Diabetic embryopathy in C57BL/6J mice. Altered fetal sex ratio and impact of the splotch allele.

Maternal diabetes (types 1 and 2) induces a broad array of congenital malformations, including neural tube defects (NTDs), in humans. One of the difficulties associated with studying diabetic embryopathy is the rarity of individual malformations. In an attempt to develop a sensitive animal model for maternal diabetes-induced NTDs, the present study uses chemically induced diabetes in an inbred mouse model with or without the splotch (Sp) mutation, a putatively nonfunctional allele of Pax3. Pax3 deficiency has been associated with an increase in NTDs. Female C57BL/6J mice, either with or without the Sp allele, were injected intravenously with alloxan (100 mg/kg), and plasma glucose was measured 3 days later. A wide range of hyperglycemia was induced, and these diabetic mice were bred to C57BL/6J males, some carrying the Sp allele. Gestational-day-18 fetuses were examined for developmental malformations. Fetuses from matings in which either parent carried the Sp allele were genotyped by polymerase chain reaction. Maternal diabetes significantly decreased fetal weight and increased the number of resorptions and malformations, including NTDs. A significant correlation was found between the level of maternal hyperglycemia and the malformation rate. The sex ratio for live fetuses in diabetic litters was significantly skewed toward male fetuses. Matings involving the Sp allele yielded litters with significantly higher percentages of maternal diabetes-induced spina bifida aperta but not exencephaly, and this increase was shown to be associated with the presence of a single copy of the Sp allele in affected fetuses. Thus, Pax3 haploinsufficiency in this murine model of diabetic embryopathy is associated with caudal but not cranial NTDs.

Histochemical localization of glycosaminoglycans during morphogenesis of the secondary palate in mice.

The hydration of hyaluronic acid (HA) accumulated in the secondary palatal processes is expected to exert an intrinsic tissue pressure that could, in part, provide the impetus for shelf reorientation. Glycosaminoglycans were histochemically localized in the A/J mouse palate during development (days 12 to 15) by specific enzymatic degradation followed by preferential staining with alcian blue under differential pH or MgCl2 concentration. The presence of HA and chondroitin sulphates A and C (CS) was demonstrated in proportions that differed regionally. At the time of reorientation (days 14 to 15) HA was the predominant staining component, being distributed according to the relative prominence of extracellular spaces (ECS). HA was present in higher concentration in the anterior than the posterior part of the palate, particularly in an area of low cell density adjoining the CS-rich mesenchyme of the maxillary process. This arrangement suggests that the maxillary process might provide a resilient incompressible structural base for the palate as its HA-rich ECS expands. Sulphated GAG, with CS being the predominant component, was localized for the most part on the oral-side mesenchyme both in the anterior and posterior palate. The most intense staining of sulphated proteoglycans occurred in association with the basal lamina along the presumptive oral-side. Mesenchymal cells along this region appeared condensed and may have been stabilized by these sulphated GAG providing structural constraints which might function in palate morphogenesis.

Substance abuse in pregnancy: teratogenesis.

Substances of abuse include those that are legal (such as alcohol) and those that are illegal (street drugs). Many of these agents produce reproductive toxicity including intrauterine growth retardation. Teratogenesis is unproven with most of these agents. Alcohol is an exception, producing the fetal alcohol syndrome. Cocaine causes marked reproductive toxicity including decreased growth and morbidity. A number of birth defects have been associated with cocaine use including genitourinary, cardiac, and limb anomalies. The reproductive toxic and putative teratogenic effects of cocaine are probably associated with its well-known pharmacologic action causing vasoconstriction. From preliminary studies, it would appear that methamphetamine also produces reproductive toxic effects similar to those of cocaine.

Role of neurotransmitters in palate development and teratologic implications.

