neurofilament 160 kDa), NCC migration (using immunostaining with anti-Cellular Retinoic Acid Binding Protein, specifically expressed during the whole culture period by the NCC [27], [28]) and rhombomere organisation (using immunostaining with anti-Hox-b1 and anti-Krox20 proteins, specifically expressed in early embryonic stages, respectively by the rhombomere 4 and by rhombomeres 3 and 5 [29]) in a whole embryo culture system.