Adenosine receptor antagonists including caffeine alter fetal brain development in mice.

Consumption of certain substances during pregnancy can interfere with brain development, leading to deleterious long-term neurological and cognitive impairments in offspring. To test whether modulators of adenosine receptors affect neural development, we exposed mouse dams to a subtype-selective adenosine type 2A receptor (A2AR) antagonist or to caffeine, a naturally occurring adenosine receptor antagonist, during pregnancy and lactation. We observed delayed migration and insertion of γ-aminobutyric acid (GABA) neurons into the hippocampal circuitry during the first postnatal week in offspring of dams treated with the A2AR antagonist or caffeine. This was associated with increased neuronal network excitability and increased susceptibility to seizures in response to a seizure-inducing agent. Adult offspring of mouse dams exposed to A2AR antagonists during pregnancy and lactation displayed loss of hippocampal GABA neurons and some cognitive deficits. These results demonstrate that exposure to A2AR antagonists including caffeine during pregnancy and lactation in rodents may have adverse effects on the neural development of their offspring.

Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses.

Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor.

A cross-species analysis of Satb2 expression suggests deep conservation across vertebrate lineages.

Mutation of SATB2 causes cleft palate in humans. To understand the role of SATB2 function in palatogenesis, SATB2 analyses in vertebrate model systems will be essential. To facilitate these analyses, we have performed a cross-species comparison of SATB2 structure and function across three vertebrate model systems: mouse, chick, and zebrafish. We find that the SATB2 transcript is highly conserved across human, mouse, chick, and zebrafish, especially within the Satb2 functional domains. Furthermore, our expression analyses demonstrate that SATB2 is likely to have similar functions in vertebrate model organisms and humans during development of the facial processes and secondary palate. Together, these data suggest an evolutionary conserved role for SATB2 during development of the face and palate across vertebrates. Moreover, expression of zebrafish satb2 in the anterior neurocranium supports the utility of the anterior neurocranium as a simplified model of amniote palatogenesis.

The anthropoid postcranial axial skeleton: comments on development, variation, and evolution.

Within-species phenotypic variation is the raw material on which natural selection acts to shape evolutionary change, and understanding more about the developmental genetics of intraspecific as well as interspecific phenotypic variation is an important component of the Evo-Devo agenda. The axial skeleton is a useful system to analyze from such a perspective. Its development is increasingly well understood, and between-species differences in functionally important developmental parameters are well documented. I present data on intraspecific variation in the axial postcranial skeleton of some Primates, including hominoids (apes and humans). Hominoid species are particularly valuable, because counts of total numbers of vertebrae, and hence original somite numbers, are available for large samples. Evolutionary changes in the axial skeleton of various primate lineages, including bipedal humans, are reviewed, and hypotheses presented to explain the changes in terms of developmental genetics. Further relevant experiments on model organisms are suggested in order to explore more fully the differences in developmental processes between primate species, and hence to test these hypotheses.

Mitochondrial ultrastructure in embryos after implantation.

Information on the morphology of mitochondria during embryogenesis is scattered in the literature, but there appears to be a consistent pattern. During early organogenesis, the embryo is in a state of relative hypoxia associated with a major decrease in terminal electron transport system activity and a marked increase in anaerobic glycolysis. Ultrastructural studies of a 14-somite monkey embryo and day 10 and 12 rat embryos, together with a review of the literature, led us to determine that this hypoxic stage is characterized by vesiculation of the mitochondrial inner membranes, or cristae. Starting in the late morula stage and continuing during early postimplantation embryogenesis, the cristae increase but appear tubular or vesicular. After the end of neurulation, and with the onset of vascular perfusion of embryonic tissues, the cristae gradually become lamellated; by the limb bud stage they appear more mature. We suggest that new cristae derive from blebs of the inner mitochondrial membrane and that with maturation these blebs collapse, giving them a lamelliform appearance. The delamellated state of the cristae might inactivate oxidative phosphorylation to protect the embryo from toxic respiratory end-products that could accumulate in an embryo before there is vascular perfusion. Consistent with this hypothesis, mitochondrial diameters in the developing heart of monkey and rat embryos were approximately twice those found in skin and neural tube.

