Morphological effects of streptomyces hyaluronidase treatment on the outgrowth of the nasal processes in mouse embryos

The role of hyaluronic acid (HA) in embryonic mouse nasal process outgrowth was assessed following administration of Streptomyces hyaluronidase, an enzyme that degrades HA. Enzyme-treated and control embryos were compared morphologically 4 and 24 hr after treatment on day 11 of gestation. After 4 hr the nasal processes of treated embryos were reduced in volume compared to controls. This size reduction was associated with a decrease in the amount of extracellular space in the nasal processes and a change in mesenchymal cell shape. Extracellular matrix material observed in controls included collagenlike fibers, 25-30-nm granules, and a delicate meshwork of 3-4-nm filaments. Basal laminae exhibited filamentous and granular material that extended to the surface of underlying cells. Similar matrix constituents were observed in treated embryos with the exception of the 3-4-nm filaments, which probably represent HA. By 24 hr after treatment, embryonic circulation had ceased and heart beat was slow. The nasal processes of these embryos were very small, but their configuration was such that fusion had often begun. Thus the presence of HA appears to be important in maintenance of the normal volume of the nasal processes and in maintenance of normal mesenchymal cell morphology, but other factors appear to contribute to the change in process shape requisite for fusion.     

Alterations in craniofacial growth induced by isotretinoin (13-cis-retinoic acid) in mouse whole embryo and primary mesenchymal cell culture

Recent evidence has demonstrated that 13-cis-retinoic acid (13-cis-RA, or isotretinoin) is responsible for various craniofacial malformations in the rodent and human embryo. Our studies have been directed toward understanding this effect using mouse whole embryo and primary cell cultures. In whole embryo culture, 13-cis-RA caused significant overall embryonic growth retardation, especially in the primary and secondary palatal processes. In embryos explanted on day 10 of gestation and exposed for 24 or 48 hr, the mesenchyme beneath the epithelium of the nasal and maxillary processes contained pyknotic nuclei as well as a dramatically reduced number of nuclei incorporating 3H-thymidine. The secondary palatal processes and the roof of the oral-nasal cavity had fewer mesenchymal cells than control embryos. The incorporation of 3H-thymidine into TCA-insoluble macromolecules was 30% less in the retinoid-treated heads. In primary cell cultures from day-12 mouse secondary palatal mesenchyme, subsequent cell growth was decreased at concentrations of 13-cis-RA greater than 1 X 10(-5) M. After a 40-hr treatment period, labeling indices in retinoid-treated cells were significantly lower than control values (25% compared with 40%). Retinoic acid also caused a significant, concentration-dependent decrease in 3H-thymidine incorporation. The inhibitory effect of 13-cis-RA on proliferation of oral-nasal mesenchymal cells appears to be related to the production of craniofacial malformations.     

Is Far a Hox mutation?

The mouse First arch mutation, Far, causes a severe syndrome of craniofacial defects described previously. All of the known defects are derived from the anterior first arch, and to a very small extent, the dorsal second arch. Recently Far has been shown to be closely linked to Ulnaless on chromosome 2, and therefore in the vicinity of the Hox-4 gene cluster. This paper reports the results of several studies focused on the development origin of the most consistently expressed dominant effect caused by Far, an abnormal major bifurcation of the maxillary nerve. Nerve-stained whole-mount preparations of day 12 embryos showed that in Far mutants the maxillary nerve appears to have a central wedge missing from the normal single-stalked fan shape, and that the nerve defect in Far/Far and +/Far may be equally severe. The effect of retinoic acid on the development of the maxillary nerve was tested. Maternal treatment with 5 mg/kg retinoic acid on day 9 of gestation had no detectable effect on the maxillary nerve of +/Far embryos, and similar treatment with a teratogenic dosage (20 mg/kg) on day 8 or 9 produced no Far-like maxillary nerve defects in genetically normal embryos. The neural crest cells that give rise to nerves and mesenchyme of the first arch originate from specific rhombomeres, discrete segments of the developing head. The rhombomeres of 15 embryos at the 14-23 somite stages, of which 75% are expected to be +/Far or Far/Far, were examined. There was no detectable defect in segmentation or morphology of the rhombomeres compared with controls. The significance of ectopic cartilage in the palate of Far/Far mutants in relation to nerve bifurcation was explored. In histological studies, five out of six Far/Far day-15 fetuses had a rod of ectopic cartilage lateral to the posterior palate, running parallel to, and morphologically similar to, Meckel's cartilage, and lying between the two trunks of the abnormally bifurcated maxillary nerve. None of six +/Far day-15 fetuses examined had detectable ectopic cartilage in this region. We hypothesize that the maxillary nerve defects in Far mutants may be explained by the presence of an ectopic precartilaginous blastema that does not always further develop into detectable cartilage. The ectopic cartilage found in Far/Far resembles the epibranchial cartilage expressed in more posterior branchial arches and in the first arch of lower organisms, and therefore may represent an atavistic posteriorization of the anterior first arch in Far mutants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Processes involved in retinoic acid production of small embryonic palatal shelves and limb defects

