The first lineage determination in mammals.

This review describes in detail the morphological, cytoskeletal and gene expression events leading to the gene regulatory network bifurcation point of trophoblast and inner cell mass cells in a variety of mammalian preimplantation embryos. The interrelated processes of compaction and polarity establishment are discussed in terms of how they affect YAP/WWTR activity and the location and fate of cells. Comparisons between mouse, human, cattle, pig and rabbit embryos suggest a conserved role for YAP/WWTR signalling in trophoblast induction in eutherian animals though the mechanisms for, and timing of, YAP/WWTR activation differs among species. Downstream targets show further differences, with the trophoblast marker GATA3 being a direct target in all examined mammals, while CDX2-positive and SOX2-negative regulation varies.

Alternative mammalian strategies leading towards gastrulation: losing polar trophoblast (Rauber's layer) or gaining an epiblast cavity.

Using embryological data from 14 mammalian orders, the hypothesis is presented that in placental mammals, epiblast cavitation and polar trophoblast loss are alternative developmental solutions to shield the central epiblast from extraembryonic signalling. It is argued that such reciprocal signalling between the edge of the epiblast and the adjoining polar trophoblast or edge of the mural trophoblast or with the amniotic ectoderm is necessary for the induction of gastrulation. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Growing cattle embryos beyond Day 8 - An investigation of media components.

The growth of viable cattle embryos in culture to stages beyond the hatching blastocyst is of interest to developmental biologists wishing to understand developmental events beyond the first lineage decision, as well as for commercial applications, because a lengthening of the culturing time allows more time for diagnostic tests on biopsies, whereas extended survival can be used as a better assay system for monitoring developmental potential. We here report on a novel extended culture medium for embryo growth until embryonic day (Day) 12. We used a non-invasive morphological characterisation system that scored viability, inner cell mass (ICM) grade, hatching and embryo and ICM diameter. The basal medium was based on published uterine fluid concentrations of amino acids, carbohydrates and electrolytes. Addition of fetal bovine serum was necessary and the additive ITSX greatly improved culture success. We tested the inclusion of a seven-growth factor cocktail consisting of Activin A, Artemin, BMP4, EGF, FGF4, GM-CSF/CSF2 and LIF, as well as omission of individual components of the cocktail. In the context of the growth factor cocktail, Artemin and BMP4 provided the greatest benefit, while FGF omission had more positive than negative effects on embryo characteristics. Lastly, replacement of ITSX by B27-additive led to the most successful culture of embryos, in all media permutations.

On the enigmatic disappearance of Rauber's layer.

The polar trophoblast overlays the epiblast in eutherian mammals and, depending on the species, has one of two different fates. It either remains a single-layered, thinning epithelium called "Rauber's layer," which soon disintegrates, or, alternatively, it keeps proliferating, contributing heavily to the population of differentiating, invasive trophoblast cells and, at least in mice, to the induction of gastrulation. While loss of the persistent polar trophoblast in mice leads to reduced induction of gastrulation, we show here that prevention of the loss of the polar trophoblast in cattle results in ectopic domains of the gastrulation marker, BRACHYURY This phenotype, and increased epiblast proliferation, arose when Rauber's layer was maintained for a day longer by countering apoptosis through BCL2 overexpression. This suggests that the disappearance of Rauber's layer is a necessity, presumably to avoid excessive signaling interactions between this layer and the subjacent epiblast. We note that, in all species in which the polar trophoblast persists, including humans and mice, ectopic polar trophoblast signaling is prevented via epiblast cavitation which leads to the (pro)amniotic cavity, whose function is to distance the central epiblast from such signaling interactions.

Building Principles for Constructing a Mammalian Blastocyst Embryo.

The self-organisation of a fertilised egg to form a blastocyst structure, which consists of three distinct cell lineages (trophoblast, epiblast and hypoblast) arranged around an off-centre cavity, is unique to mammals. While the starting point (the zygote) and endpoint (the blastocyst) are similar in all mammals, the intervening events have diverged. This review examines and compares the descriptive and functional data surrounding embryonic gene activation, symmetry-breaking, first and second lineage establishment, and fate commitment in a wide range of mammalian orders. The exquisite detail known from mouse embryogenesis, embryonic stem cell studies and the wealth of recent single cell transcriptomic experiments are used to highlight the building principles underlying early mammalian embryonic development.

