Search

The germinal zones of the embryonic macaque neocortex comprise the ventricular zone (VZ) and the subventricular zone (SVZ). The mammalian SVZ is subdivided into an inner SVZ and an outer SVZ, with the outer SVZ being particularly large in primates. The existence of distinct precursor cell types in the neocortical proliferative zones was inferred over 100 years ago and recent evidence supports this concept. Precursor cells exhibiting diverse morphologies, patterns of transcription factor expression, and fate potential have been identified in the neocortical proliferative zones. Neurogenic precursor cells are thought to exhibit characteristics of glial cells, but the existence of neurogenic precursor cells that do not share glial specific properties has also been proposed. Therefore, one question that remains is whether neural precursor cells in the prenatal neocortex belong within the astroglial cell class, as they do in neurogenic regions of the adult neocortex, or instead include a diverse collection of precursor cells belonging to distinct cell classes. We examined the expression of astroglial markers by mitotic precursor cells in the telencephalon of prenatal macaque and human. We show that in the dorsal neocortex all mitotic cells at the surface of the ventricle, and all Pax6+ and Tbr2+ mitotic cells in the proliferative zones, express the astroglial marker GFAP. The majority of mitotic cells undergoing division away from the ventricle express GFAP, and many of the GFAP-negative mitoses express markers of cells derived from the ventral telencephalon or extracortical sites. In contrast, a markedly lower proportion of precursor cells express GFAP in the ganglionic eminence. In conclusion, we propose that the heterogeneity of neural precursor cells in the dorsal cerebral cortex develops within the GFAP+ astroglial cell class.

Morphological analyses of zonule conjugated with lens capsule were performed on the developmental change in eyes from the age of fetus to 7 years old of the rhesus macaques (Macaca fuscata). The zonule was filamentous network in late fetus. After birth, the zonular microfibrils originated from the nonpigmented epithelium of the ciliary process. On the extending path toward the lens capsule through the chamber, microfibril assembled with neighbor fibril and also cohered with one another forming bundle. With growth, these bundles bifurcated into anterior and posterior groups on the equatorial region of capsule. The developmental distribution of bundles in the capsule was characteristic on anterior group, that is, bifurcation into radial and circumferential extension. On the other hand, the posterior bundle undivided but radially extended within short distance from the equator. In the process of fixating with capsule, bundles untangled into fibrils and penetrated circumferentially into the superficial layer and radially into deep apical layer of the capsule. Zonule was composed fibrillin 1 microfibrils and on the extending path toward the lens capsule through the chamber, microfibril self-assembled with neighbored fibril in composition of fascicle and also cohered with one another forming bundle. Each bundle had alternating pale and dense horizontal bands in the intracapsular extension and the stripe pattern changed in flaccid or extensive tension of zonule between capsule and process. Zonular fibril intermingled with collagen fibril of capsule with interlacing molecule of laminin. At the base of ciliary muscle, elastin-positive connective tissue intercalated circumferentially between ciliary processes. The developmental changes of the intralamellar distribution and extension of zonule with striped pattern informed the functional role upon the elasticity in coordination with the lens capsule micromolecules., (Copyright © 2013 Wiley Periodicals, Inc.)

Thyroid hormone (TH) is an essential regulator of both fetal development and energy homeostasis. Although the association between subclinical hypothyroidism and obesity has been well studied, a causal relationship has yet to be established. Using our well-characterized nonhuman primate model of excess nutrition, we sought to investigate whether maternal high-fat diet (HFD)-induced changes in TH homeostasis may underlie later in life development of metabolic disorders and obesity. Here, we show that in utero exposure to a maternal HFD is associated with alterations of the fetal thyroid axis. At the beginning of the third trimester, fetal free T(4) levels are significantly decreased with HFD exposure compared with those of control diet-exposed offspring. Furthermore, transcription of the deiodinase, iodothyronine (DIO) genes, which help maintain thyroid homeostasis, are significantly (P < 0.05) disrupted in the fetal liver, thyroid, and hypothalamus. Genes involved in TH production are decreased (TRH, TSHR, TG, TPO, and SLC5A5) in hypothalamus and thyroid gland. In experiments designed to investigate the molecular underpinnings of these observations, we observe that the TH nuclear receptors and their downstream regulators are disrupted with maternal HFD exposure. In fetal liver, the expression of TH receptor β (THRB) is increased 1.9-fold (P = 0.012). Thorough analysis of the THRB promoter reveals a maternal diet-induced alteration in the fetal THRB histone code, alongside differential promoter occupancy of corepressors and coactivators. We speculate that maternal HFD exposure in utero may set the stage for later in life obesity through epigenomic modifications to the histone code, which modulates the fetal thyroid axis.

In nonhuman primates, we previously demonstrated that a maternal high-fat diet (MHFD) induces fetal nonalcoholic fatty liver disease (NAFLD) and alters the fetal metabolome. These changes are accompanied by altered acetylation of histone H3 (H3K14ac). However, the mechanism behind this alteration in acetylation remains unknown. As SIRT1 is both a lysine deacetylase and a crucial sensor of cellular metabolism, we hypothesized that SIRT1 may be involved in fetal epigenomic alterations. Here we show that in utero exposure to a MHFD, but not maternal obesity per se, increases fetal H3K14ac with concomitant decreased SIRT1 expression and diminished in vitro protein and histone deacetylase activity. MHFD increased H3K14ac and DBC1-SIRT1 complex formation in fetal livers, both of which were abrogated with diet reversal despite persistent maternal obesity. Moreover, MHFD was associated with altered expression of known downstream effectors deregulated in NAFLD and modulated by SIRT1 (e.g., PPARΑ, PPARG, SREBF1, CYP7A1, FASN, and SCD). Finally, ex vivo purified SIRT1 retains deacetylase activity on an H3K14ac peptide substrate with preferential activity toward acetylated histone H3; mutagenesis of the catalytic domain of SIRT1 (H363Y) abrogates H3K14ac deacetylation. Our data implicate SIRT1 as a likely molecular mediator of the fetal epigenome and metabolome under MHFD conditions.

Intrauterine gene therapy (IUGT) potentially enables the treatment and possible cure of monogenic -diseases that cause severe fetal damage. The main benefits of this approach will be the ability to correct the disorder before the onset of irreversible pathology and inducing central immune tolerance to the vector and transgene if treatment is instituted in early gestation. Cure has been demonstrated in small animal models, but because of the significant differences in immune ontogeny and the much shorter gestation compared to humans, it is unlikely that questions of long-term efficacy and safety will be adequately addressed in rodents. The nonhuman primate (NHP) allows investigation of key issues, in particular, the different outcomes in early and late-gestation IUGT associated with different stages of immune maturity, longevity of transgene expression, and delayed-onset adverse events in treated offspring and mothers including insertional mutagenesis. Here, we describe a model based on the Macaca fascicularis using ultrasound and fetoscopic approaches to systemic vector delivery and the processes involved in vector administration and longitudinal analyses.

