Sites of serotonin uptake in epithelia of the developing mouse palate, oral cavity, and face: possible role in morphogenesis.

With the method of whole mouse embryo culture, together with immunocytochemistry with an antiserum to serotonin (5-HT), sites of 5-HT uptake were found to be transiently expressed in the epithelia of the developing palate, tongue, nasal septum, and maxillary and mandibular prominences during the period of active morphogenesis (embryonic days 12-14; or E12-14). These sites had the ability to take up 5-HT when added to the culture medium in the presence of the MAO inhibitor nialamide and an antioxiant, L-cysteine (NC), and could also be seen after exposure of embryos to the 5-HT precursor L-tryptophan (L-TRP) + NC. These sites were also visible after culturing embryos without any additives, which may have been due to the presence of L-TRP in one component of the culture medium (DMEM) or to 5-HT itself, which is present in relatively high amounts in fetal calf serum. At E12-13, the appearance of 5-HT immunoreactivity (IR) at these sites after treatment with 5-HT + NC was blocked by the 5-HT uptake inhibitor fluoxetine, providing further evidence that these are true sites of 5-HT uptake. However, fluoxetine did not completely block the appearance of these sites in E14 embryos after 5-HT + NC or L-TRP + NC although it was effective with NC alone. This finding could mean that at E14 5-HT uptake into these sites occurs by mechanisms not completely blocked by fluoxetine or that there is some limited capacity for 5-HT synthesis. Taken together with results from previous studies where 1) 5-HT has been reported to stimulate palatal shelf reorientation and palatal mesenchyme cell motility in vitro [Wee et al., J Embryol Exp Morphol 53:75-90, 1979; Zimmerman et al., J Craniofac Genet Dev Biol 3:371-385, 1983] and 2) long-term culturing of mouse embryos in the presence of 5-HT or fluoxetine has been shown to cause malformations of the craniofacial region (Lauder, Thomas, and Sadler, in preparation), the results of the present study suggest that 5-HT could act as a developmental signal in the palate, oral cavity, and face during the period of active morphogenesis.

Glucocorticoid receptor-mediated teratogenesis and cell proliferation in the limbs and face of the chick embryo.

The susceptibility of the face region of the chick embryo to the teratogenic action of intraamniotically injected hydrocortisone contrasts with the resistance of the limbs to its action while at the same time their dysmorphogenesis may be induced by other agents. Since glucocorticoid receptors were shown to mediate face teratogenesis, their development was investigated in freshly dissected limb buds of 3-, 3.5-, and 4-day-old chick embryos in comparison with the face region. The specific binding of 3H-dexamethasone to molybdate-stabilized glucocorticoid receptors was estimated by the dextran-coated charcoal method and complemented by cytologic analysis of mitotic activity in control and hydrocortisone-treated embryos. The glucocorticoid receptors were found in both organ anlagen already on day 3 when their concentration in femtomoles per microgram DNA was significantly higher in the face region. Accordingly, on day 3 intraamniotic hydrocortisone inhibited the mitotic activity in the face without affecting the developing limbs. On days 3.5 and 4 the concentration of glucocorticoid receptors was similar in both organ anlagen. Administration of hydrocortisone on day 4 induced mitotic depression in the face as well as in the limbs. However, the degree of inhibition appeared to be dependent upon the actual mitotic rate. In the face region where the mitotic activity culminated at that time, the inhibition was much deeper and longer-lasting than in the developing limbs characterized by continuous decrease of proliferation rate in controls. These findings are consistent with a view that glucocorticoid receptors are a prerequisite, but not the only factor in receptor-mediated teratogenesis.

Distribution of glycosaminoglycans in the developing facial region of mouse embryos.

Synthesis and distribution of glycosaminoglycans in the developing facial region of mouse embryos were investigated during stages of development when the embryonic facial processes form and expand. Facial regions of 11th and 12th day embryos were either 1) labeled with 3H-glucosamine and newly synthesized glycosaminoglycans determined on the basis of specific enzyme sensitivity, or 2) sectioned and stained with Alcian Blue coupled with enzyme digestion in order to localize glycosaminoglycans. On the basis of enzyme sensitivity over half of the newly synthesized glycosaminoglycans during this time period were identified as hyaluronate, with only 12-15% representing chondroitin sulfate. Hyaluronate was found to be the primary component of the extracellular matrix. Basal laminae exhibited prominent chondroitin sulfate staining and also appeared to contain hyaluronate and heparan sulfate. It is suggested that hyaluronate may play a structural role in maintaining the shape and orientation of the facial processes.