Sites of aspecific ATP-phosphohydrolase activity in the branchial region of chick embryos, with particular emphasis on the myogenic cell population.

The distribution pattern of unspecific ATP-phosphohydrolase (ATP-PH) activity was studied in serial sections through the branchial region of chick embryos from HH stage 14 to 27. A selective reaction was found in the prospective myogenic mesoderm of voluntary muscles of the first three branchial arches. This property initially characterized uncondensed myogenic cell populations in the mandibular and hyoid arches and appeared later on in the third arch. In each case, the positive premuscular mesoderm was closely apposed to the lateral wall of the corresponding aortic arch. Myogenic primordia retained a strong ATP-PH activity up to stage 27 when they began to segregate into smaller premuscular units. Discrete changes observed in the mandibular and hyoid blastemas suggested that non reacting cells probably of neural crest origin invade their proximal core and contribute to the subsequent pathfinding of muscular nerves. Other selective sites of ATP-PH reactivity were detected in the odd-numbered rhombomeres, rhombencephalic floorplate, notochord, arterial endothelial cells, prospective dermis and perichondrium.

A comparative review of the sites of histoenzymatic dephosphorylating activities and of gene expression in developing limb buds of mammalian and avian species.

The sites of dephosphorylating activities histochemically demonstrated in developing limb buds of mammalian and avian species have been reviewed and compared with the pattern of gene expression or of other biochemical properties reported at similar stages in the corresponding sites. Alkaline phosphatase, acid phosphatase, 5' nucleotidase and ATP-phosphohydrolase reactions were studied in mouse, rat and chick embryos. Alkaline phosphatase only was detected in mole limb buds whereas only 5' nucleotidase and ATP-phosphohydrolase were revealed in limb rudiments of macacus rhesus embryos. Five decisive periods or events of limb morphogenesis have been considered successively: (1) the early stages during which the prospective limb constituents acquire limb forming properties and give rise to the young limb buds, (2) the invasion of the limb bud mesoderm by myogenic cells of somitic origin, (3) the ectoderm-mesoderm interactions with particular emphasis on the properties displayed by the apical ectodermal ridge and by the underlying subridge mesoderm of the progress zone, (4) the period of growth and pattern formation along the proximo-distal, anterior-posterior and dorso-ventral axes, with special attention to the properties of the zone of polarizing activity, and (5) the period of tissular predifferentiation particularly as concerns prospective skeletal, musculo-tendinous and connective tissues, with brief comments about growing nerves and blood vessels. At least during the morphogenetic period, most dephosphorylating properties appear independently associated with gene expression or other regional biochemical properties. Many sites of dephosphorylating activity may therefore be considered as interesting markers of ungoing morphogenetic events among which tissue interaction and signalling are frequently concerned.

Further analysis of developmental potentialities expressed by the footplate of 11-day mouse embryo hindlimb buds explanted in organ culture.

The developmental potentialities expressed in vitro by hindlimb buds or excised footplates of 11-day mouse embryos have been analyzed using different kinds of organ culture procedures. As compared with unsatisfactory results obtained with explants transferred in submerged culture, others cultured on Nuclepore membranes following the Trowell technique exhibited growth and pattern formation performances which could be correlated with the CR length of their donor embryos. According to the case, their distal segment appeared hypotrophic and strongly necrotic, triangular shaped with 2 or 3 converging digital rays, four toed shaped with 3 or 4 digital rays, and pentadactylous. Insufficient distal morphogenesis observed in younger limb buds could be related to selective cell death in the A.E.R. and subridge mesoderm, as well as to early dilation of the marginal vein. Prospective area of digit 1 proved to be particularly sensitive to culture conditions. However, observations made in explants excised from potentially polydactylous embryos showed that the X-linked polydactyly (Xpl) phenotype can be expressed in vitro. Three technical variants contributed to improve the quality of pattern formation in the distal segment: the use of collagen-coated nuclepore membranes and the addition of bFGF or TGF-beta in the culture medium. The presence of bFGF seems to provide a protection against mesodermal cell death; that of TGF-beta appeared to promote digital outgrowth. The preskeletal pattern achieved in cultured footplates was in good agreement with maps of prospective areas previously established for the mouse hindlimb bud.

Abnormal cutaneous nerve outgrowth: a secondary effect of methyl triazene teratology in developing mouse limb buds.