We cross-sectionally investigated prenatal ontogeny of craniofacial shape in the two subspecies of the Japanese macaque (Macaca fuscata fuscata and Macaca fuscata yakui) using a geometric morphometric technique to explore the process of morphogenetic divergence leading to the adult morphological difference between the subspecies. The sample comprised a total of 32 formalin-fixed fetal specimens of the two subspecies, in approximately the second and third trimesters. Each fetal cranium was scanned using computed tomography to generate a three-dimensional surface model, and 68 landmarks were digitized on the external and internal surface of each cranium to trace the growth-related changes in craniofacial shape of the two subspecies. The results of our study demonstrated that the two subspecies generally shared the same craniofacial growth pattern. Both crania tend to exhibit relative contraction of the neurocranium in the mediolateral and superoinferior directions, a more superiorly positioned cranial base, a more vertically oriented occipital squama, and a more anteriorly positioned viscerocranium as the cranial size increased. However, distinctive subspecific differences, for example relatively narrower orbital breadth, higher orbit, higher position of the nuchal crest, and more protrudent snout found in Macaca fuscata yakui were already present during the prenatal period. This study demonstrated that morphological differentiation in the craniofacial shape may occur at a very early stage of the fetal period even between closely related subspecies of the Japanese macaque.

By using the developing monkey brain as a model for human development, we investigated the expression pattern of the FOXP2 gene, a member of the FOX family of transcription factors in the developing monkey brain, and compared its expression pattern with transcription factors PBX3, MEIS2, and FOXP1. We observed FOXP2 mRNA expression in several brain structures, including the striatum, the islands of Calleja and other basal forebrain regions, the cerebral cortex, and the thalamus. FOXP2 mRNA was preferentially expressed in striosomal compartments during striatal development. The striosomal expression was transient and developmentally down-regulated in a topographical order. Specifically, during the perinatal state, striosomal FOXP2 expression was detected in both the caudate nucleus and the putamen, although expression was more prominent in the caudate nucleus than in the putamen. Striosomal FOXP2 expression declined during the postnatal period, first in the putamen and later in the caudate nucleus. During the same period, we also detected PBX3 mRNA in the striosomal compartment of the developing monkey striatum. FOXP2, as well as PBX3 and MEIS2, was expressed in the islands of Calleja and other cell clusters of the basal forebrain. FOXP2, in combination with PBX3 and MEIS2, may play a pivotal role in the development of striosomal neurons of the striatum and the islands of Calleja., ((c) 2008 Wiley-Liss, Inc.)

The role of the non-human primate (NHP) oocyte and embryo in translational research is considered here including both in vitro activities directly involving oocytes or embryos as well as animal studies that impact reproductive function. Reasons to consider NHPs as animal research models along with their limitations are summarized. A case is made that in limited instances, such as in the development and application of the assisted reproductive technologies or in the study of embryonic stem cells, the human oocyte and embryo have acted as models for the monkey. The development of strategies for the preservation of fertility is used as an example of ongoing research in the non-human primate that cannot be conducted in women for ethical reasons. In animal studies, monitoring reproductive potential, responses to embryonic stem cell transplantation, along with translational research in the field of contraceptive development for women are considered as subjects that benefit from the availability of a NHP model.

Excessive preformed vitamin A (VA) intake is contraindicated during pregnancy because of teratogenic concerns. Recent studies have provided biochemical and histologic evidence of chronic hypervitaminosis A in captive Old World monkeys consuming laboratory diets containing high concentrations of retinyl acetate. To investigate the effects of maternal chronic overconsumption of preformed VA on VA storage in early fetal liver, we analyzed monkey fetal livers ranging from 35 to 93 d gestational age (comparable with mid-first to late second trimester in humans) for VA (n = 19) and retinoic acid (n = 9). Retinyl esters were identified in all fetal livers, and retinol, on a percentage basis, was more abundant in younger fetuses. Liver VA concentration increased with gestational age, ranging from 0.0011 to 0.26 micromol/g in the youngest (35 d) and oldest fetuses (93 d), respectively. Liver VA concentrations (mean +/- 1 standard deviation) were 0.023 +/- 0.008 micromol/g in early gestation and 0.19 +/- 0.06 micromol/g in midgestation fetuses. All-trans retinoic acid concentrations were higher in early gestation (99.2 +/- 57.0 pmol/g, n = 6) than in midgestation (18.2 +/- 6.1 pmol/g, n = 3) but were variable. Liver VA concentrations from midgestation fetuses were higher than those in fetal human and monkey livers from later stages of development, when growth and VA accumulation rates are assumed to be highest. Therefore, excessive intake of preformed VA by the mothers results in amplified early fetal liver retinyl ester storage.

In the rodent, arcuate nucleus of the hypothalamus (ARH)-derived neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons have efferent projections throughout the hypothalamus that do not fully mature until the second and third postnatal weeks. Since this process is likely completed by birth in primates we characterized the ontogeny of NPY and melanocortin systems in the fetal Japanese macaque during the late second (G100), early third (G130) and late third trimesters (G170). NPY mRNA was expressed in the ARH, paraventricular nucleus (PVH), and dorsomedial nucleus of the hypothalamus (DMH) as early as G100. ARH-derived NPY projections to the PVH were initiated at G100 but were limited and variable; however, there was a modest increase in density and number by G130. ARH-NPY/agouti-related peptide (AgRP) fiber projections to efferent target sites were completely developed by G170, but the density continued to increase in the postnatal period. In contrast to NPY/AgRP projections, alphaMSH fibers were minimal at G100 and G130 but were moderate at G170. This study also revealed several significant species differences between rodent and the nonhuman primate (NHP). There were few NPY/catecholamine projections to the PVH and ARH prior to birth, while projections were increased in the adult. A substantial proportion of the catecholamine fibers did not coexpress NPY. In addition, cocaine and amphetamine-related transcript (CART) and alpha-melanocyte stimulating hormone (alphaMSH) were not colocalized in fibers or cell bodies. As a consequence of the prenatal development of these neuropeptide systems in the NHP, the maternal environment may critically influence these circuits. Additionally, because differences exist in the neuroanatomy of NPY and melanocortin circuitry the regulation of these systems may be different in primates than in rodents.

To improve efficiency and assess variation in nuclear transfer techniques in non-human primates, we investigated the following factors: type of donor cell, interval between enucleation and cell injection, activation after electrical pulsing and cytokinesis inhibitors. An average of 16.4 oocytes were recovered from 91 retrievals; however, 15 (14%) additional retrieval attempts yielded no oocytes due to a failure of follicular stimulation. Oocyte maturation rates at 36, 38 and 40 h post-hCG were 46.2, 52.6 and 61.2%, respectively. The MII spindle could be seen clearly using polarized microscopy in 89.1% (614/689) of oocytes. Nuclei were seen in 42% of the NT couplets, 53% of those cleaved to the 2-cell stage and 63% of the 2-cell embryos developed to the 8-cell stage by Day 3. There was no difference in the occurrence of nuclear formation between couplets created using fibroblasts or cumulus cells, although embryos were more reliably produced with fibroblasts. The interval (2, 3 and 4 h) between enucleation and cell injection did not affect NT efficiency. Ethanol treatment after electrical pulses yielded more 2-cell NT embryos than did treatment with ionomycin, but the frequency of nuclear formation and development to the 8-cell stage was not different. Treatment of couplets with cycloheximide and cytochalasin B for 5 h after activation had no impact on NT efficiency.