All-trans-retinoic acid (RA) is teratogenic to the embryonic mouse, producing malformations in many developing systems, including the limb bud and palate. High incidences of limb defects and cleft palate are induced at doses which are not maternally toxic and do not increase resorptions. Exposure to RA on gestational day (GD) 10 results in small palatal shelves, which fail to make contact on GD 14. The formation of small shelves could be a consequence of increased cell death, reduced proliferation, a combination of these effects, or some other effect such as inhibition of extracellular matrix production. After exposure to 100 mg RA/kg on GD 10, proliferation in mesenchymal cells of the palatal shelves was not reduced from GD 12 to GD 14 and the levels of cell death in control and treated shelves did not differ when observed by light and electron microscopy. The present study examines the effects of RA on cell death and proliferation from GDs 10-12 and compares the effects in palatal shelves and limb buds. Embryonic mice were exposed to RA suspended in corn oil (100 mg/kg on GD 10), a dose that was teratogenic but not maternally toxic or embryolethal. Embryos were collected at 4, 12, 24, 36, or 48 hr postexposure, and tissues which form the palate or limb were dissected from the embryos, stained by a modified Feulgen procedure, and whole mounted on slides. Mitotic index (MI) and percentage dead cells were determined for mesenchymal cells of the first visceral arch, maxillary process, or palatal shelf (depending on stage of development) and forelimb buds. In the palatal tissues from GD 10 to GD 12, RA did not significantly alter MI and percentage dead cells was significantly increased only at 4 hr postexposure. Some whole embryos were prepared for scanning electron microscopy (SEM). At 48 hr (GD 12) a reduction in the size of the shelves was not apparent on SEM. In the limb buds, RA did not increase percentage dead cells, but MI was significantly decreased. A decreasing rate of proliferation was detected in control facial tissues as development progressed, and this agrees with findings in rat and chick. Thus it appears that mesenchymal cell death and reduced proliferation are not responsible for the small palatal shelves seen on GD 14. RA did not increase cell death but inhibited proliferation in the limb bud, and this effect may contribute to the retarded development and malformations occurring in the limb.     

Antifungal triazole derivative triadimefon induces ectopic maxillary cartilage by altering the morphogenesis of the first branchial arch

BACKGROUND: The triazole derivative, triadimefon (FON), induces branchial arch abnormalities in post-implantation rat embryos cultured in vitro, and cranio-facial malformations in mouse fetuses. Ectopic maxillary cartilage has been also described as a typical FON-related malformation. This work studies the morphogenesis of the ectopic cartilage in rat embryos and fetuses exposed in vivo to FON during the early postimplantation period. METHODS: Pregnant rats were treated with 0, 250, and 500 mg/kg FON on Day 9.5 of pregnancy (D9.5) and sacrificed at term (D20), during the early fetal period (D17) or at different embryogenetic periods (D10, D11, D12). The skeleton was examined after stain of bone and cartilage or of cartilage alone respectively at term or at D17. The neural crest cell (NCC) migration and compaction was investigated at D10 and D11 and the cranial nerve organization described at D12. RESULTS: Triadimefon is teratogenic in rats under the chosen experimental conditions. The malformations were at the level of the cranio-facial and axial skeleton at term and of the hindbrain nerves in embryos. A NCC abnormal migration and compaction was observed at the level of the first branchial arch: in FON-exposed embryos NCC were detected at the level of both maxillary and mandibular processes, whereas control embryos showed the immunostained tissue only at the level of the mandibular bud. CONCLUSIONS: The pathogenic pathway, proposed to explain the ectopic cartilage, is the displacement of part of the NCC-derived tissues at the maxillary region of the first branchial arch.     