MicroRNA expression in bovine preimplantation embryos.

We profiled 98 mature microRNAs (miRNAs) using a stem-loop reverse transcription polymerase chain reaction assay array based on human miRNAs. We demonstrated that one, but not two, base-pair changes in the miRNA recognition sequence at the 3' end only marginally affected copy number estimates. Absolute levels of miRNAs were measured in matured cattle oocytes, eight-cell embryos and normal and parthenogenetic blastocysts and Day-14 trophoblast. Most miRNA concentrations were below the expected functional threshold required for effective repression of moderately to highly abundant target RNA. In oocytes and peri-embryonic genome activation embryos, miRNA 320, a member of the Dgcr8/Drosha-independent class of miRNAs, was expressed at greater than 1000 copies per embryo. miRNAs were more abundant at the eight-cell than the oocyte stage. miRNA concentrations per cell increased from the eight-cell to the blastocyst stage. Both the number of miRNA species and their expression levels were reduced in trophoblast tissue at Day 14. The parthenogenetic samples were more related in their miRNA expression profiles to each other than to their wild-type (in vitro-produced cultured) counterparts. miRNAs 299 and 323, which have been shown to be maternally expressed in other species, were also more than 4-fold overexpressed in the cattle parthenogenetic samples.

A mathematical model of the interaction between bovine blastocyst developmental stage and progesterone-stimulated uterine factors on differential embryonic development observed on Day 15 of gestation.

A complex interaction between the developing bovine embryo and the growth potential of the uterine milieu it inhabits results in an embryo capable of developing past the maternal recognition stage and on to a successful pregnancy. Previously, we observed variation in the lengths of embryos recovered 8 d after bulk transfer of Day 7 in vitro-produced (IVP) blastocysts into the same uterus. Potential causes of the differential embryonic growth were examined and modeled using 2 rounds of bulk (n = 4-6) IVP transfers and recovery of these embryos 8 d later. Morphological and gene expression measurements of the embryos were determined and the progesterone concentration of the cows was measured throughout the reproductive cycle as a reflection of the status of the uterine environment. These data were used to develop and evaluate a model that describes the interaction between the uterine environment and the growth rate of the developing embryo. Expression of 6 trophectoderm genes (IFNT, TKDP1, PAG11, PTGS2, DKK1, and PDPN) was correlated with conceptus length. The model determined that if the embryo develops to blastocyst stage, the uterine environment, driven by progesterone, is a more important component than blastocyst size in the stimulation of embryonic growth rate to ensure adequate interferon tau (IFNT) for pregnancy recognition. We detected an effect of Day 7 progesterone on the expression of all 6 genes, embryonic disc size, and trophectoderm length on Day 15. We also found effects of embryo transfer size on trophectoderm length and expression of IFNT and PAG11 on Day 15. Lower energy balance over the period from transfer to recovery was associated with reduced embryo growth to Day 15, and this effect was independent of progesterone. Energy balance also affected expression of PDPN and TKDP1 on Day 15. We observed an effect of energy balance from transfer to recovery on embryo survival in cows with partial embryo losses, where embryo factors dominate embryo survival, with cows with greater energy balance having lower embryo losses. This effect was independent of energy balance 40 d before transfer and suggests that energy balance has direct, immediate effects on the embryo and maternal environment during this period. Furthermore, energy balance effects on embryo survival in cows with partial embryo losses were largely mediated by expression of TKDP1, PAG11, and PDPN. These results provide candidate signaling pathways for the effect of progesterone and energy balance on embryo growth and survival.

Gene expression analysis of bovine embryonic disc, trophoblast and parietal hypoblast at the start of gastrulation.