In macaque monkeys the foveal depression forms between fetal day (Fd) 105 and birth (Fd 172 of gestation). Before this, the incipient fovea is identified by a photoreceptor layer comprising cones almost exclusively, a multilayered ganglion cell layer (GCL), and a "domed" profile. Vessels are absent from the central retina until late in development, leading to the suggestion that the GCL in the incipient fovea may be transitorily hypoxic. Vascular endothelial growth factor (VEGF), expressed by both glial and neuronal cells and mediated by the hypoxia-inducible transcription factor (HIF)-1, is the principal factor involved in blood vessel growth in the retina. We examined VEGF expression in macaque retinas between Fd 85 and 4 months postnatal. Digoxygenin-labeled riboprobes were generated from a partial-length human cDNA polymerase chain reaction fragment, detected using fluorescence confocal microscopy, and quantified using Scion Image. High levels of VEGF mRNA were detected in astrocytes associated with developing vessels. We also detected strong expression of VEGF mRNA in the GCL at the incipient fovea prior to Fd 105, with peak labeling in the incipient fovea that declined with distance in nasal and temporal directions. By Fd 152 peak labeling was in two bands associated with development of the inner nuclear layer (INL) capillary plexus: in the inner INL where Müller and amacrine cell somas are located, and in the outer INL where horizontal cells are found. The findings suggest that at the incipient fovea the GCL is hypoxic, supporting the hypothesis that the adaptive significance of the fovea centralis is in ensuring adequate oxygen supply to neuronal elements initially located within the avascular region., (Copyright 2003 Wiley-Liss, Inc.)

To date, two subtypes of TrkB, a BDNF receptor, have been described. One is full-length TrkB (TK+), which has a tyrosine kinase-containing intracellular domain. The other is truncated TrkB (TK-), which has a short intracellular domain lacking the tyrosine kinase. In this study, we investigated the dimerization of TrkB subtypes in the developing monkey prefrontal cortex by means of cross-linking. At embryonic day 120, the TK+/TK+ and the 100 kDa/100 kDa homodimers were observed with BDNF stimulation. At the newborn stage, the TK+/TK+ and the TK-/TK- homodimers were observed with BDNF stimulation. At the adult stage, the TK-/TK- homodimer and the TK+/TK- heterodimer were formed by BDNF stimulation. The levels of all dimers increased in proportion to the concentration of BDNF. Moreover, the dimers were clearly formed within 5 min of treatment with BDNF. BDNF and NT-4/5 induced the dimers, whereas NT-3 formed slight dimers but NGF did not. Furthermore, anti-BDNF antibody inhibited the TrkB dimerization. Moreover, the intercellular binding proteins of TrkB were not cross-linked by BS3. Therefore, these results suggest that the change in dimerization among TrkB subtypes occurs during development of the monkey prefrontal cortex., (Copyright 2001 Wiley-Liss, Inc.)

Purpose: To characterize the spatial and temporal expression of key structural and phototransduction cascade proteins in the monkey cone outer segment (OS)., Methods: Retinas from Macaca monkeys from ages fetal day (Fd) 89 through adulthood were double labeled using immunofluorescence for short (S) or long/medium (L/M) wavelength-sensitive cone opsin and either a structural protein (peripherin) or a phototransduction cascade protein (alpha-transducin [alpha-T], phosphodiesterase [PDE], or rhodopsin kinase [RK]). The spatial and temporal patterns of expression for each protein at each age were determined and graphed as a percentage of retinal coverage., Results: In both cone types, opsins and phototransduction proteins appear first in the fovea and last at the retinal edge. Peripherin appears concomitantly with opsin in both S and L/M cones, but S cones express peripherin and opsin 1 to 3 weeks before neighboring L/M cones. Alpha-T, PDE, and RK are expressed together in the L/M cone OS shortly after L/M opsin appears. Phototransduction proteins are not expressed in S cones until 1 to 3 weeks after the appearance of S opsin and at the same time that neighboring cones are expressing both L/M opsin and phototransduction proteins., Conclusions: The concomitant appearance of opsin and peripherin strongly suggests roles in promoting the structural integrity of the developing OS. Phototransduction cascade proteins appear in the developing OS at the same time as one another, but after opsin. The significant lag between their expression and that of S cone opsin indicates that phototransduction proteins are not essential for OS formation, nor does opsin expression trigger their expression. The different temporal but similar spatial expression patterns of phototransduction proteins within S and L/M cones suggests that some local signal(s) coordinates their appearance.

In situ hybridization histochemistry was used to examine the expression of 10 GABA(A) receptor messenger RNAs corresponding to the alpha1-alpha5, beta1-beta3, gamma1 and gamma2 subunits in primary somatosensory and visual areas of macaque monkey cerebral cortex from embryonic day (E) 125 to postnatal day (P) 125. Results were compared with expression patterns in adults. In the sensorimotor cortex at E125, overall levels of all subunit transcripts were low. At E137, there was a major lamina-specific increase in all subunit messenger RNAs except gamma1. For alpha1, alpha2, alpha4, beta2, beta3 and gamma2 subunit transcripts, this increase was highest in areas 3a and 3b, particularly in layers III/IV and VI. Postnatally, there were significant decreases in all transcripts. Alpha1, alpha5, beta2 and gamma2 subunit transcripts, while still at significantly lower levels than at E137, remained expressed at levels higher than other transcripts. Unlike in rodents, there was no obvious "switch" in the major subunits expressed in fetal and adult cortex, alpha1, alpha5, beta2 and gamma2 remaining highest throughout. In area 17, the most prominently expressed subunits at earliest ages were alpha2, alpha5, beta1, beta2, beta3 and gamma2, especially in layers II/III and VI. At E150, expression for alpha2, alpha3, beta1 and beta3 subunit transcripts in these layers decreased, but levels for alpha1, alpha4, alpha5, beta2, gamma1 and gamma2 transcripts increased, particularly within layer IV. The increase at E150 was particularly marked for alpha5 transcripts, which were expressed at levels more than four times those of other transcripts. Alpha1, beta2 and gamma2 remain highest into aduthood. Fetal area 17 displayed lamina-specific patterns of expression not found in adult animals. In particular, alpha3 messenger RNAs were present in layer IVA and gamma1 transcripts were present in layer IVC at E150, despite a lack of expression in these layers in the adult. These data demonstrate increased expression of GABA(A) receptors during the period of establishment of thalamocortical and intracortical connections, and a temporal regulation that may be associated with the period of developmental plasticity.

