Evolutionary divergence of embryo implantation in primates.

Implantation of the conceptus into the uterus is absolutely essential for successful embryo development. In humans, our understanding of this process has remained rudimentary owing to the inaccessibility of early implantation stages. Non-human primates recapitulate many aspects of human embryo development and provide crucial insights into trophoblast development, uterine receptivity and embryo invasion. Moreover, primate species exhibit a variety of implantation strategies and differ in embryo invasion depths. This review examines conservation and divergence of the key processes required for embryo implantation in different primates and in comparison with the canonical rodent model. We discuss trophectoderm compartmentalization, endometrial remodelling and embryo adhesion and invasion. Finally, we propose that studying the mechanism controlling invasion depth between different primate species may provide new insights and treatment strategies for placentation disorders in humans. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Non-human primates as a model for human development.

Human development has been studied for over a century, but the molecular mechanisms underlying human embryogenesis remain largely unknown due to technical difficulties and ethical issues. Accordingly, mice have been used as a model for mammalian development and studied extensively to infer human biology based on the conservation of fundamental processes between the two species. As research has progressed, however, species-specific differences in characteristics between rodents and primates have become apparent. Non-human primates (NHPs) have also been used for biomedical research, and are now attracting attention as a model for human development. Here, we summarize primate species from the evolutionary and genomic points of view. Then we review the current issues and progress in gene modification technology for NHPs. Finally, we discuss recent studies on the early embryogenesis of primates and future perspectives.

Origin and function of the yolk sac in primate embryogenesis.

Human embryogenesis is hallmarked by two phases of yolk sac development. The primate hypoblast gives rise to a transient primary yolk sac, which is rapidly superseded by a secondary yolk sac during gastrulation. Moreover, primate embryos form extraembryonic mesoderm prior to gastrulation, in contrast to mouse. The function of the primary yolk sac and the origin of extraembryonic mesoderm remain unclear. Here, we hypothesise that the hypoblast-derived primary yolk sac serves as a source for early extraembryonic mesoderm, which is supplemented with mesoderm from the gastrulating embryo. We discuss the intricate relationship between the yolk sac and the primate embryo and highlight the pivotal role of the yolk sac as a multifunctional hub for haematopoiesis, germ cell development and nutritional supply.

Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos.

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing.

Primate embryogenesis predicts the hallmarks of human naïve pluripotency.

Naïve pluripotent mouse embryonic stem cells (ESCs) resemble the preimplantation epiblast and efficiently contribute to chimaeras. Primate ESCs correspond to the postimplantation embryo and fail to resume development in chimaeric assays. Recent data suggest that human ESCs can be 'reset' to an earlier developmental stage, but their functional capacity remains ill defined. Here, we discuss how the naïve state is inherently linked to preimplantation epiblast identity in the embryo. We hypothesise that distinctive features of primate development provide stringent criteria to evaluate naïve pluripotency in human and other primate cells. Based on our hypothesis, we define 12 key hallmarks of naïve pluripotency, five of which are specific to primates. These hallmarks may serve as a functional framework to assess human naïve ESCs.

Prenatal Brain-Body Allometry in Mammals.

Variation in relative brain size among adult mammals is produced by different patterns of brain and body growth across ontogeny. Fetal development plays a central role in generating this diversity, and aspects of prenatal physiology such as maternal relative metabolic rate, altriciality, and placental morphology have been proposed to explain allometric differences in neonates and adults. Primates are also uniquely encephalized across fetal development, but it remains unclear when this pattern emerges during development and whether it is common to all primate radiations. To reexamine these questions across a wider range of mammalian radiations, data on the primarily fetal rapid growth phase (RGP) of ontogenetic brain-body allometry was compiled for diverse primate (np = 12) and nonprimate (nnp = 16) mammalian species, and was complemented by later ontogenetic data in 16 additional species (np = 9; nnp = 7) as well as neonatal proportions in a much larger sample (np = 38; nnp = 83). Relative BMR, litter size, altriciality, and placental morphology fail to predict RGP slopes as would be expected if physiological and life history variables constrained fetal brain growth, but are associated with differences in birth timing along allometric trajectories. Prenatal encephalization is shared by all primate radiations, is unique to the primate Order, and is characterized by: (1) a robust change in early embryonic brain/body proportions, and (2) higher average RGP allometric slopes due to slower fetal body growth. While high slopes are observed in several nonprimate species, primates alone exhibit an intercept shift at 1 g body size. This suggests that primate prenatal encephalization is a consequence of early changes to embryonic neural and somatic tissue growth in primates that remain poorly understood.

Targeted genome editing in primate embryos.

Mosaic mutations and off-target effects caused by CRISPR/Cas9 have led to concerns about the efficiency and specificity of this new technique in non-human primates and other large animals. Here we discuss recent findings from primate embryos, with a focus on the technical issues CRISPR/Cas9 faces before producing non-human primate models of human diseases.

Adaptive evolution at immune system genes and deep pregnancy implantation in primates.