Metabolism of very low density lipoproteins in rats with isotretinoin (13-cis retinoic acid)-induced hyperlipidemia

A significant rise in plasma triacylglycerols from the control level of 0.89 mmol/l to 1.88 mmol/l (P less than 0.001) was observed in male Sprague-Dawley rats treated for 11 days with isotretinoin (oral dosing; 10 mg/day). This rise was due to an increased level of plasma very low density lipoproteins (VLDL). When VLDL from untreated rats were labeled with 125I-labeled tyramine-cellobiose and injected intravenously into rats treated for 10 days with isotretinoin (n = 6) and in control rats (n = 6), it was found that the disappearance of radioactivity from the blood was dramatically retarded in the treated animals. The disappearance could be divided into two phases, a rapid (alpha) phase dominated the first 5 min and was followed by a slower (beta) phase. The half-life of the beta-phase increased significantly from 53 +/- 7 min in the controls, to 120 +/- 62 min after isotretinoin. VLDL prepared from isotretinoin-treated animals (n = 6) had about the same half-life in control animals (62 +/- 8 min) as had ordinary VLDL. The elimination of tracer from the blood was mainly due to uptake by the liver. The amount of radioactivity in the liver after 30 min of circulation was significantly reduced from 34 +/- 7% of injected dose in controls to 24 +/- 5% in the isotretinoin group (P = 0.013). The uptake in other organs was less than 3% per organ and was essentially unaffected by the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parameters determining isotretinoin teratogenicity in rat embryo culture

At the in vitro threshold serum concentration of 500 ng/ml, isotretinoin induces defects of visceral arch development in 9.5-day rat embryos grown in culture for 48 h. Experiments were performed to determine the minimum period of exposure necessary to induce these arch defects and whether an increase in concentration of isotretinoin could compensate for reduced exposure time. The results showed that a minimum 6-h exposure to 500 ng/ml immediately prior to cranial neural crest migration was necessary to induce severe defects of the second visceral arch in a majority of embryos. Maximal increase in isotretinoin concentration to 16,000 ng/ml did not compensate for shorter exposure periods. These results suggest that to cause malformations of the visceral arches, the embryo must be exposed to isotretinoin for a minimum period of time regardless of the concentration of isotretinoin above the threshold.     

A quantitative and qualitative study of craniofacial development in the rat embryo aged 302 hours to 374 hours (days 12.5 to 15.5)

One-hour mated Sprague-Dawley rats underwent a normal pregnancy, which was terminated at hours 302, 326, 350, and 374. After appropriate processing and scanning electron microscopy of the embryos, absolute measurements of the craniofacial area, especially of the snout, maxillary process, eye, lower face, and brain were taken in the 302- and 326-hr embryos. Additionally, growth ratios were established in all stages. The greatest growth and developmental changes occurred between 302 and 326 hr, when 1) the snout developed from the nasal anlagen and the maxillary process, 2) the eye lens replaced the eyepit, and 3) the hindlimb developed. Between 326 and 350 hr, the optic pit developed. The results of the present study can markedly enhance precision in interpretation of results of teratological studies in the rat. The detailed quantitative data established allow reporting of subtle departures from the normal hitherto impossible.     

Morphological observations in normal primary palate and cleft lip embryos in the Kyoto collection