Previous studies have demonstrated that axonal arbors specific for the four main cortical layers - 2/3, 4, 5, and 6 - develop precisely from the outset using activity-independent cues. In macaque primary visual cortex (V1), layer 2/3 is subdivided into layers named 2/3A, 3B, 4A, and 4B, and layer 4 is subdivided into 4Calpha and 4Cbeta. Individual neurons in V1 of mature macaques have axonal arbors that are highly specific for these sublayers. We have studied the prenatal development of laminar and sublaminar specificity of local circuits in macaque V1. Two-hundred thirty-eight neurons were labeled intracellularly in living brain slices prepared from V1 of five prenatal macaque monkeys aged 100 to 145 d postconception (E100-E145). Axonal and dendritic arbors of labeled neurons were reconstructed to assess their relationships to the cortical layers. We find that developing spiny neurons in layers 2-4B and layer 5 specifically target superficial and deep layers without forming "incorrect" branches in layer 4C. Similarly, layer 6 pyramidal neurons that target layer 4C do not form "incorrect" branches in layer 5. These results indicate that specific projections to the main cortical layers develop with a high degree of selectivity, as in other species. However, the development of sublayer-specific projections was not always precise from the outset. Unlike postnatal animals, axons of some prenatal layer 4Cbeta spiny neurons branch in layer 4B. At similar ages, many pyramidal neurons in the upper half of layer 6 have axonal branches in layer 4Calpha as well as 4Cbeta; these projections are specific for 4Cbeta in more mature animals. Also, there is similar "exuberance" in axonal arbors of other layer 6 cell types. Transient projections were also observed in the subplate and to the white matter for cells from all layers, except 4Cbeta. These observations indicate that at least some sublayer-specific projections emerge by elimination of exuberant axonal branches and suggest that they may use activity-dependent mechanisms to identify "correct" target layers. Such cues could be provided by laminar differences in the patterns of spontaneous prenatal activity in the retino-geniculo-cortical network.

The primate brain was thought to contain only the GnRH known as mammalian GnRH (mGnRH). This study investigates whether a second form of GnRH exists within the primate brain. We found that brain extracts from adult stumptail and rhesus monkeys contained two forms of GnRH that were similar to mGnRH and chicken GnRH-II (cGnRH-II) based on the elution position of the peptides from HPLC and on cross-reactivity with antisera that are specific to mammalian or chicken GnRH-II in RIAs. The fetal brain of rhesus monkeys also contained mGnRH and a cGnRH-II-like peptide by the same criteria. Immunocytochemistry with a cGnRH-II-specific antiserum in adult and fetal rhesus monkeys showed immunopositive neurons generally scattered in the periaqueductal region of the midbrain, with a few positive cells in the posterior basal hypothalamus. Neurons immunopositive for cGnRH-II were fewer in number and smaller in size, with less defined nuclei and thinner neurites compared with those for mGnRH. Administration of synthetic cGnRH-II to adult rhesus monkeys resulted in a significant increase in the plasma LH concentration during the luteal phase of the menstrual cycle, but not during the midfollicular phase. We conclude that the primate brain contains mGnRH and a cGnRH-II-like molecule, although the function of the latter is unknown.

In both human and Old World primates visual information is conveyed by two parallel pathways: the magnocellular (M) and parvocellular (P) streams that project to separate layers of the lateral geniculate nucleus and are involved primarily in motion and color/form discrimination. The present study provides evidence that retinal ganglion cells in the macaque monkey embryo diverge into M and P subtypes soon after their last mitotic division and that optic axons project directly and selectively to either the M or P moieties of the developing lateral geniculate nucleus. Thus, initial M projections from the eyes overlap only in prospective layers 1 and 2, whereas initial P projections overlap within prospective layers 3-6. We suggest that the divergence of the M and P pathways requires developmental mechanisms different from those underlying competition-driven segregation of initially intermixed eye-specific domains in the primate visual system.

Purpose: To determine the temporal and spatial pattern of rod opsin appearance in Macaca monkey retina., Methods: Frozen sections from fetal day (Fd) 55 to adulthood (birth = Fd168) containing the entire horizontal meridian were stained using Rho4D2 monoclonal antiserum visualized with immunofluorescent labeling. At Fd66, Fd79, and Fd89, retinal samples taken at known eccentricities were studied from the opposite eye using standard electron microscope methods., Results: Rod opsin was detected at Fd66 in or near the fovea, and a second focus appeared at Fd75 to Fd77 near the optic disc in the nasal rod ring. The earliest opsin appeared in the apical stubs, which resembled the apical connecting cilium in the electron microscope. Staining of the entire cell body membrane, including the synaptic spherule, was present 4 to 7 days later. Opsin expression had a nasal bias with rods at the nasal ora labeled at Fd140, whereas temporal ora was not labeled until Fd155. Cell body labeling disappeared by Fd132 across central retina but persisted into the first postnatal year in far peripheral retina. Outer segment (OS) length measurements showed that rods in the rod ring had the longest OS between Fd115 and postnatal week 9. Rod OS at all retinal eccentricites continued to elongate between 11 months of age and adulthood., Conclusions: Rod opsin expression follows a foveal-to-peripheral gradient beginning at Fd66 and ending near birth. Rod opsin is detected first in the connecting cilium and slightly later in the entire cell membrane, and then cell membrane labeling disappears as the heavily labeled OS elongates. Although the first OS appear on rods near the fovea, these OS still are short at birth and do not reach adult length until after 2 years of age. The longest OS at birth are found on rods at the rod ring, suggesting that this region could have higher scotopic sensitivity than central retina at birth.

The distribution of integrin subunits alpha 6 and beta 1, and the alpha 6 beta 1 integrin ligand, laminin, was examined during somitogenesis in developmental stages 11, 13, and 16 in the long-tailed macaque, using peroxidase immunocytochemistry. Within differentiating somites in stage 11, alpha 6 expression was observed in the sclerotome, basal surface of dermamyotomal cells adjacent to the basal lamina and on scattered cells throughout the dermamyotome. In further advanced somites in stages 13 and 16, alpha 6 immunoreactivity became restricted to the myotome. alpha 6 was expressed on mesenchymal core cells within the myocele of undifferentiated epithelioid somites and the ventromedial wall of somites commencing differentiation at each stage. beta 1 distribution resembled that of alpha 6 in stage 11 somitic tissue, however, it remained present on myotome and sclerotome cells in the later stages, and was also expressed on dermatomal cells in stage 16. Laminin immunoreactivity, while more intense and prevalent than alpha 6 and beta 1 in each stage examined, occurred on the same somite cell populations as the 2 integrin subunits. These results show a defined distribution of alpha 6 on somitic tissue, and suggest this integrin is involved in somite differentiation. They also support a possible role for alpha 6 in myoblast formation and migration. Overlapping of beta 1 and laminin immunoreactivity with that of alpha 6 further suggests that alpha 6 pairs with beta 1 as a functional heterodimer for laminin in defined somitic regions.