A major evolutionary change in the lineage ancestral to humans, chimpanzee and gorilla (HCG) has been the embedding of the embryo into maternal tissue. Thus, the first layer of cells (trophoblast) to differentiate after fertilization must adapt to invade the uterus. Such event would likely leave signatures of positive selection at genes with roles in embryo implantation. Here, 163 pregnancy implantation genes are tested for evidence of adaptive diversification in the ancestral lineage to HCG. Two immune system genes, HLA-E and KIR2DL4 showed evidence of positive selection. Some of the positive selected sites involve amino acid substitution with predicted damaging effects on protein function, thus highlighting the possibility of antagonistic pleiotropic effects. Selection at a gene coding for a receptor expressed in uterine cells (KIR) that interacts with trophoblast human leukocyte antigen (HLA) genes suggests a main role for immunological adaptations in embryo deep invasion of the maternal endometrium.

Septomarginal trabecula and anterior papillary muscle in primate hearts: developmental issues.

The septomarginal trabecula is present in all human hearts as well as in the hearts of other primates. It usually connects the interventricular septum with the anterior papillary muscle, although there are many variations in how this is achieved. The object of the analyses was to estimate the bilateral topography of the septomarginal trabecula and the anterior papillary muscle in the context of the ontogeny and phylogeny of primates. A total of 138 hearts were examined from number of different non-human primates. The presence of the septomarginal trabecula was confirmed in 94.9% of cases, although not in the hearts of Lemur varius. Four configurations could be distinguished by defining the location of the septomarginal trabecula and its relation to the anterior papillary muscle.For the hearts of the Strepsirrhini and the majority of Platyrrhini neither structure was related, whereas in all examined representatives of Homino idea they had fused and created morphologically varying forms. On the basis of these results,a concept was developed for the sequence of changes which the topography of the septomarginal trabecula and the anterior papillary muscle undergo during ontogeny and phylogeny.

Evolution of columns, modules, and domains in the neocortex of primates.

The specialized regions of neocortex of mammals, called areas, have been divided into smaller functional units called minicolumns, columns, modules, and domains. Here we describe some of these functional subdivisions of areas in primates and suggest when they emerged in mammalian evolution. We distinguish several types of these smaller subdivisions. Minicolumns, vertical arrays of neurons that are more densely interconnected with each other than with laterally neighboring neurons, are present in all cortical areas. Classic columns are defined by a repeating pattern of two or more types of cortex distinguished by having different inputs and neurons with different response properties. Sensory stimuli that continuously vary along a stimulus dimension may activate groups of neurons that vary continuously in location, producing "columns" without specific boundaries. Other groups or columns of cortical neurons are separated by narrow septa of fibers that reflect discontinuities in the receptor sheet. Larger regions of posterior parietal cortex and frontal motor cortex are parts of networks devoted to producing different sequences of movements. We distinguish these larger functionally distinct regions as domains. Columns of several types have evolved independently a number of times. Some of the columns found in primates likely emerged with the first primates, whereas others likely were present in earlier ancestors. The sizes and shapes of columns seem to depend on the balance of neuron activation patterns and molecular signals during development.

The von Economo neurons in the frontoinsular and anterior cingulate cortex.

The von Economo neurons (VENs) are large bipolar neurons located in the frontoinsular cortex (FI) and limbic anterior (LA) area in great apes and humans but not in other primates. Our stereological counts of VENs in FI and LA show them to be more numerous in humans than in apes. In humans, small numbers of VENs appear the 36th week postconception, with numbers increasing during the first 8 months after birth. There are significantly more VENs in the right hemisphere in postnatal brains; this may be related to asymmetries in the autonomic nervous system. VENs are also present in elephants and whales and may be a specialization related to very large brain size. The large size and simple dendritic structure of these projection neurons suggest that they rapidly send basic information from FI and LA to other parts of the brain, while slower neighboring pyramids send more detailed information. Selective destruction of VENs in early stages of frontotemporal dementia (FTD) implies that they are involved in empathy, social awareness, and self-control, consistent with evidence from functional imaging.

Evolution of invasive placentation with special reference to non-human primates.

It is now possible to view human placentation in an evolutionary context because advances in molecular phylogenetics provide a reliable scenario for the evolution of mammals. Perhaps the most striking finding is the uniqueness of human placenta. The lower primates have non-invasive placentae and even tarsiers and New World monkeys show restricted trophoblast invasion. Moreover, a truly villous placenta occurs only in Old World monkeys and great apes. The two latter groups of haplorhine primates show varying degrees of trophoblast-uterine interaction, including differences in the extent of decidualization, formation and disintegration of a cytotrophoblastic shell, degree of interstitial trophoblast invasion and depth of trophoblast invasion into spiral arteries. Recently, the occurrence of human-like deep invasion was confirmed in gorillas and chimpanzees. As the still enigmatic disease of pre-eclampsia also occurs in these species, such information may reveal the evolutionary roots of this disease of impaired maternal-fetal interaction.

Primate maternal placental angiography.

