Defects in cervical vertebrae in boric acid-exposed rat embryos are associated with anterior shifts of hox gene expression domains.

BACKGROUND: Previously, we showed that prenatal exposure to boric acid (BA), an industrial agent with large production, causes alterations of the axial skeleton in rat embryos, reminiscent of homeotic transformations. Indeed, Sprague-Dawley rats exposed in utero to BA on gestation day 9 (GD 9) had only six, rather than the normal seven, cervical vertebrae. This finding, observed in 91% of GD 21 fetuses, suggests posterior transformations of vertebrae. The present study attempts to determine if these skeletal alterations could be explained by modifications of the hox code, involved in the establishment of positional information along the craniocaudal axis of the embryo. METHODS: Pregnant rats were treated by gavage with BA (500 mg/kg, twice) on GD 9. Embryos were collected on GD 11 or GD 13.5 and processed for in situ hybridization. Several hox genes were selected according to the position of their cranial limit of expression in the cervical and thoracic region. RESULTS: At GD 13.5, we detected a cranial shift of the anterior limit of expression of hoxc6 and hoxa6. We observed no difference between control and treated embryos in the location of the cranial limit of expression of the other genes: hoxd4, hoxa4, hoxc5, and hoxa5. CONCLUSIONS: Our results demonstrate that following in utero exposure to BA on GD 9, a disturbance of the expression of hox genes involved inthe specification of most anterior vertebrae is observed at GD 13.5. Based on their expression domain and on their implication in the definition of the cervicothoracic vertebral boundary, it is likely that the anteriorization of hoxc6 and hoxa6 reported here is correlated to the morphological phenotype observed in BA-exposed fetuses at GD 21

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

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

The skeleton : biochemical, genetic and molecular interactions in development and homeostasis. : Effects of boric acid on Hox gene expression and the axial skeleton in the developing rat.

Gestational exposure to boric acid (BA) causes reduced incidences of supernumerary ribs and shortening/absence of the 13th rib in the progeny of multiple laboratory species. To further explore this, Sprague-Dawley rats received 500 mg/kg b.i.d. on gestation days (GD) 6, 7, 8, 9, 10, or 11 (plug day = GD 0). GD-21 fetuses were stained for skeletal examination. BA's most noteworthy effects were apparent homeotic shifts in the axial skeleton; i.e., a given vertebra anatomically resembled an adjacent vertebra, thus leading to altered numbers of cervical, thoracic, or lumbar vertebrae. Whereas most groups generally had no such effect, about 90% of the GD-9 exposed fetuses had only six cervical vertebrae. Deficiencies in the C3-C5 region, C6, or C7 were observed in 67%, 1%, and 23% of the exposed fetuses, respectively. In contrast, GD-10 treatment caused agenesis of a thoracic/lumbar vertebra in over 60% of the fetuses. In these fetuses, the deficient region was usually T11. In view of the 90% incidence of six-cervical vertebrae in GD-9 exposed fetuses, we used this exposure regimen (500 mg/kg b.i.d. on GD 9) as an experimental model for the study of homeotic shifts. We sought to determine if these skeletal alterations could be explained by modifications of the hox code, involved in the establishment of positional information along the cranio-caudal axis of the embryo. Embryos were collected on GD 13.5 and processed for in situ hybridization. Several hox genes were selected according to the position of their cranial limit of expression in the cervical and thoracic region. A cranial shift in the cranial limit of expression of hoxc6 and hoxa6 was evident in the prevertebrae, whereas no difference was observed between control and treated embryos in the expression of hoxd4, hoxa4, hoxc5, and hoxa5. Anteriorization of the expression domain of hoxc6 and hoxa6 is consistent with the posterior transformation of cervical vertebrae, and may partially account for the phenotype observed on GD 21 in BA-exposed fetuses