It is hypothesized that neuropharmacologic agents are more teratogenic to humans. Since many neuropharmacologic agents function through neurotransmitter mechanisms, then neurotransmitters should function to regulate embryonic development. Evidence has been obtained that neurotransmitters do indeed function as biological signals in palate development. It has been shown that palate reorientation is modulated by neurotransmitters with a wide range of diversity, similar to the CNS. Thus serotonin and acetylcholine stimulate and GABA inhibits the reorientation process. Spatial diversity is also observed: serotonin functions at the anterior and acetylcholine at the posterior end, and GABA functions more efficiently at either end in different inbred strains. Many criteria for functioning neurotransmitters have been obtained. Both serotonin and GABA have been measured in the palate and developmental changes observed. Physiologic responses to serotonin have been monitored. Serotonin has been shown to stimulate palate cell motility as well as protein carboxyl methylation and cyclic GMP. The serotonin effects on protein carboxyl methylation and cyclic GMP could function to stimulate palate reorientation by modulating cell contractility and protein secretion. Further support for the hypothesis that neuropharmacologic agents could be teratogenic by perturbation of neurotransmitter mechanisms comes from studying GABA and diazepam. Evidence has been obtained that diazepam induces cleft palate by mimicking GABA in a functional GABAergic system in palate development. A significant finding is that genetic differences in both diazepam teratogenesis and in a GABAergic system have been observed. Comparing the SWV and AJ strains, the SWV mouse showed (1) a greater sensitivity to diazepam-induced cleft palate, (2) a greater sensitivity to GABA and diazepam inhibition of palate reorientation in embryo culture, (3) a greater concentration of palatal GABA and (4) a more efficient GABA uptake system. These results are suggestive that diazepam may be more teratogenic in individuals of both the mouse and human who possess a more developed GABAergic mechanism. If such a result would obtain in the human, these genotypic differences in GABA would probably not influence the individual to a significant degree in most situations, except for the pregnant woman being at high risk when treated with diazepam.

Neuropharmacologic teratogenesis and neurotransmitter regulation of palate development.

Neuropharmacologic agents may be more teratogenic to human beings by perturbing neurotransmitter mechanisms that regulate embryonic development. There is evidence that neurotransmitters function in mouse palate development: Serotonin and acetylcholine stimulate and GABA inhibits shelf reorientation. Both serotonin and GABA have been measured in the palate and uptake systems monitored. Serotonin stimulates cell motility, protein carboxyl methylation, and cyclic GMP. Diazepam (Valium) could cause cleft palate by mimicking GABA. Genetic differences in both diazepam teratogenesis and in a GABAergic system of the mouse have been observed. If human beings were to show genetic differences in a GABAergic system, some pregnant women could be at high risk when treated with diazepam.

GABA uptake in embryonic palate mesenchymal cells of two mouse strains.

To obtain further evidence that the inhibitory neurotransmitter GABA functions in palate development, the presence of an active GABA uptake mechanism was sought using primary cultures of embryonic palate mesenchymal cells. Uptake was compared from cells of two inbred mouse strains in which the SWV strain shows greater sensitivity than the AJ strain to effects of GABA on palate morphogenesis and of diazepam in producing cleft palate. Palate cells were capable of accumulating [3H]GABA by saturable uptake mechanisms characteristic of a high and low affinity active transport as indicated by temperature, Na+ ion and carrier dependence as well as Km and Vmax values that were comparable to other biological systems. The Vmax of the high-affinity uptake system from cells of the SWV strain was 1.8 fold higher than that of the AJ. GABA uptake was also observed in fibroblasts from various sources including embryonic mouse limb cells, human skin fibroblasts and 3T3 cells. When active GABA uptake was measured in skin fibroblasts from the mouse SWV and AJ strains, the rate of uptake from SWV cells under high affinity conditions was also 1.8 fold greater than in AJ cells. Thus active GABA uptake appears to be genetically regulated in non-neural cells which may contribute to differential responses to GABA.

Chloride transport in embryonic cells: effect of ethanol and GABA.