Early development of the spinal cord was studied in macaque monkey embryos, using light- and electron microscopy, Golgi impregnation and [3H]thymidine radioautography. All neurons engaged in the first reflex arc are generated before E27 in the 165 day gestation period. The earliest-generated cells differentiate into motoneurons while the commissural and association neurons are generated later. Synaptogenesis starts at E27 in the basal plate, and 2 days later in the alar plate. The first synapses appear as symmetrical junctions situated on the neuronal perikarya and proximal dendrites. Closure of the first spinal reflex are is established within 2 days and follows an antidromic pattern as related to the physiological spread of nerve impulses: synapses on motoneuronal somata and primary dendrites in the basal plate appear first and are followed by synapses in the alar plate, between dorsal root axon collaterals and somata of borderline (commissural/association) neurons. Commissural axons grow towards the floor plate, cross the midline and proceed caudo-rostrally, while association fibers remain ipsilateral. The first wave of apoptosis (programmed cell death) thins out dense populations of nerve and glial cells by E30. Some of the early-generated borderine cells that form commissural and association interneurons, seem to play the role of transient target cells and die once the definitive axonal pathways are established. Since transient cells form numerous synapses, deprivation from the afferent impulses is not a likely cause of their elimination. The present results indicate that the initial developmental events, including formation of the first reflex arc in the primate spinal cord, occur considerably earlier in respect to birth than in other mammals, but that the schedule of cellular events and cellular mechanisms seem to be the same.

The segregation of callosal and association pathways in the developing visual cortex of the monkey was studied using the retrograde tracers fast blue and diamidino yellow. Quantitative analysis of the laminar distribution of labeled callosal and association neurons made it possible to reveal the shifting pattern of connections that characterizes the development of these two pathways. In the adult, callosal neurons are restricted to supragranular layers, where they are concentrated at the bottom of layer 3. Association neurons are located both in infra- and supragranular layers. Supragranular layer association neurons are concentrated in layer 2, with limited spread into layer 3 so that there is little overlap with callosal neurons. In the immature brain, callosal neurons are characterized by a tangential distribution that is more widespread than in the adult, while their laminar distribution undergoes little developmental change. Association neurons show two types of changes in their laminar distribution: (1) in the early fetus, there is a large excess of association neurons in supragranular layers, the adult distribution being achieved some time after birth; and (2) during maturation there is a selective elimination of at least 50% of the projections originating from the lower part of layers 2/3. Hence, the adult radial segregation of association and callosal pathways is achieved in part by regressive phenomena. The developmental reduction of bihemispheric projections is largely independent of changes in the organization of association neurons. Quantitative analysis of the morphology and spatial location of neurons sending axon collaterals to both hemispheres suggests that they constitute a subset of callosal neurons and that their frequency is determined by factors that regulate directly this population. These results are discussed with respect to the specification of visual cortical pathways during ontogenesis.

Time of cell origin in the retina of the rhesus monkey (Macaca mulatta) was studied by plotting the number of heavily radiolabeled nuclei in autoradiograms prepared from 2- to 6-month-old animals, each of which was exposed to a pulse of 3H-thymidine (3H-TdR) on a single embryonic (E) or postnatal (P) day. Cell birth in the monkey retina begins just after E27, and approximately 96% of cells are generated by E120. The remaining cells are produced during the last (approximately 45) prenatal days and into the first several weeks after birth. Cell genesis begins near the fovea, and proceeds towards the periphery. Cell division largely ceases in the foveal and perifoveal regions by E56. Despite extensive overlap, a class-specific sequence of cell birth was observed. Ganglion and horizontal cells, which are born first, have largely congruent periods of cell genesis with the peak between E38 and E43, and termination around E70. The first labeled cones were apparent by E33, and their highest density was achieved between E43 and E56, tapering to low values at E70, although some cones are generated in the far periphery as late as E110. Amacrine cells are next in the cell birth sequence and begin genesis at E43, reach a peak production between E56 and E85, and cease by E110. Bipolar cell birth begins at the same time as amacrines, but appears to be separate from them temporally since their production reaches a peak between E56 and E102, and persists beyond the day of birth. Müller cells and rod photoreceptors, which begin to be generated at E45, achieve a peak, and decrease in density at the same time as bipolar cells, but continue genesis at low density on the day of birth. Thus, bipolar, Müller, and rod cells have a similar time of origin. The maximal temporal separation of cell birth is between cones and amacrine cells so that cell generation exhibits two relatively distinct phases: the first phase gives rise to ganglion, horizontal, and cone cells, and the second phase to amacrine, bipolar, rod, and Müller cells. In addition, cells of the first phase are generated faster than the second phase cells, and there are differences in the topography of spread of labeled cells between the two phases. Each cell class displays a central-to-peripheral gradient in genesis, although the spatiotemporal characteristics of the gradients differ between the classes.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult monkey sensorimotor cortex consists of several structurally and functionally distinct areas. The developmental sequence through which the characteristic architectonic features and the borders of these areas become resolved was examined in a series of fetal, postnatal and adult monkeys by using Nissl staining, cytochrome oxidase and acetylcholinesterase histochemistry, and immunocytochemistry for GABA and the neuropeptides somatostatin, neuropeptide Y, substance P and cholecystokinin. At the youngest fetal age examined (E110), the pre- and postcentral gyri possess six clearly delineated cellular layers; populations of GABA- and neuropeptide-immunoreactive cells can be identified, but their somatic sensory cortex at E110 lacks areal cytoarchitectonic parcellation. Despite the apparent homogeneity in the cytoarchitecture of the somatic sensory cortex, incipient areal borders are revealed by staining for cytochrome oxidase and acetylcholinesterase activity, and by staining immunocytochemically for several neuropeptides. The motor cortex at E110 differs from that in adults by the presence of a prominent layer IV; a clear cytoarchitectonic border between areas 3a and 4 is detectable at E110, which is also revealed by chemoarchitectonic markers. With increasing age, the characteristic architectonic features gradually emerge and areal cytoarchitectonic borders appear, becoming adult-like by early postnatal ages. The gradual changes in cytoarchitecture are paralleled by redistributions of GABA- and neuropeptide-immunoreactive cells and fiber plexuses. The data demonstrate that the progressive refinement in cytoarchitectonic features and in the distributions of neurotransmitter- and peptide-containing cells occurs primarily during the latter third of gestation. The major changes are temporally coincident with the ingrowth of afferent axonal systems, suggesting that the establishment of connectivity may be capable of modulating finer details of structural or molecular phenotype, particularly intra-areal cytoarchitectonic features and neurotransmitter or peptide expression.