In cattle early gastrulation-stage embryos (Stage 5), four tissues can be discerned: (i) the top layer of the embryonic disc consisting of embryonic ectoderm (EmE); (ii) the bottom layer of the disc consisting of mesoderm, endoderm and visceral hypoblast (MEH); (iii) the trophoblast (TB); and (iv) the parietal hypoblast. We performed microsurgery followed by RNA-seq to analyse the transcriptome of these four tissues as well as a developmentally earlier pre-gastrulation embryonic disc. The cattle EmE transcriptome was similar at Stages 4 and 5, characterised by the OCT4/SOX2/NANOG pluripotency network. Expression of genes associated with primordial germ cells suggest their presence in the EmE tissue at these stages. Anterior visceral hypoblast genes were transcribed in the Stage 4 disc, but no longer by Stage 5. The Stage 5 MEH layer was equally similar to mouse embryonic and extraembryonic visceral endoderm. Our data suggest that the first mesoderm to invaginate in cattle embryos is fated to become extraembryonic. TGFß, FGF, VEGF, PDGFA, IGF2, IHH and WNT signals and receptors were expressed, however the representative members of the FGF families differed from that seen in equivalent tissues of mouse embryos. The TB transcriptome was unique and differed significantly from that of mice. FGF signalling in the TB may be autocrine with both FGFR2 and FGF2 expressed. Our data revealed a range of potential inter-tissue interactions, highlighted significant differences in early development between mice and cattle and yielded insight into the developmental events occurring at the start of gastrulation.

Morphological and Gene Expression Changes in Cattle Embryos from Hatched Blastocyst to Early Gastrulation Stages after Transfer of In Vitro Produced Embryos.

A detailed morphological staging system for cattle embryos at stages following blastocyst hatching and preceding gastrulation is presented here together with spatiotemporal mapping of gene expression for BMP4, BRACHYURY, CERBERUS1 (CER1), CRIPTO, EOMESODERMIN, FURIN and NODAL. Five stages are defined based on distinct developmental events. The first of these is the differentiation of the visceral hypoblast underlying the epiblast, from the parietal hypoblast underlying the mural trophoblast. The second concerns the formation of an asymmetrically positioned, morphologically recognisable region within the visceral hypoblast that is marked by the presence of CER1 and absence of BMP4 expression. We have termed this the anterior visceral hypoblast or AVH. Intra-epiblast cavity formation and the disappearance of the polar trophoblast overlying the epiblast (Rauber's layer) have been mapped in relation to AVH formation. The third chronological event involves the transition of the epiblast into the embryonic ectoderm with concomitant onset of posterior NODAL, EOMES and BRACHYURY expression. Lastly, gastrulation commences as the posterior medial embryonic ectoderm layer thickens to form the primitive streak and cells ingress between the embryonic ectoderm and hypoblast. At this stage a novel domain of CER1 expression is seen whereas the AVH disappears. Comparison with the mouse reveals that while gene expression patterns at the onset of gastrulation are well conserved, asymmetry establishment, which relies on extraembryonic tissues such as the hypoblast and trophoblast, has diverged in terms of both gene expression and morphology.

Elf5 and Ets2 maintain the mouse extraembryonic ectoderm in a dosage dependent synergistic manner.

The ETS superfamily transcription factors Elf5 and Ets2 have both been implicated in the maintenance of the extraembryonic ectoderm (ExE) of the mouse embryo. While homozygous mutants of either gene result in various degrees of ExE tissue loss, heterozygotes are without phenotype. We show here that compound heterozygous mutants exhibit a phenotype intermediate to that of the more severe Elf5-/- and the milder Ets2-/- mutants. Functional redundancy is shown via commonalities in expression patterns, in target gene expression, and by partial rescue of Elf5-/- mutants through overexpressing Ets2 in an Elf5-like fashion. A model is presented suggesting the functional division of the ExE region into a proximal and distal domain based on gene expression patterns and the proximal to distal increasing sensitivity to threshold levels of combined Elf5 and Ets2 activity.

Elf5 counteracts precocious trophoblast differentiation by maintaining Sox2 and 3 and inhibiting Hand1 expression.