Normal developmental events during human primary palate formation and alterations associated with cleft lip remain poorly defined. The purpose of this study was to analyze serially sectioned human embryos to identify morphological changes during normal palatal closure and alterations associated with failure of palatal formation. Normal and cleft embryos from the histological collection at the Congenital Anomaly Research Center at the University of Kyoto were studied and photographed for detailed evaluation. Seven serially sectioned cleft lip embryos of stages shortly after primary palate formation (Streeter-O'Rahilly stages 19, 20, and 22) with unilateral or bilateral clefts with varying degrees of clefting were studied. In the normal Kyoto embryos, initial nasal fin (epithelial seam) formation was observed between the medial nasal process and the lateral nasal and maxillary processes at stage 17. During stages 18 and 19, the nasal fin epithelium was replaced by an enlarging mesenchymal bridge, as the maxillary processes united with the medial nasal processes to form the primary palate. The most prominent features observed in the cleft embryos were a reduced thickness of mesenchymal bridging between the medial nasal and maxillary processes, with an excessive amount of epithelium at the junctions between these processes. With ingrowth of the maxillary processes, greater cell dispersion and apparent extracellular matrix accumulation were observed in the medial nasal region. During closure of the primary palate, terminal branches of the maxillary nerve crossed the mesenchymal bridge to the medial nasal region. The partial clefts had reduced maxillary ingrowth and smaller union areas with the medial nasal process. Detailed studies of experimental animal models are required to identify regional growth required for contact between the facial prominences, to clarify the mechanisms of mesenchymal ingrowth and epithelial displacement during palatal formation, and to identify local and/or general factors causing alterations that lead to primary palatal clefting.     

Ventricular blood pressure and cardiac output changes in epinephrine- and metoprolol-treated chick embryos

The effects of a teratogenic dose (5 micrograms) of epinephrine on mean ventricular blood pressure (MVBP) and cardiac output (CO) at one and two hours after treating stage 24 chick embryos were investigated. Previous work demonstrated that a differential response in terms of cardiac rhythm during the first hour after epinephrine treatment was related to pathogenesis of two contrasting types of aortic arch malformations. Absence of one or more aortic arches occurred more frequently in embryos which developed a characteristic dysrhythmia, while persistence of the left fourth aortic arch (PL4AA) occurred more frequently in nondysrhythmic embryos. In this study, dysrhythmic epinephrine-treated embryos exhibited reductions in both MVBP and CO at one hour after treatment when compared to control values. Nondysrhythmic epinephrine-treated embryos exhibited elevated MVBP and no change in CO at one hour after treatment. MVBP and CO in recovered dysrhythmic and nondysrhythmic embryos were similar to control values at two hours following epinephrine treatment. MVBP and CO measurements were obtained from embryos which were pretreated with metoprolol and then subsequently treated with epinephrine. Metoprolol is a beta 1-adrenoreceptor antagonist which was previously shown to block the teratogenic effects of epinephrine and other catecholamines with beta 1-adrenoreceptor agonist properties. Pretreating embryos with metoprolol in this study reduced the dysrhythmogenic potential of epinephrine and also blocked the MVBP and CO changes observed in embryos treated with epinephrine alone. We conclude that pathogenesis of 1) abnormally absent aortic arches is related to dysrhythmogenesis, reduced MVBP, and reduced CO, and 2) an abnormally persistent left fourth aortic arch is related to elevated MVBP in the epinephrine model.     

Subdivision of mouse vibrissae on an embryological basis, with descriptions of variations in the number and arrangement of sinus hairs and cortical barrels in BALB/c (nu/+; nude, nu/nu) and hairless (hr/hr) strains.

Development of vibrissae was studied in dd/y mouse embryos by scanning electron microscopy. Arrangement of vibrissae and cortical barrels were also studied by light microscopy in adult dd/y, BALB/c(nu/+), nude (BALB/c, nu/nu) and hairless (hr/hr) mice to find genetic or epigenetic variations. Rudiments of vibrissae first appear on Day 12 of pregnancy as longitudinal ridges on the developing muzzle, and each hair rudiment is represented by a dome on the ridges. The dorsal two rows (A and B; Woolsey and Van der Loos, '70) of mystacial vibrissae are on the lateral nasal prominence, while the ventral three (C, D and E) are on the maxillary prominence. Smaller hairs of mystacial vibrissae appear at the labial part of the maxillary prominenceon Day 13. The rudiments of rhinal hairs also appear at this stage on the part of the muzzle derived from the medial nasal prominence. Thus the so-called mystacial vibrissae should be subdivided into three (or 4, including the rhinal) groups on an embryological basis. They are the lateral nasal, the maxillary and the labial. A supernumerary sinus hair and a corresponding barrel was observed between D and C rows uni-or bilaterally in one third of individuals of BALB/c, nude and hairless mice. It is suggested that supernumerary hairs tend to occur between the groups of hairs as defined above. In nude and hairless mice small barrels representing labial hairs are diminished in number. The number of hair follicles, however, is normal.     