Developmental changes in the fibre composition of elbow, knee, and ankle extensor muscles in cercopithecid monkeys.

Developmental changes of muscle fibre composition in the various heads of the elbow, knee, and ankle extensors have been studied in three genera of cercopithecid monkeys. In order to circumvent the technical hindrances of usual histoenzymological procedures (fresh muscles need to be frozen at once at -80 degrees C), immunofluorescence methods were used and technical adjustments were successfully carried out to make the study of formaldehyde-preserved muscles possible. Clear responses to antibodies against adult fast myosin in newborn macaques demonstrated that, at birth, adult myosins have already replaced the fetal isoforms, thus providing a reliable marker for the study of postnatal evolution of the muscle fibre composition. For each one of the three joints, from birth to adulthood, the percentage of slow, fatigue-resistant fibres increases only in that head of the extensor muscle groups which is specialized in maintaining posture by counteracting gravity (the 'postural' head). Hence, the question is raised of the relationships between such cytological evolution, developmental changes in postural behaviour, and body weight increase.

Growth cone distribution patterns in the optic nerve of fetal monkeys: implications for mechanisms of axon guidance.

The distribution of growth cones was studied in the optic nerve of monkeys during the first half of prenatal development using quantitative electron microscopic methods. Our aim was to test the hypothesis that ganglion cell growth cones extend predominantly along the surfaces of the nerve, just beneath the pia mater. A complete census of growth cones in cross sections of the nerve during the early phase of axon ingrowth, from embryonic day 39 (E39) to E41, demonstrates that growth cones are scattered within the majority of fascicles, even those located far from the surface of the nerve. By E45, growth cones are concentrated around the nasal, dorsal, and ventral edge of the optic nerve. They are less concentrated in the core and around the temporal edge. However, even as late as E49, virtually all fascicles in the nerve, whether deep or superficial, contain growth cones. Growth cones are dispersed within single fascicles and are often located far from glia. Thus, the newest fibers penetrate deep parts of the pathway and push through centers of densely packed bundles of older axons. This finding is consistent with the vagrant paths of growing axons reported in previous work on embryonic monkey optic nerve (Williams and Rakic, 1985). Our data challenge the hypotheses that growth cones extend selectively along the basal lamina, the pia mater, or glial end feet. Gradients found at later stages of development in the nerve are not due to a particular affinity of growth cones for non-neuronal substrata. The pattern we observed is much more likely to result from central-to-peripheral gradients in ganglion cell generation and possible associations between growth cones originating from the same regions of the retina.

Variation in the pattern of cranial venous sinuses and hominid phylogeny.

In 1967 Tobias noted that Australopithecus boisei cranium O.H.5 exhibited a cranial venous sinus pattern in which the occipital sinus and the marginal sinuses of the foramen magnum appeared to have replaced the transverse-sigmoid sinuses as the major venous outflow track. Specimens of A. robustus and several more recently recovered A. boisei crania also show evidence of enlarged occipital-marginal sinuses. In contrast, A. africanus and H. habilis retain a dominant transverse-sigmoid system that characterizes the great majority of extant apes and modern human cadaver samples. Pliocene A. afarensis exhibits a high frequency of occipital-marginal drainage systems. An examination of several series of precontact North American Indian crania shows that the frequency distribution of the occipital-marginal sinus pattern is spatiotemporally disjunct , ranging from 7.5% to 28%. The Late Pleistocene sample from P redmost , Czechoslovakia, also shows a very high incidence of occipital-marginal sinus patterns (approximately 45%). These observations suggest that occipital-marginal and transverse-sigmoid sinus patterns are adaptively equivalent character states. This conclusion is supported by the fact that enlarged occipital-marginal and transverse-sigmoid sinus systems often coexist on the same and/or contralateral sides of the head. It is well known that the frequencies of such adaptively neutral traits are often heavily influenced by population-specific epistatic interactions. The utilization of such traits in phylogenetic reconstruction entails a substantial risk of mistaking parallelism for synapomorphy . It is concluded that using functional-adaptive criteria in the definition of morphologic characters is a more reliable method to guide phylogeny reconstruction. In light of this, the distribution of venous sinus variants in Plio -Pleistocene hominids gives little or no basis for revising the phylogenetic scheme of Johanson and White (1979), or the functional-adaptive interpretation offered by White et al. (1981).