In mice the transcription factor Elf5 is necessary for correct trophoblast development. Upon knockdown of Elf5, TS cells display neither a decrease in proliferation nor an increase in cell death but rather an increased propensity to differentiate. Such cells rapidly lose Sox2 and 3 expression, while transiently upregulating the giant cell differentiation determinant gene Hand1. Other genes affected within 24h of Elf5 knock-down, many of which have not previously been implicated in trophoblast development, exhibited in vivo expression domains and in vitro expression responses consistent with Elf5 having a role in counteracting trophoblast differentiation. In an ES to TS differentiation assay using Cdx2 overexpression with Elf5 loss of function cell lines, it was shown that Elf5 is necessary to prevent terminal trophoblast differentiation. This data thus suggest that Elf5 is a gatekeeper for the TS to differentiated trophoblast transition thereby preventing the precocious differentiation of the undifferentiated extraembryonic ectoderm.

Specific epiblast loss and hypoblast impairment in cattle embryos sensitized to survival signalling by ubiquitous overexpression of the proapoptotic gene BAD.

Early embryonic lethality is common, particularly in dairy cattle. We made cattle embryos more sensitive to environmental stressors by raising the threshold of embryo survival signaling required to overcome the deleterious effects of overexpressing the proapoptotic protein BAD. Two primary fibroblast cell lines expressing BAD and exhibiting increased sensitivity to stress-induced apoptosis were used to generate transgenic Day 13/14 BAD embryos. Transgenic embryos were normal in terms of retrieval rates, average embryo length or expression levels of the trophectoderm marker ASCL2. However both lines of BAD-tg embryos lost the embryonic disc and thus the entire epiblast lineage at significantly greater frequencies than either co-transferrred IVP controls or LacZ-tg embryos. Embryos without epiblast still contained the second ICM-derived lineage, the hypopblast, albeit frequently in an impaired state, as shown by reduced expression of the hypoblast markers GATA4 and FIBRONECTIN. This indicates a gradient of sensitivity (epiblast > hypoblast > TE) to BAD overexpression. We postulate that the greater sensitivity of specifically the epiblast lineage that we have seen in our transgenic model, reflects an inherent greater susceptibility of this lineage to environmental stress and may underlie the epiblast-specific death seen in phantom pregnancies.

Trophoblast development.

This review summarises current knowledge about the specification, commitment and maintenance of the trophoblast lineage in mice and cattle. Results from gene expression studies, in vivo loss-of-function models and in vitro systems using trophoblast and embryonic stem cells have been assimilated into a model seeking to explain trophoblast ontogeny via gene regulatory networks. While trophoblast differentiation is quite distinct between cattle and mice, as would be expected from their different modes of implantation, recent studies have demonstrated that differences arise much earlier during trophoblast development.

Elf5 regulation in the trophectoderm.

Mouse Elf5 is expressed exclusively in the trophectoderm from the late blastocyst stage to postgastrulation. We demonstrate here that the proximal promoter is used for trophectoderm expression but is not sufficient on its own. In transgenic assays, deletion of a differentially methylated region (DMR) within the promoter has no effect on the activation and maintenance of trophectoderm expression and does not result in ectopic activity. Two redundant enhancers drive Elf5 expression to the extraembryonic ectoderm and ectoplacental cone. The enhancers, located in the 5' half of intron 1 and 3' half of intron 2, require the presence of 1.8kbp, although not the DMR, of the endogenous proximal promoter for optimal activity. These trophectoderm enhancers are mouse specific. A cattle Elf5 BAC reporter transgene is not expressed in mouse trophectoderm although it is expressed in skin, a known foetal domain of mouse Elf5 expression. The established importance of Elf5 for mouse trophectoderm at pre- and perigastrulation stages is not a conserved mammalian feature as Elf5 expression localises to embryonic as opposed to trophectodermal ectoderm in cattle.

The microenvironment patterns the pluripotent mouse epiblast through paracrine Furin and Pace4 proteolytic activities.

The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell-cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

Trophectoderm lineage determination in cattle.