The cytological organization and the timetable of emergence and dissolution of the transient subplate zone subjacent to the developing visual and somatosensory cortex were studied in a series of human and monkey fetal brains. Cerebral walls processed with Nissl, Golgi, electron-microscopic, and histochemical methods show that this zone consists of migratory and postmigratory neurons, growth cones, loosely arranged axons, dendrites, synapses, and glial cells. In both species the subplate zone becomes visible at the beginning of the mid-third of gestation as a cell-poor/fiber-rich layer situated between the intermediate zone and the developing cortical plate. The subplate zone appears earlier in the somatosensory than in the visual area and reaches maximal width at the beginning of the last third of gestation in both regions. At the peak of its size the ratio between the width of the subplate zone and cortical plate in the somatosensory cortex is 2:1 in monkey and 4:1 in man while in the occipital lobe these structures have about equal width in both species. The dissolution of the subplate zone begins during the last third of gestation with degeneration of some subplate neurons and the relocation of fiber terminals into the cortex. The subplate zone disappears faster in the visual than in the somatosensory area. The present results together with our previous findings support the hypothesis that the subplate zone may serve as a "waiting" compartment for transient cellular interactions and a substrate for competition, segregation, and growth of afferents originated sequentially from the brain stem, basal forebrain, thalamus, and from the ipsi- and contralateral cerebral hemisphere. After a variable and partially overlapping time period, these fibers enter the cortical plate while the subplate zone disappears leaving only a vestige of cells scattered throughout the subcortical white matter. A comparison between species indicates that the size and duration of the subplate zone increases during mammalian evolution and culminates in human fetuses concomitantly with an enlargement of cortico-cortical fiber systems. The regional difference in the size, pattern, and resolution of the subplate zone correlates also with the pattern of cerebral convolutions. Our findings indicate that, contrary to prevailing notions, the subplate may not be a vestige of the phylogenetically old network but a transient embryonic structure that expanded during evolution to subserve the increasing number of its connections.

Aromatase, 5 alpha-reductase and cytosolic androgen receptor levels were measured in the medial basal hypothalamus (MHB), amygdala (AMG), cerebellum and cerebral cortex of male and female fetal rhesus monkeys on day 70 of gestation. Higher aromatase activities were noted in the MBH and AMG of male than female fetuses. In contrast, no sex differences were found for 5 alpha-reductase and androgen receptor levels. These data suggest that at this early stage of development, differentiation of the MBH and AMG of the male fetus may be more susceptible to androgen modification, by way of aromatization to estrogens, than corresponding areas in the female fetus. Moreover, based upon a comparison of the current data to that published previously for later stages of development, it is suggested that the sex differences in aromatase activity are not the result of androgen stimulation.

Testosterone secreted by the testis of the macaque fetus is thought to influence certain aspects of the brain's subsequent development which may be responsible for the ontogeny of sexually dimorphic patterns of behavior. To compare the interactions between testosterone and the receptors for androgens and estrogens in brain cell nuclei in the two sexes, 7 intact female fetuses and 5 intact male fetuses were injected in utero at about 120 days of gestation with [3H]-testosterone (250 microCi i.v. or 500 microCi s.c.). One hour later, fetuses were delivered by cesarean section, and samples of brain and peripheral tissues were homogenized and separated into purified nuclear and supernatant fractions. Fractions were analyzed by high performance liquid chromatography to measure levels of [3H]-testosterone and its metabolites. Concentrations of radioactivity extracted from cell nuclei were significantly higher in the hypothalamus-preoptic area than in other brain areas (p less than 0.001); [3H]-estradiol represented 65.0 +/- 5.7% of this radioactivity and nuclear concentrations of this metabolite were 73% lower in males than in females (p less than 0.001). Nuclear concentrations of [3H]-testosterone in the pituitary gland (68.9 +/- 8.8% of extracted radioactivity) were 48% lower in males than in females (p less than 0.001). There was no evidence of a sex difference in the tissue uptake of radioactive steroids from blood, but in males, levels of endogenous plasma testosterone (599.8 +/- 208.2 ng/100 ml) were significantly higher than in females (37.7 +/- 28.5 ng/100 ml; p less than 0.01), and the specific activity of [3H]-testosterone in blood was consequently lower in males than in females.(ABSTRACT TRUNCATED AT 250 WORDS)

The time of origin of the neurons that comprise the magnocellular basal forebrain nuclei in rhesus monkeys was determined by using [3H]thymidine autoradiography. Thirteen pregnant animals received an injection of [3H]thymidine between embryonic days 27 (E27) and E50 of their 165 day gestation, and their offspring were sacrificed during the early postnatal period. Neurons within this region were generated in a biphasic pattern. An initial burst of [3H]thymidine-labeled magnocellular neurons was first observed throughout short quiescent period, cells of the remaining anterior basal forebrain (inclusive of magnocellular neurons comprising the vertical limb of the diagonal band and the anteromedial and anterolateral regions of the nucleus basalis) were generated between E36 and E45 with a peak of neurogenesis seen on E40-E43. The intermediate division of the nucleus basalis was generated about the same time, but the peak period of neurogenesis in this region occurred slightly earlier (E36 and E40) and was completed by E43. During the second phase of neurogenesis, neurons within the posterior division of the basal forebrain were generated first, with their genesis virtually completed between E33 and E36. The genesis of all neurons comprising the magnocellular basal forebrain nuclei was completed by E48 of gestation. A general caudal to rostral gradient of neurogenesis was observed within this telencephalic region. In contrast, a neurogenic gradient was not discerned in the radial direction. The present data demonstrate that neurons comprising the basal forebrain magnocellular nuclei in monkeys are generated early in gestation with two peak times of neuronal genesis. These nuclei are among the earliest to be generated in the entire telencephalon, which, like neurons of the thalamus and cortical neurons giving rise to cortical-cortical connections, places them in a strategic position to potentially influence their target neurons within the cortical mantle that are generated later in gestation.

Using the avidin-biotin-peroxide immunohistochemical method, we studied the distributions of somatostatin (SOM)-, cholecystokinin-8 (CCK-8)- and substance P (SP)-like immunoreactivities in the cerebellum of macaque monkeys at embryonic day 120 (E120), E140, newborn, postnatal day 60 (P60) and adults. During the embryonic stages, there were many SOM-, CCK- and SP-immunoreactive structures in the external granular layer, Purkinje cell layer and white matter, SP-immunoreactive mossy fibers and their terminals were distributed in the granular layer and white matter. During these stages, there were SOM-immunoreactive Purkinje cells, Golgi cells and a few cells in the molecular layer, and CCK-immunoreactive Purkinje cells and cells in the molecular layer. At the newborn stage, all of the immunoreactivities in the external granular layer decreased and the number of immunoreactive fibers increased in the white matter. At P60 stage, SOM- and CCK-immunoreactive fibers were observed around Purkinje cells, which seem to be the fiber terminals of basket cells. Many SOM, CCK and SP fibers were distributed in the white matter. In the adult stage, we observed little neuropeptide-immunoreactivity in the cerebellum. The high concentration of the neuropeptide-immunoreactive fibers and cells in the earlier stages suggests that the neuropeptides may be involved in the development of the primate cerebellar cortex.

Digital skin from fetal and neonatal primates was examined using light and electron microscopic techniques to document the development of the Meissner corpuscle. Generation of the receptor was initiated early in the third trimester by fine neurites which left the superficial dermal nerve plexus, ascended the dermal papillae and entered the basal epidermis. As maturation of the Meissner corpuscle continued, the axons were confined to the apex of the dermal papilla, where they were oriented parallel to the surface to the surface of the skin and terminated among cytoplasmic extensions of presumptive lamellar cells. During late fetal life the complexity of the lamellar cell stacking increased and the lamellar cell bodies were found solely at the base of the receptor. Numerous axon terminals were evident between the cytoplasmic lamellae. The appearance of the neonatal Meissner corpuscle was indistinguishable from that of the adult, indicating that the complete cycle of development is concluded before birth.