Ethanol and GABA (gamma-aminobutyric acid) and their interaction on 36Cl-influx were analyzed in cultured embryonic palate and limb mesenchymal cells in order to determine whether ethanol exerts its teratogenic action through a GABA receptor involved in embryogenesis. Cl- transport in secondary cultures of C57BL/6 palate mesenchymal cells was shown to consist of three systems including the electroneutral Cl-/HCO3- exchange (50%) and Na+/K+/Cl- cotransport (30%) pathways and the voltage-dependent Cl- channel (20%). Treatment with DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) or SITS (4-acetamido-4'-isocyano-stilbene-2,2' disulfonic acid) in SWV palate cells inhibited the Cl-/HCO3- exchange pathway, while treatment with DIDS and bumetanide inhibited both the exchange and cation cotransport pathways, the residual Cl- influx inferred to be the electrogenic pathway. Inhibition of Cl- transport by anthracene-9-carboxylic acid confirmed the presence of the electrogenic Cl- pathway. It was observed that the rate of Cl- transport was significantly greater in palate cells of C57BL/6 mice than those of SWV mice. Also the rate of Cl- transport was significantly greater in secondary cultures of palate cells from C57BL/6 mice than from primary cultures of limb cells from the same strain. No evidence could be obtained that ethanol (10 to 100 mM) or GABA (3 X 10(-5) M) or their combination stimulated total Cl- influx in either C57BL/6 or SWV palate mesenchymal cells, putative voltage-dependent Cl- influx in C57BL/6 palate cells, or total Cl- influx in primary cultures of C57BL/6 limb mesenchymal cells.

Involvement of GABA in palate morphogenesis and its relation to diazepam teratogenesis in two mouse strains.

Previous studies have indicated that serotonin and acetylcholine stimulate palate shelf reorientation. The present studies were undertaken to determine whether gamma-aminobutyric acid (GABA) functions as an inhibitory neurotransmitter in the palate and whether diazepam mimics GABA to inhibit shelf reorientation and cause cleft palate. First, it was shown that 10(-4) M GABA inhibits palate shelf reorientation in day 14.5 AJ embryos cultured for 2 hours. Anterior palate reorientation stimulated by 10(-5) M serotonin was decreased by GABA; 10(-5) M picrotoxin (GABA antagonist) stimulated anterior shelf reorientation and reversed the effect of GABA. Diazepam (10(-4) M) partially inhibited palate shelf reorientation and that stimulated by 10(-5) M serotonin. Diazepam (400 mg/kg) was administered to AJ mice at day 13.5 of gestation and embryos were cultured at day 14.5. The inhibition produced by diazepam was significantly reduced by 10(-5) M picrotoxin. The teratogenic effect of diazepam was compared with AJ and Swiss-Webster Vancouver (SWV) inbred strains. Diazepam produced greater clefting in SWV mice (57% net) than in the AJ (18% net) when compared to their water- and food-starved controls. The greater sensitivity of the SWV strain than the AJ strain to diazepam, as well as to GABA, was also observed in embryo culture. GABA (10(-5) M) markedly inhibited posterior palate reorientation and reversed the stimulation produced by bethanechol in SWV mice. The inhibitory effects of GABA on the posterior palate were partially reversed by picrotoxin. Furthermore, diazepam inhibited palate reorientation either when administered to the pregnant dam or added in embryo culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Palate cell motility and substrate interaction.

To elucidate the mechanism by which palate shelves reorient during embryogenesis, migration of palate mesenchymal cells has been studied employing various substrates. When palate explants were cultured in a hydrated collagen lattice, it was observed that bipolar spindle-shaped cells migrated out of each explant toward the other. These cells were aligned parallel to each other and to the fibrous tracks that formed. The cells appeared to move along and through the fibrous tracks. Cell migration was dependent on the presence of serum and fibronectin. The fibrous tracks viewed by phase microscopy were sensitive to collagenase. Scanning electron microscopy revealed that the collagen fibers of the hydrated lattice had coalesced into larger bundles. Pretreatment of explants with serotonin stimulated cell migration out of the explant into the hydrated collagen lattice. This effect was specific, since the antagonist methysergide blocked the stimulation produced by serotonin. Employing other substrates, it was noted that palate cells migrating out of double explants toward each other produced large wrinkles in a polysiloxane substratum. Similarly, cultured monolayer cells also produced wrinkles that disappeared as cells rounded up after trypsin treatment. Finally, monolayer cells pulled on and distorted collagen films when cultured on the substrate. It is concluded that migrating palate cells can interact with their substrate producing tractional forces. Serotonin-induced modulation of cell motility and its relationship to palate reorientation are discussed.