The cutaneous branches originating from the superficial distal division of the ulnar nerve exhibit abnormal developmental features in 70.2% of the forelimb buds from embryos submitted 2 or 3 days earlier to methyl triazene administered to their pregnant mother. Similar abnormalities characterize the preaxial ventral cutaneous nerve of the thumb in 17.8% of forelimb primordia. The affected nerves undergo anticipated growth with respect to the normal schedule, follow abnormal pathways through areas of extensive cell death, and finally reach the apical ectoderm where they run in close contact with the basement membrane without forming a plexus. Histological observations gathered in pyronin-methyl green stained serial sections as well as in whole limb buds after cholinesterase method suggest that three factors probably contribute to modify nerve outgrowth: (1) a discrepancy between the rate of nerve progression and that of mesodermal growth in the prospective zeugopod territory which is preferentially affected by the teratogen; (2) facilitated nerve pathfinding into areas strongly hit by triazene-induced mesodermal cell death; and (3) alteration or abolition of some unknown ectodermal influence necessary to stimulate selective guidance of terminal sensory afferents and to maintain them transiently at some distance from the epidermis.

Histological analysis of limb defects induced in developing limb buds of NMRI mouse embryos after oral administration of 3-3-dimethyl-1-phenyltriazene (DMPT) to their mother on day 10 of gestation.

A single oral dose of DMPT was given to pregnant NMRI mice on day 10 of gestation and the subsequent histological changes were studied in serial sections of affected limb buds isolated from 13-, 12- and 11-day treated embryos. Differences in abnormal skeletal patterns observed between fore- and hindlimb buds as well as between embryos from different litters provided clear evidence that the teratogen hits preferentially undifferentiated preskeletal mesoderm just before blastema formation. Absence or severe reduction of skeletal rudiments characterizes selectively the girdle and stylopod of hindlimbs and the zeugopod and autopod in forelimbs. In embryos slightly more advanced at the time of drug administration, the defects shifted in the zeugopod and distal segment of the posterior limb and in the distal segment only of the anterior limbs. In all cases, defects in the two distal segments displayed a postaxial predominance. Extensive cell death detected in the undifferentiated mesoderm of the affected limb parts of 11-day embryos similarly exhibited a postaxial predominance with the maximal damage in the ZPA territory. Together with the regular genesis of a postaxial subectodermal bleb in that area, followed by local involution of the AER, this observation strongly suggests that the teratogenic injury might involve an early impairment of ZPA and AER properties. In addition, predictive signs of hyperphalangy of digit I and distal duplication of the IId to e could be correlated with a transient reactional hyperplasia restricted to the preaxial part of the AER.

Fetal esophagus: normal sonographic appearance.

Our objective was to describe the sonographic appearance of the normal fetal esophagus and to determine the rate of its visualization. The fetal esophagus has a tubular echogenic appearance. Two patterns of the tubular structure were observed: two parallel echogenic lines or several parallel echogenic lines (the so-called "multilayered pattern"). A cannula was placed in the stomach of three stillborns and removed under ultrasonographic monitoring, confirming its location. In 155 consecutive pregnancies examined, the thoracic part of the fetal esophagus was easy to visualize (about 90% of cases); the cervical and abdominal segments were more difficult to visualize (19 and 30%, respectively). The multilayered pattern was observed in 49% of cases before and in 87% of cases after 26 weeks' gestation. A direct sonographic demonstration of the fetal esophagus is possible and could help to exclude esophageal malformations.

A new interpretation of the necrotic changes occurring in the developing limb bud paddle of mouse embryos based upon recent observations in four different phenotypes.

Degenerative changes occurring in the apical ectodermal ridge (a.e.r.) and undifferentiated distal mesoderm of developing limb buds were studied macro- and microscopically in day-11 to day-13 mouse embryos displaying the normal (+/+), oligosyndactylous (Os/+), polydactylous (Xpl/+) and hybrid (Os/+/Xpl/+) phenotypes. Isolated limb buds were submitted either to supravital staining with Nile blue sulfate or to lectin binding staining in serial paraffin sections, taking advantage of strong binding affinites of macrophage cells for peanut agglutinin after neuraminidase treatment and for ricinus communis agglutinin. Necrotic changes detected in three definite areas of the distal mesoderm of normal limb buds exhibit characteristic spatial temporal relationships with earlier cytolytic changes affecting the pre- and postaxial parts of the a.e.r. Two of them, known as the primary preaxial site (fpp) and the anterior marginal necrotic zone (AMNZ) appeared deeply modified in mutant embryos as compared to the posterior marginal necrotic zone (PMNZ) which remained unaffected. Macrophage cells loaded with cell debris appear in advance and in excessive number in the fpp of Os/+ limb buds. Conversely, they were found absent or locally reduced in number in the fpp and AMNZ of Xpl/+ limb buds which otherwise develop in the same area a preaxial protrusion covered with a healthy portion of the a.e.r. Hybrid Os/+/Xpl/+ limb buds expressing both mutant genes develop a smaller and macrophage-free preaxial protrusion which coexists with residual and locally excessive necrotic changes in its immediate surrounding and is covered with a normally necrotic portion of the a.e.r. Microscopic observations collected in the limb buds of all phenotypes, though more frequently in Os/+ limb buds, strongly suggest that in all three necrotic sites examined, macrophage cells of vascular origin somehow contribute to the clearance of ectodermal necrotic debris and eventually return in the blood stream through the marginal vein and its affluents.