The order of appearance of the carpals, tarsals and secondary epiphyseal centers in the hand-wrist and foot-ankle regions of the fetal and infant pigtail macaque (Macaca nemestrina) was reconstructed using a cross-sectional sample of 127 animals and a longitudinal one of 14 animals. Comparison of the most common sequence in the monkey with that described in the human studies showed major interspecific differences in the order of appearance of several groups of centers, particularly those involving the thumb and great toe. The monkey displays a degree of sequence variability equivalent to that documented by the human longitudinal data, with certain centers markedly variable in timing and/or order of appearance. The evidence from related animals suggests a strong genetic component underlying some of the variable sequences observed, as has been reported in the human studies. The demonstrated variability in both timing and sequence precludes the use of specific centers in the determination of gestational age in fetuses of unknown conception date.

Groups of fetal monkeys were studied at 50, 75, 100, 125 and 150 days gestational age, and postnatal monkeys were studied at 175 days gestational age and in adult life (full term gestation is 168 plus or minus 4 days). The ratios between the various tissues of which lung is composed were determined by light and electronmicroscopy. Characteristic changes in ratios were noted at each developmental age. Primitive bronchioles increased from 50 to 75 days, decreased as permanent bronchioles, alveolar ducts and alveoli developed, and were absent after 150 days. Mesenchymal tissue decreased progressively from 50 to 150 days. Alveoli were present at 100 days gestational age; alveolar lumens accounted for approximately 1/2 the volume of peripheral lung by 150 days, and 2/3 the volume of lung in adult animals. By 150 days alveolar epithelial cells could be identified as Type I (membranous) or Type II (granular) on the basis of the characteristic cytoplasmic lamellar inclusion bodies of the latter. The ratios of the Type II/Type I cells were .21-.23 at 150 and 175 days, and fell to approximately half that value in adult lungs.

The present study examines the sequence of maturation of sensory nerve terminals that can be identified on primate facial guard hairs. At birth, the sensory innervation of both guard and vellus hairs is mature in that lanceolate, Ruffini, and free nerve ending (FNE) terminals can be identified and resemble those of the adult. Presumptive lanceolate terminals can be identified at the beginning of the third trimester of gestation, and other axons present are either Ruffini or FNEs, but definite identification is not possible. In the latter half of the second trimester only axons arranged circumferentially are present, resembling FNE or Ruffini terminals. Some of these axons directly abut the hair follicle and might eventually become lanceolate endings. The earliest nerve terminals associated with hairs cytologically resemble FNE or Ruffini terminals. At the onset of hair differentiation in the early part of the second trimester, nerves were always associated with developing epithelial hair placodes. Branching Schwann cells in the axons radiating toward the epidermis in these youngest embryos studied were best identified by electron microscopy. The early development of afferent nerve fibers in hairy skin provides an anatomical substrate for the known reflexogenic activity of primate embryos. Adequate cytologic criteria are thus available for the identification of sensory terminals in growing, differentiating, and presumably regenerating axons in primate hairy skin.

Previous investigations of the effect of glucocorticoids on fetal lung development have suggested that changes in lung structural properties may be of greater functional significance than changes in lung surfactant. In this study we examined the mechanism of the glucocorticoid-induced change in lung structure and compared it with the changes that occur during normal development. Rhesus monkeys (Macaca Mulatta) were treated with betamethasone prior to 133 days gestational age and then delivered by cesarian section either at 133 days or near term at 160 days. Our results show that normal fetal lung development over the last month of gestation occurs with a 45% increase in lung dry weight and a 56% increase in the maximal air volume per gram lung (Vmax/g). The immediate effect of the steroid was to cause a similar percentage increase in Vmax/g but no increase in lung weight. In fact, there were significant decreases in lung weight at both 133 and 160 days in the treated animals. In addition, we found no significant changes in the mean alveolar size either with normal development or with the steroid treatment. We thus conclude that in the rhesus monkey, the lung maturation process involves both an increase in lung mass and an increase in the number of alveoli per gram of that mass. The betamethasone treatment results in accelerated recruitment of alveoli coupled with impaired growth of lung tissue.

The present study documents the early innervation of the epidermis prior to the onset of differentiation of the papillary (sweat duct) ridge in glabrous digital skin of rhesus monkey embryos measuring 45, 50 and 55 mm (crown-rump) length. We observed small papillary ridges, spaced at a distance of approximately 40 microns, projecting into the dermis in the center of the distal glabrous digital pad of digits 2-5 in the 55-mm embryo. The other digital pads lacked any sign of ridge formation. A two-dimensional, approximately hexagonal grid of afferent nerves was present in the superficial dermis of all digital and palmar pads. At regular intervals of approximately 40 microns, afferent nerves ascended from the superficial dermal nerve plexus and innervated the overlying epidermis. By electron microscopy, axonal growth cones were identified contacting Merkel cells that projected several microns down into the superficial dermis in the digital pad of digit 3. Thus, the earliest wave of differentiated dorsal root ganglion neuroblasts innervates Merkel cells. Schwann cells partially encircled these growing axon tips and could be identified by the presence of rough endoplasmic reticulum and free ribosomes. The youngest embryo studied had no sign of ridge formation; however, axons ascended from the superficial dermal nerve net at 30-40-microns intervals to innervate the epidermis. We conclude that afferent nerve fibers provide a two-dimensional grid that could modulate the spacing and arrangement of the papillary or sweat duct ridges of successive digits. Such an interaction is possible between digits based on the overlapping dermatotopic maps of each rete ridge. An abnormal fingerprint could thus reflect abnormal dorsal root ganglion neuroblasts expressed through mesenchyme and epidermis.

C17-20Lyase and 21-hydroxylase activities were measured during late gestation in the rhesus monkey (Macaca mulatta) fetal adrenal. Activities were assessed in 10,000 x g supernatants with 17-hydroxyprogesterone and NADPH as substrates. Although conversion of [14C]17-hydroxyprogesterone to [14C]androstenedione was noted, activity was often nonlinear and far less than the rate of hydroxylation which together prevented an accurate estimation of lyase rate, Km and Vmax. 21-Hydroxylase activity was characterized; the mean reaction rate was 1.6 x 10(-3) mumoles NADPH oxidized/min. x mg-1 protein with an apparent Km of 3.6 x 10(-7) M and a Vmax of 2.2 x 10(-3) mumoles/min. x mg-1 protein. These values were similar to data obtained in adrenals from adult monkeys. A relatively high level of hydroxylase activity in the fetal gland might lead to an inadequate supply of precursors for the synthesis of dehydroepiandrosterone sulfate (DHEAS) in the adrenal if it also contained 3 beta-hydroxysteroid dehydrogenase (3 beta-hsdh). However, the fact that the fetal adrenal reportedly is deficient in 3 beta-hsdh may serve to protect both DHEAS and corticoid synthesis.