The effects of dexamethasone on palate mesenchymal cell phospholipase activity.

Corticosteroids will induce cleft palate in mice. One suggested mechanism for this effect is through inhibition of phospholipase activity. This hypothesis was tested by measuring the effects of dexamethasone, a synthetic corticosteroid, on phospholipase activity in cultures of palate mesenchymal cells. Palate mesenchymal cells were prelabeled with [3H]arachidonic acid. The cells were subsequently treated with various concentrations of dexamethasone. Concurrently, cultures of M-MSV-transformed 3T3 cells were prepared identically. After treatment, phospholipase activity was stimulated by the addition of serum or epidermal growth factor (EGF), and radioactivity released into the medium was taken as a measure of phospholipase activity. Dexamethasone (1 X 10(-5) or 1 X 10(-4) M) could inhibit serum-stimulated phospholipase activity in transformed 3T3 cells after 1 to 24 hr of treatment. However, no inhibition of activity was measured in palate mesenchymal cells following this period of treatment. Not until 120 hr of treatment with dexamethasone (1 X 10(-4) M) was any significant inhibition of serum-stimulated phospholipase activity observed in palate mesenchymal cells. When EGF was used to stimulate phospholipase activity, dexamethasone (1 X 10(-5) M) caused an increase in phospholipase activity in palate mesenchymal cells. These observations suggested that phospholipase in transformed 3T3 cells was sensitive to inhibition by dexamethasone. However, palate mesenchymal cell phospholipase is only minimally sensitive to dexamethasone, and in certain instances can be enhanced. These results cannot support the hypothesis that corticosteroids mediate their teratogenic effect via inhibition of phospholipase activity.

The influence of the epithelium on palate shelf reorientation.

The intrinsic forces necessary for directing the reorientation of the secondary palate appear to reside in the anterior two thirds of the palate or presumptive hard palate. The hard palate could reorient regardless of whether it was intact or separated from the posterior third or presumptive soft palate. The soft palate could only reorient if the palate shelves are left intact. These intrinsic forces, within the hard palate, may be mediated by the mesenchymal cells, their extracellular matrix, or the epithelium surrounding the shelves. This latter possibly was tested by removing the epithelium, from either the presumptive oral or nasal surface followed by measurement of reorientation in vitro. Only after removal of the oral epithelium was a significant inhibition in reorientation observed. The treatment used to remove the epithelium, EDTA and scraping, was shown to remove 41% of the oral epithelium leaving the majority of the basement membrane intact. The observed inhibition of reorientation did not appear to be a consequence of wound healing. Creation of wounds twice the area that was observed after treatment with EDTA and scraping inhibited reorientation minimally. These results suggest that the epithelium and particularly the anterior oral epithelium plays a major role in the reorientation of the murine secondary palate.

Sialyltransferase: regulation of alpha-foetoprotein microheterogeneity during development.

The temporal accumulation of the electrophoretic components of mouse alpha-foetoprotein in foetal plasma and amniotic fluid is reported. To explain the progressive appearance of the sialylated alpha-foetoproteins, the activity of sialyltransferase in foetal liver and yolk sac was measured. These results indicate that the increase in sialyltransferase activity in these tissues is responsible for the increased sialylation of alpha-foetoprotein.

Developmental changes in microheterogeneity of foetal plasma glycoproteins of mice.