Experimental alteration of fetal growth in animals.

I have reviewed some of the experimental data related to nutritional and vascular experiments designed to produce intrauterine growth retardation in animals. While considerable effect of maternal nutritional deprivation can be seen in lower mammals, these effects tend to diminish or completely disappear in the nonhuman primate and possibly in the human. This does not mean that there are not nutritional influences that can be measured in the offspring, but that there appears to be a larger reserve in the higher order species. It is likely that the final common pathway for acquired intrauterine growth retardation is related to a compromise in the vascular supply at the uterine, placental, or umbilical levels resulting in a net decrease in the transport of oxygen as well as nutrients. Certain aspects of each of these experimental animal preparations such as the timing of the brain growth spurt in relation to the insult; the number of fetuses in the litter; the relative variability in growth parameters; the capacity for recovery and the specific end points examined, will influence the interpretation of results and allow for cautious extrapolation to the human problem of altered fetal growth. With the improvement in experimental techniques and equipment there should be more information forthcoming on blood flow studies and nutrient transport in a variety of chronic animal preparations.

The prenatal development of the cat's retinogeniculate pathway.

The prenatal development of connections between the retina and the lateral geniculate nucleus (LGN) was studied by means of the anterograde axonal transport of 3H-amino acids or horseradish peroxidase injected intraocularly in fetal cats older than embryonic day 27 (E27) and in newborn cats. (Gestation is 65 days.) A retinothalamic pathway exists as early as E28, when label can be seen in both ipsilateral and contralateral optic tracts. Afferents from the contralateral eye are the first to invade the anlage of the LGN by E32 with those from the ipsilateral eye following about 3 days later. Initially, the pattern of labeling within the nucleus is uniform, suggesting that the two sets of afferents must share a good deal of territory at early ages. By E47, however, gaps appear in the labeling pattern contralaterally, indicating that afferents from the two eyes are beginning to segregate from each other. Segregation continues so that by E54 it is possible to identify unambiguously regions of the LGN destined to comprise ipsilateral and contralateral eye layers. By birth, afferent input appears adult-like in organization, with the two sets of afferents almost completely segregated from each other into their appropriate layers. Cellular lamination of the nucleus has just commenced, however, thereby lagging the onset of afferent segregation by about 2 weeks. Prenatal development could be followed much more easily in the horizontal than in the coronal plane of section due to the finding here that the LGN is displaced approximately 90 degrees in the horizontal plane between E40 and E60. Measurements of the area occupied by the ipsilateral and contralateral afferents within the LGN indicated that even prior to segregation, the two sets of afferents are not completely intermixed within the LGN. On the contrary, those from the contralateral eye retain almost exclusive control of some territory throughout development. This detail contrasts with development in primates, in which intermixing of afferents from the two eyes is thought to be complete early on (Rakic, P. (1976) Nature 261: 467-471). Nevertheless, in the cat, as in other mammals, development of the retinogeniculate pathway is broadly characterized by an initial period of overlap followed by a period of segregation that gives rise to the adult pattern of afferent input.

Fertilization in vitro of Saimiri sciureus follicular oocytes.

An in vitro fertilization system was developed for the squirrel monkey. Oocytes recovered from follicles of gonadotropin-treated females and cultured in vitro with spermatozoa resulted in fertilized ova capable of developing to the four-cell stage in culture. In total, 264 follicular oocytes were recovered from 922 follicles in 38 Saimiri sciureus. Seventy-nine developed to the first polar body stage, and 32 of the 79 were fertilized in vitro.