Radioautographic studies were conducted on early developmental stages of the inner ear in monkey (Macaca mulatta) embryos ranging in age from stage 11 to stage 20 (25 to 39 days' gestation) and labeled for one hour with [3H] thymidine. The most active period of proliferation occurred at stage 13, at which time all regions of the otocyst, including the endolymphatic duct, were heavily labeled. In subsequent stages the dorsal portion of the endolymphatic duct failed to incorporate label, whereas proliferation continued in the ventral portion of the duct near its communication with the medial wall of the otocyst. The duct thus appeared to grow upward as a result of cell division from below. Mitotic activity continued in the remainder of the otocyst although there was a progressive temporal decrease in the labeling index. Spatial gradients also occurred, with dorsal and lateral regions less active than ventral and medial ones. As the otocyst differentiated, the cells in various areas became attenuated. These flattened cells were still capable of undergoing cell division, as indicated by the presence of labeled nuclei in all regions of the utricle, saccule, semicircular ducts, and cochlear duct.

To determine whether individual optic fibers grow along constant sets of neighboring fibers, a group of 160 axons and 25 axonal growth cones were traced through a set of 500 serial electron micrographs of an optic nerve taken from a 39-day-old monkey embryo (Macaca mulatta). In single transverse sections, growth cones contact an average of 7.9 fibers, whereas axons contact 5.3 other fibers. The particular set of fibers in contact with one another changed rapidly, and, on average, growth cones and axons lost half of their original neighbors over a distance of only 8-10 micron. Between the first and last sections of the series, 92% of all initial contacts were lost. Individual axons moved freely between fiber fascicles, and the distance separating initial neighbors increased progressively. Most remarkably, the sets of fibers touched by the tips and the shanks of growth cones had no common neighbors in 17 out of 25 cases. These results demonstrate that, in primates, fibers in the optic nerve do not retain a particular set of immediate neighbors during their outgrowth.

Temporal changes in the ultrastructure of cranial neuroepithelial cells and their developmental interactions with adjacent chordamesodermal cells were analyzed during early neurogenesis in rhesus monkey embryos at stages 8-12 (19-28 days of gestation). In the neural plate and early neural fold stages, microtubules and apical microfilaments are not as prominent in the neuroepithelial cells as they are after the neural tube closes, whereas gap junctional vesicles are more evident prior to closure. Initially, the association between neuroepithelial cells and the presumptive notochordal cells is relatively closer than that between neuroepithelial cells and mesenchymal cells. After neural tube closure, the notochord becomes more distant. Cytoplasmic processes from neuroepithelial cells, notochordal cells, and mesenchymal cells interact via the neural basal lamina. In the rhesus monkey embryo, the ultrastructural features of neuroepithelial cells and their interactions with adjacent chordamesodermal cells are similar to those which have been observed in the mouse at comparable stages of gestation.

The metabolic interrelationship between cortisol (F) and cortisone (E) was studied in four long term catheterized rhesus monkey fetuses in utero during the last third of gestation. The MCR of E (50.8 +/- 5.4 liters/day) was greater than that of F (22.4 +/- 2.1, P less than 0.005) as was the plasma concentration (187.9 +/- 5.0 vs 86.1 +/- 2.5 ng/ml, P less than 0.001). The production rate of E (9.6 +/- 1.4 mg/day) was several-fold greater than for F (1.9 +/- 0.2, P less than 0.005). Of all fetal F, 79.5 +/- 7.0% was metabolized to E, and 43.4 +/- 3.9% originated from E within the fetal circulation. A significant mass of F was infused in these experiments because of the low specific activity of [14C]F. Nevertheless, the fetus was able to maintain F concentrations in the normal range. The MCR of F was similar to that which we previously found using trace amounts of [3H]F. This indicates that the fetus regulates the amount of F in the fetal compartment, probably by decreasing fetal; adrenal secretion rate. We conclude that F in the primate fetus is extensively oxidized to E. We conclude also that E is produced and metabolized much more extensively than is F. Reduction of E back to F could be an important source of fetal F, and increasing activity of this pathway, if present, could contribute to the increase in fetal F levels observed in late gestation in the primate.

This study has examined the fine structure and some cytochemical characteristics of the endodermal and mesothelial cells of the rhesus monkey yolk sac between 25 and 66 days of gestation. The endodermal cells were characterized by abundant granular endoplasmic reticulum, some agranular endoplasmic reticulum, a well-developed Golgi apparatus, and numerous large mitochondria. During the earlier part of the period studied, endodermal cells had a few acid phosphatase and arylsulfatase-positive lysosomes and moderate numbers of catalase-positive microperoxisomes. During the later stages of development, large granules (believed to be lysosomes) with a heterogeneous content were numerous in the cytoplasm. Mesothelial cells showed fewer development changes. Throughout this period they were usually flattened cells with long microvilli, small mitochondria, and limited amounts of granular endoplasmic reticulum. The mesothelial cells had acid phosphatase reaction product in the Golgi region and occasional large vesicles, but were negative for arylsulfatase and catalase. One specimen was incubated at 37 degrees C in the presence of horseradish peroxidase in order to examine endocytosis. Both the mesothelial cells and endodermal cells internalized the peroxidase into a variety of cytoplasmic vesicles. Based on their cytology, the endodermal cells may function in the synthesis of serum proteins during this period, as has been suggested in other species. They may also be involved in lipid metabolism. The mesothelial cells appeared less synthetically active, but evidence suggested that they may be involved in collagen and extracellular matrix production. The endocytic activity displayed by both cell types may indicate a role in fluid and metabolite transfer across the epithelia. The cytology of both cell types was very similar to that described for human yolk sacs, suggesting that the rhesus monkey may be a useful species in which to study the maturation of yolk sac function.

Fetal metabolic conversion and clearance of hypoxanthine was investigated in five pregnant rhesus monkeys. [14C]hypoxanthine injected into the fetal circulation disappeared rapidly and its metabolic break-down products uric acid and allantoin appeared in fetal plasma and erythrocytes. In amniotic fluid only hypoxanthine could be demonstrated. When [14C] hypoxanthine was infused into the fetal circulation at a constant rate and metabolic conversion to uric acid and allantoin was inhibited with allopurinol, a constant level of hypoxanthine was obtained within 30 min. No uric acid or allantoin was demonstrated, but inosine monophosphate and adenine nucleotides appeared in the fetal erythrocytes. The concentration of adenine nucleotides in the fetal red cells showed a steady increase during the experiments. Total fetal hypoxanthine clearance, calculated in the steady-state from the maternal and fetal arterial concentrations and the amount infused per unit of time varied between 4.2 and 8.4 ml min-1. In two separate experiments maternal blood was incubated with [14C] hypoxanthine and [14C]adenosine. The erythrocytes appeared to be unable to convert hypoxanthine into adenine nucleotides, but adenosine was readily converted into ADP and ATP. There appears to be no preferential uptake of hypoxanthine by the fetal erythrocytes which makes it unlikely that they could serve as carriers in the transport of hypoxanthine.