[Teratogenic effects of acetazolamide in mouse embryos]. / Les effets tératogènes de l'acétazolamide chez l'embryon de souris.

The observations gathered in mouse embryos collected 4, 24 and 48 hours after the administration of a teratogenic doses of acetazolamide to their pregnant mothers strongly suggest that the resulting postaxial defects in the anterior limbs can be the result of a selective perturbation of the inductive process responsible for the genesis of the apical ectodermal ridge, probably secondary to a transient acidosis. The vascular stasis provoked by the treatment provides an explanation for the selective localization of the injury in the forelimb buds and even for its preferential occurrence on the right side.

Lectin binding sites in developing mouse limb buds.

The binding sites of the following biotinylated lectins were demonstrated in serial paraffin sections of fore- and hindlimb buds from day-9 to day-16 mouse embryos with the Avidin-Biotin-Peroxidase Complex (ABC) procedure: Concanavalin A (Con A), Soybean Agglutinin (SBA), Wheat Germ Agglutinin (WGA), Peanut Agglutinin (PNA), Ricinus Communis Agglutinin I (RCA), Ulex Europaeus I Agglutinin (UEA), and Dolichos Biflorus Agglutinin (DBA). Alternating neighbouring sections were used to compare the distribution of PNA staining, PNA staining after neuraminidase treatment (N-PNA) and the autoradiographic sites of [35S]-sulphate uptake. Unspecific binding sites common to all lectins tested were observed in periderm and chondrocytes. Several lectin affinities were seen in the undifferentiated mesoderm (Con A, WGA, RCA), blood vessels (WGA, PNA, N-PNA, RCA, UEA, DBA) and macrophages (Con A, WGA, N-PNA, RCA). A very selective and mainly extracellular affinity to N-PNA was demonstrated in the condensed preskeletal mesoderm, where it characterizes indistinct prospective chondrogenic, perichondral and pre-articular areas. Comparison with the distribution pattern of [35S]-sulphate uptake and other previously published histochemical data suggests that N-PNA staining occurs at the late blastema stage, i.e. after the stage of cell condensation and before the earliest deposit of stainable matrix in chondrogenic areas. This property later disappears from the chondrifying rudiments, and is maintained in perichondral and pre-articular tissues. Surprisingly, only the pre-articular areas bind PNA without pretreatment with neuraminidase. A transient RCA binding probably related to terminal morphogenesis was detected in the undifferentiated distal part of the predigital columns of day-12 and day-13 limb buds. From the day-13 stage onwards, diverse new lectin affinities appeared in differentiating tissues, such as pretendinous rudiments, perichondrium and prospective periosteum, muscular connective tissue, myotubes, superficial fasciae and prospective dermis. A strong SBA and PNA staining was also detected in the extracellular matrix associated with the epithelial septa separating the roots of the digits in day-15 and day-16 limb buds.

Interactions between chronic murine Trypanosoma cruzi infection and pregnancy: fetal growth retardation.

Fetal growth, reproductive capacity, and parasitemia were studied in three groups of BALB/c mice: pregnant and chronically infected with Trypanosoma cruzi, non-pregnant but similarly infected, and pregnant but noninfected. The pregnant mice were killed on day 17 of pregnancy. Comparisons of the two pregnant groups showed significant differences in fetal weights and x18 magnified ossification lengths of radius and cubitus, whereas placental weights were not modified. The results indicate that intrauterine growth retardation occurs during chronic murine T. cruzi infection. No difference was noted between the reproductive capacities of the two pregnant groups. Parasitemias were similar in infected pregnant and control groups. Mice of all groups survived infection until killing. Pregnancy, therefore, does not influence chronic murine T. cruzi infection. Parasites were never found in fetal blood, indicating a very low, if any, frequency of transplacental transmission of parasite during the chronic phase of infection.

[Echographic evaluation of non-immunologic feto-placental anasarca. A series of 24 cases]. / Evaluation échographique des anasarques foeto-placentaires non immunitaires. Une série de 24 cas.