BACKGROUND: In humans, it is known that blood flow is directed to the gravid uterus from two (right and left) pelvic uterine arteries. The extent of supply from the tubo-ovarian anastomosis (joining of the ovarian and uterine arteries) is unknown. The aim of this study was to delineate the arterial blood supply to the placenta via systematic angiography in normal pregnancies in a non-human primate, the baboon (Papio hamadryas). METHODS: The assessment of the distribution of blood supply with single-shot 3-vessel angiography (aorta, right and left common iliac arteries), allowed assessment of bilateral supply and possible ovarian supply (n=9). In 2-vessel pictures (aorta and left or right iliac), the contralateral supply was determined by subtraction of the ipsilateral supply from the total supply (n=7). The studies were all approved by the Institutional animal welfare committee and were conducted as part of a broader project investigating preeclampsia. RESULTS: The animals were 9 years of age and 140 days of gestation for the 3 vessel study and 154 days of gestation for the 2 vessel study. The angiograms were more likely to have cotyledons perfused by the left uterine artery (p=0.012) than the right. Overall, 55% of placentae had 5-44% of supply overlapping and 22% had 10-15% ovarian contribution to blood supply. DISCUSSION: This study demonstrates the variation in primate uteroplacental blood flow including the contribution of ovarian arteries and left and right collateralization. Similarity to human vascular anatomy strengthens the use of primate species as a model of human placentation.

Cloning of non-human primates: the road "less traveled by".

Early studies on cloning of non-human primates by nuclear transfer utilized embryonic blastomeres from preimplantation embryos which resulted in the reproducible birth of live offspring. Soon after, the focus shifted to employing somatic cells as a source of donor nuclei (somatic cell nuclear transfer, SCNT). However, initial efforts were plagued with inefficient nuclear reprogramming and poor embryonic development when standard SCNT methods were utilized. Implementation of several key SCNT modifications was critical to overcome these problems. In particular, a non-invasive method of visualizing the metaphase chromosomes during enucleation was developed to preserve the reprogramming capacity of monkey oocytes. These modifications dramatically improved the efficiency of SCNT, yielding high blastocyst development in vitro. To date, SCNT has been successfully used to derive pluripotent embryonic stem cells (ESCs) from adult monkey skin fibroblasts. These remarkable advances have the potential for development of human autologous ESCs and cures for many human diseases. Reproductive cloning of nonhuman primates by SCNT has not been achieved yet. We have been able to establish several pregnancies with SCNT embryos which, so far, did not progress to term. In this review, we summarize the approaches, obstacles and accomplishments of SCNT in a non-human primate model.

Fetal programming of adrenal androgen excess: lessons from a nonhuman primate model of polycystic ovary syndrome.

Adrenal androgen excess is found in adult female rhesus monkeys previously exposed to androgen treatment during early gestation. In adulthood, such prenatally androgenized female monkeys exhibit elevated basal circulating levels of dehydroepiandrosterone sulfate (DHEAS), typical of polycystic ovary syndrome (PCOS) women with adrenal androgen excess. Further androgen and glucocorticoid abnormalities in PA female monkeys are revealed by acute ACTH stimulation: DHEA, androstenedione and corticosterone responses are all elevated compared to responses in controls. Pioglitazone treatment, however, diminishes circulating DHEAS responses to ACTH in both prenatally androgenized and control female monkeys, while increasing the 17-hydroxyprogesterone response and reducing the DHEA to 17-hydroxyprogesterone ratio. Since 60-min post-ACTH serum values for 17-hydroxyprogesterone correlate negatively with basal serum insulin levels (all female monkeys on pioglitazone and placebo treatment combined), while similar DHEAS values correlate positively with basal serum insulin levels, circulating insulin levels may preferentially support adrenal androgen biosynthesis in both prenatally androgenized and control female rhesus monkeys. Overall, our findings suggest that differentiation of the monkey adrenal cortex in a hyperandrogenic fetal environment may permanently upregulate adult adrenal androgen biosynthesis through specific elevation of 17,20-lyase activity in the zona fasciculata-reticularis. As adult prenatally androgenized female rhesus monkeys closely emulate PCOS-like symptoms, excess fetal androgen programming may contribute to adult adrenal androgen excess in women with PCOS.

Phylogenetic proximity revealed by neurodevelopmental event timings.

Statistical properties such as distribution and correlation signatures were investigated using a temporal database of common neurodevelopmental events in the three species most frequently used in experimental studies, rat, mouse, and macaque. There was a fine nexus between phylogenetic proximity and empirically derived dates of the occurrences of 40 common events including the neurogenesis of cortical layers and outgrowth milestones of developing axonal projections. Exponential and power-law approximations to the distribution of the events reveal strikingly similar decay patterns in rats and mice when compared to macaques. Subsequent hierarchical clustering of the common event timings also captures phylogenetic proximity, an association further supported by multivariate linear regression data. These preliminary results suggest that statistical analyses of the timing of developmental milestones may offer a novel measure of phylogenetic classifications. This may have added pragmatic value in the specific support it offers for the reliability of rat/mouse comparative modeling, as well as in the broader implications for the potential of meta-analyses using databases assembled from the extensive empirical literature.