Distribution of F-actin during mouse facial morphogenesis and its perturbation with cytochalasin D using whole embryo culture.

Histological and experimental studies were performed in mouse embryos to elucidate possible roles of actin filaments in the nasal epithelium during facial morphogenesis. C57BL/6 mouse embryos (8.5-11.5 days of gestation) were fixed and frozen sections were stained with rhodamine-phalloidin. Before formation of the nasal placode, there was no specific localization of F-actin. After the nasal placode was formed, intense staining of F-actin was observed at the apical side of the placode. Conversely, it was located at the basal side of the epithelium of developing nasal prominences. By using the whole embryo culture system, perturbation experiments were conducted with cytochalasin D (CD), which inhibits the polymerization of actin filaments. When day-10 embryos were exposed to CD at several concentrations for 24 hr, fusion of nasal prominences was inhibited in a dose-dependent manner. Treatment with a high dose of CD for 2 hr also prevented the same development irreversibly. In contrast, when day-9 embryos were exposed to CD at several concentrations for 24 hr, invagination of the nasal placode was not perturbed at all. The results suggest that apical F-actin plays an essential role in maintaining the close apposition state of the nasal prominences and in the following fusion. During the invagination stage, F-actin might be important in maintaining the epithelial structure, but is not crucial to the initiation of placode invagination.     

Developmental deficiencies of the upper facial skeleton due to partial elimination of mesencephalic neural crest cells in the chick embryo

With the aim to test the hypothesis that cells derived from the mesencephalic portion of the neural crest, are involved in the process of differentiation of various upper facial bones, in 41 chick embryos of the 6-somite stage (approx. 26 hours of incubation) the anterior and middle thirds of this part of the neural crest were partially eliminated by micro-laser irradiation, either unilaterally or bilaterally. Of the 14 embryos sacrificed at the age of 12 days, a number of 6 proved to have developed harelip and/or cleft palate conditions. In these embryos, in addition a reduction or absence of the maxillary, palatal, jugale and quadrato-jugale was observed. On the contrary, other facial bones as well as the first and second branchial arch cartilages proved to have developed normally. From these results the conclusion may be drawn that (a sufficient number of) cells from the anterior and middle thirds of the mesencephalic neural crest are indispensable for a normal differentiation of the maxillary, palatal, jugale and quadrato-jugale.     

Teratogenic effects of alcohol and isotretinoin on craniofacial development: an analysis of animal models.

The teratogens alcohol and isotretinoin cause different patterns of facial dysmorphogenesis in the human. For isotretinoin the pattern is consistent with interference with the normal development of the cranial neural crest, particularly that destined for the second visceral arch. In vitro studies in the rat indicate that, at threshold levels of exposure to isotretinoin, the development of the second arch crest represents the most sensitive process of organogenic development. For alcohol, the facial abnormalities result from exposure very early in development, during the gastrulation process. There is no evidence that this is a peculiarly sensitive stage of development with respect to alcohol; animal studies indicate that other processes in the organogenic period are equally or more vulnerable. The emphasis given to the abnormal facial features in the fetal alcohol syndrome is considered a phenomenon associated with the exclusivity of syndromes.     

Alteration of hepatic lipidperoxide levels during cataract formation caused by glucocorticoids in developing chick embryos

The level of hepatic lipidperoxides in chick embryos was determined during cataract formation resulting from glucocorticoid treatment. When 15 day old chick embryos were administered 0.25 mumol of hydrocortisone acetate their hepatic lipidperoxide level, determined by thio-barbituric acid, increased after a lag time of 20 hr and reached approximately 8-fold of control at 48 hr after the treatment. These studies indicate that the peroxidation of lipid in tissues should be considered in elucidating mechanisms of action or adverse effects of glucocorticoids.     