Changes in microheterogeneity of foetal plasma glycoproteins during development of mouse embryos were investigated. Analysis of foetal plasma by polyacrylamide-gel electrophoresis indicated three major zones of proteins: (1) transferrins, (2) alpha-foetoproteins and (3) albumin. Three transferrins (Tr1, Tr2, Tr3) and five alpha-foetoproteins (Fp1, Fp2, Fp3, Fp4, Fp5) were resolved. Evidence for the presence of transferrins was the binding of (59)Fe to the three electrophoretic variants. By day 15.5 of gestation, there was a marked increase in the more-acidic components (Tr3, Fp4, Fp5) and a decrease in the less-acidic ones (Tr1, Tr2, Fp1, Fp2, Fp3). Treatment of foetal plasma with neuraminidase at this time of development converted the more acidic components into Tr1 and Tr2 and Fp1, Fp2 and Fp3. Furthermore, it was shown that early in development (day 12.5) only the less-acidic components of transferrin and alpha-foetoprotein were synthesized; at the later time in development (day 14.5) new synthesis of the acidic components of both groups occurred. That these more-acidic components of alpha-foetoprotein (Fp4, Fp5) were in fact electrophoretic variants of the less-acidic alpha-foetoproteins was shown by the immunoprecipitation of labelled Fp4 and Fp5 with anti-Fp1, anti-Fp2 and anti-Fp3. From these results it is postulated that the plasma glycoproteins that are synthesized later in development contain increased amounts of sialic acid and that the observed changes in microheterogeneity of these proteins represent regulation of glycoprotein biosynthesis at the level of carbohydrate attachment.

Palate morphogenesis: II. Contraction of cytoplasmic processes in ATP-induced palate rotation in glycerinated mouse heads.

It has been previously shown that non-muscle contractile system(s) exist in mouse palate mesenchyme underlying the palatal epithelium before shelf rotation. In order to obtain evidence that the non-muscle contractile system(s) function to elevate the palate, glycerinated heads have been incubated with ATP. It was shown that 5 mM ATP and a 30 min incubation at 25 degrees C stimulated palate rotation optimally. Elevation of the anterior end of the palate was nearly complete (PSI = 3.90, p less than 10(-6)). Although rotation of the posterior end was significant (p less than 0.02), movement was limited (PSI = 1.70). Light microscopy of the palate revealed that ATP caused a marked condensation of the cytoplasmic processes of the mesenchymal cells. The contraction of the processes of the mesenchymal cells induced by ATP increased roughly with increased palate shelf rotation and was greater at the peripheral than at the internal mesenchyme. Cytochalasin B pretreatment at 40 microM completely blocked the ATP-induced rotation at the anterior end. The effect of other nucleotides on palate rotation was tested. GTP caused a significant stimulation of anterior shelf rotation (p less than 0.005), which was less than ATP, while ADP and CTP were ineffective. Low temperature (6 degrees C) prevented the ATP-induced shelf rotation. These results suggest that the non-muscle contractile cells in the mesenchyme play a role in palate elevation and that contraction of the actomyosin containing microfilaments supplies the motive force.

The effect of reducing ATP levels on reorientation of the secondary palate.

The force for directing palate shelf reorientation appears to be associated with elements of the presumptive hard palate (Brinkley & Vickerman, 1979; Bulleit & Zimmerman, 1985). The palatal elements that mediate this process do not require palate cells to be metabolically active for expression of the force. This contention was demonstrated using an in vitro system that allows substantial reorientation of the hard palate to occur. ATP levels were reduced by treatment with metabolic inhibitors and the degree of reorientation was measured 1 h following pretreatment with inhibitors. Treatment of cultured embryonic heads under anoxic conditions with 2,4-dinitrophenol or KCN had no effect on the degree of reorientation occurring in vitro. These agents reduced ATP levels by 71% and 62%, respectively. Treatment of cultured heads with 2-deoxy-D-glucose under anoxia also had no effect on reorientation. This treatment reduced ATP levels in embryonic heads by 92-94%. A similar reduction was observed if ATP levels were measured in palate tissue alone. The treatment of cultured heads with 2-deoxy-D-glucose and anoxia not only reduced levels of ATP but also reduced CTP, GTP and UTP. These results indicate that palate shelf reorientation is independent of cellular metabolic activity and supports the hypothesis that reorientation is dependent on a pre-existing infrastructure within the palate shelves.

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.

Role of neurotransmitters and teratogens on palate development.

Neurotransmitters regulate palate shelf reorientation. Acetylcholine and serotonin stimulate, whereas GABA inhibits reorientation. Serotonin stimulates cell movement in an in vitro chemotactic system. Diazepam may cause cleft palate by mimicking GABA. Diazepam sensitivity may be caused by genotypic differences in a GABA-ergic system in the embryo.