24 cases of non-immune feto-placental hydrops which were diagnosed by ultrasonography are presented. The precise etiology was possible in 21 of the cases using chromosome analyses, radiological assessments and carefully conducted post mortem examinations. The cause was maternal in 3 cases and due to fetal pathology in 18 cases, of which 5 were due to chromosome abnormalities. Three recessive autosomal syndrome, 9 malformations and in particular cardiac malformations and one twin pregnancy with transfusion from one fetus to the other were diagnosed. These features, which can be compared with those already in the literature, show that most of these cases of hydrops are due to genetic fetal defects and demonstrate how important it is to carry out chromosome analyses and systematic anatomo-pathological investigations. The results of these tests will indicate the way the doctors should look after the mother during pregnancy and how to deliver these mothers in subsequent pregnancies.

Diaminobenzidine histochemistry in the rat limb bud.

Diaminobenzidine (DAB) histochemistry was used to determine enzymatic or non enzymatic peroxidase activity in the rat limb bud on days 12 and 13 of gestation. The optimal technical conditions were determined. Light microscopy revealed the presence of positive intracellular granules in the apical ectodermal ridge (A.E.R.) and in the condensed mesoderm. Their distribution in the A.E.R. shows a caudo-cephalic gradient. Electron microscopy, red oil red, and supravital Nile blue staining suggest the lipidic nature of these granules. The hypothesis of an endogenous pseudo- or peroxidase activity are discussed.

Histological changes induced in developing limb buds of C57BL mouse embryos submitted in utero to the combined influence of acetazolamide and cadmium sulphate.

Microscopic defects in limb buds of C57BL mouse embryos after the combined teratogenic action of acetazolamide plus cadmium sulphate administered on day 9 of gestation were studied in serial sections. Postaxial deficiencies observed in 12-15-day embryos and affecting preferentially the right forelimbs were classified in nine morphological types according to increasing amounts of missing parts. Type X defect consists of a nearly complete amelia in which all four limbs are represented only by the girdle and proximal end of the stylopod. Type XI abnormality appears as an intermediate reduction affecting the area of digit IV. In addition to modifications of the forelimb bud shape detected from the 10-day stage onwards, observations made 24 and 48 hr after treatment confirmed that the postaxial defects result from an absolute lack of postaxial mesoderm occurring without cell necrosis as a consequence of a postaxial shortening of the apical ectodermal ridge (aer). In 10-day embryos, the latter appears shortened and hypertrophied; it is later fragmented into alternate thick and thin portions in 11-day affected limb buds. These ectodermal changes might account for the genesis of all types of defects observed. Untreated 9-day embryos with 12-25 pairs of somites display a number of asymmetries between their right and left forelimb territories: Until the 19-somite stage, the vascular supply to that area is provided exclusively by the umbilical vein, which is larger on the right side; the initial amount of somatopleural limb mesoderm is greater in the right rudiment and the genesis of its aer is slightly protracted as compared to the left one. These asymmetries might contribute to the right side predominance of the forelimb defects induced by acetazolamide and cadmium.

Histogenesis in 11-day mouse embryo limb buds explanted in organ culture.

Fore- and hindlimb buds from 11-day mouse embryos with 40 to 52 somites (including the four occipital pairs) were explanted in organ culture and submitted to systematic histological analysis. Chondrogenesis occurs normally in culture in all preskeletal rudiments which were already represented by condensed blastemas before explantation. In the proximal territories, the progress of cartilage differentiation occurs according to the normal pattern and can be revealed histologically much earlier than in bulk preparations. In all explanted hindlimbs as well as in forelimbs from embryos with less than 50 somites, a primary coalescence occurs between the IId and IIId digital rays, leading to various fusions from soft tissue syndactyly to oligosyndactyly. This is the result of two combined unfavorable effects of the culture conditions: the lack of simultaneous volumetric growth of the foot- or handplate, which normally would provide the necessary space for the laying down of a pentadactylous pattern, and a loss of cells resulting from abnormal cell death affecting selective mesodermal sites in the zeugopod and in the marginal subridge area, the latter being more severely affected in hindlimb buds. Several observations suggest that the preferential sensitivity of the marginal mesoderm might be related to early changes in the apical ectoderm, which itself becomes excessively necrotic and rapidly looses its pseudostratified configuration. The forelimb buds from embryos with 50 somites and more usually develop a pentadactylous pattern with a better individuation of digital structures. In all explants, the prospective mesoderm of digit I exhibits stronger regulatory tendencies.

Lethal multiple pterygium syndrome.

We report on two fetuses with a lethal form of multiple-pterygium syndrome born to first cousins. The two pregnancies aborted spontaneously in the 2nd trimester, the fetuses, 10-11 weeks of embryonic age, showing multiple pterygia and multiple cartilaginous fusions. One had cleft lip and palate. In both cases the microscopic anatomy of the placenta showed villi with scalloped border and intravillous trophoblastic invaginations.