Retinoic Acid Treatment Induces Cell Death and the Protein Expression of Retinoic Acid Receptor β in the Mesenchymal Cells of Mouse Facial Primordia in Vitro

We isolated mesenchymal cells from individual facial primordia of mouse embryos on 11 days post coitum and examined the effects of retinoic acid (RA) on chondrogenesis, induction of cell death, and the protein expression of retinoic acid receptor (RAR) β and γ in micromass culture. Under the control condition, cells of both medial and lateral nasal prominences (MNP and LNP) displayed high chondrogenic potential, while those of maxillary and mandibular prominences (Mx and Md) had constant growth activity and low chondrogenic potential. Though none of the cells expressed detectable levels of the RAR β protein, RAR γ was expressed in the cells of all the facial primordia. One μM RA inhibited the chondrogenesis, and induced cell death accompanied with the induction of the RAR β protein in LNP, MX and Md cells within 6 hr. On the contrary, both cell death and RAR β protein induction were detected in the MNP cells treated with RA for 24 hr. These results suggest that the RAR β is involved in the process of the cell death induced by the RA treatment in the mesenchymal cells of the mouse facial primordia.     

Nasal pit formation in the hamster: a transmission and scanning electron microscopic study

The surface morphology of cells comprising the nasal placode and adjacent body surface was examined by transmission and scanning electron microscopy during nasal pit formation in hamster embryos ranging in age from $\text{8}\text{1}\text{4}$ to $\text{9}\text{1}\text{2}$ days post coitum. A sharp distinction between the apical surface appearance of cells of the nasal epithelium and cells of the surrounding periderm develops at the periphery of the nasal placode. Periderm cells increase in surface area, exhibit a change in the distribution of surface microvilli, and many acquire a single cilium per cell. Cells of the nasal placode retain a dense surface coat of microvilli and exhibit relatively smaller apical surface areas. Olfactory rods can be positively identified on the basis of their ultrastructure at the nasal groove stage. The ventral margin of the nasal groove does not initially depend on the maxillary process, but is bounded by the lateral and medial nasal processes. From their earliest development the oral and nasal cavities appear to be separated in this species.     

Acrylonitrile interaction with testicular DNA in rats

In the present study we report the in vivo interaction of acrylonitrile (VCN) with testicular tissue in rats. Covalent binding of radioactivity to testicular tissue DNA was examined for a period of 72 hr after a single oral dose (46.5 mg/kg) of [2, 3-¹⁴C] VCN. Maximal covalent binding was observed at 0.5 hr (8.9 μmol VCN equivalent/mol nucleotide). Binding decreased gradually thereafter but was still detected (2.5 μmol VCN equivalent/mol nucleotide) at 72 hr following VCN administration. Further, we examined the effects of VCN on DNA synthesis and repair in the testes of rats following a single oral dose (46.5 mg/kg) of VCN to clarify the impact of the covalent binding observed on the testicular genetic material. A significant decrease in DNA synthesis (80% of control) was observed at 0.5 hr after treatment. At 24 hr following acrylonitrile administration, testicular DNA synthesis was severely inhibited (38% of control). Testicular DNA repair was increased 1.5-fold at 0.5 hr and more than 3.3-fold at 24 hr following treatment with VCN. These results suggest that VCN can act as a multipotent genotoxic agent by alkylating DNA in testicular tissue and may affect the male reproductive function by interfering with testicular DNA synthesis and repair processes.     

Retinoic acid inhibits migration of cranial neural crest cells in the cultured mouse embryo

Clinical observations have demonstrated that isotretinoin (13-cis-retinoic acid; cis-RA) is a human teratogen causing primarily heart and craniofacial malformations. Isotretinoin exposure to the early postimplantation mouse embryo in culture results in specific defects in craniofacial development that may be due to an interference in the early migration of cranial neural crest (CNC) cells [Goulding and Pratt, 1986]. The present study was designed to test this hypothesis by examining the migration of these cells in whole embryo culture. Day 8 CD-1 mouse embryos were cultured for 6-48 hr in the presence or absence of cis-RA at 2 X 10(-6) to 2 X 10(-5) M. Embryos either were fixed for light microscopy using Nichols' method for localization of CNC cells or were processed for scanning and transmission electron microscopy. At the light microscopic level, CNC cells in the mid-brain region of control embryos had migrated to the region of the first and second visceral arches after 6 hr in culture. Cis-RA interfered with this migration; CNC cells in treated embryos either did not leave the neuroepithelium (NE) or were aggregated near the NE. Autoradiographic studies indicated that cis-RA did not affect the overall viability or DNA synthesis of the CNC cells. However, at the TEM level, there was a dramatic increase in the number of cellular blebs in the CNC cells. Our results demonstrate a direct effect of 13-cis-RA on the CNC cells and suggest that this effect is due to alterations in the cell surface.