The trophectoderm (TE) and inner cell mass (ICM) are committed and marked by reciprocal expression of Cdx2 and Oct4 in mouse late blastocysts. We find that the TE is not committed at equivalent stages in cattle, and that bovine Cdx2 is required later, for TE maintenance, but does not repress Oct4 expression. A mouse Oct4 (mOct4) reporter, repressed in mouse TE, remained active in the cattle TE; bovine Oct4 constructs were not repressed in the mouse TE. mOct4 has acquired Tcfap2 binding sites mediating Cdx2-independent repression-cattle, humans, and rabbits do not contain these sites and maintain high Oct4 levels in the TE. Our data suggest that the regulatory circuitry determining ICM/TE identity has been rewired in mice, to allow rapid TE differentiation and early blastocyst implantation. These findings thus emphasize ways in which mice may not be representative of the earliest stages of mammalian development and stem cell biology.

Nuclear transfer-specific defects are not apparent during the second week of embryogenesis in cattle.

Somatic cell nuclear transfer (NT)-specific effects on postblastocyst early cattle embryogenesis were investigated by comparison to in vitro-produced (IVP) embryos grown under identical conditions to embryonic days (E) 14 and 15. Recipient effects were excluded by transferring mixed batches of NT and IVP embryos into each cow. Embryo recovery rates, proportions with an epiblast and embryo, as well as epiblast dimensions did not differ between NT and IVP embryos. A developmental expression profile was determined for nine trophoblast markers, two inner cell mass (ICM)/epiblast markers, and E-cadherin at nine time points between E7 and E26, providing a molecular gene signature assay for developmental progression. Gene expression levels for these genes (Cdx2, Elf5, Mash2, Ifn-tau, Furin, Kunitz1, Pag11, Gata3, Oct4 and Ifitm3) were equal in NT and IVP embryos of equivalent length. Furthermore, the average residual deviation of all 10 genes did not differ significantly suggesting an overall "normality" in gene expression of E14/15 NT embryos. The absence of NT-specific defects during the second, highly selective, week of cattle embryogenesis is interpreted as supportive for the view that NT-associated defects are predominantly of an epigenetic nature.

The Ets transcription factor Elf5 specifies mammary alveolar cell fate.

Hormonal cues regulate mammary development, but the consequent transcriptional changes and cell fate decisions are largely undefined. We show that knockout of the prolactin-regulated Ets transcription factor Elf5 prevented formation of the secretory epithelium during pregnancy. Conversely, overexpression of Elf5 in an inducible transgenic model caused alveolar differentiation and milk secretion in virgin mice, disrupting ductal morphogenesis. CD61+ luminal progenitor cells accumulated in Elf5-deficient mammary glands and were diminished in glands with Elf5 overexpression. Thus Elf5 specifies the differentiation of CD61+ progenitors to establish the secretory alveolar lineage during pregnancy, providing a link between prolactin, transcriptional events, and alveolar development.

Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts.

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4, Otx2, Ifitm3, GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (beta-actin, beta-tubulin and GAPDH) in 21 NT blastocysts with that in genetically half-identical in vitro produced (IVP, n=19) and in vivo (n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts. Ifitm3 expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NT and IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP or in vivo embryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer. Oct4 levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed that in vivo embryos resembled each other much more than did NT and IVP blastocysts. Furthermore, in vivo embryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivo versus in vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

Postnatal development of the murine cerebellar cortex: formation and early dispersal of basket, stellate and Golgi neurons.

The cerebellar cortex consists of a small set of neuronal cell types interconnected in a highly stereotyped way. While the development of cerebellar cortical projection neurons, i.e. Purkinje cells, and that of granule cells has been elucidated in considerable detail, that of cerebellar cortical inhibitory interneurons is still rather fragmentarily understood. Here, we use mice expressing green fluorescent protein (GFP) from the Pax2 locus to analyse the ontogenesis of these cells. Numbers of Pax2-positive inhibitory interneuronal precursors increase following a classical sigmoidal growth curve to yield a total of some 905.000 +/- 77.000 cells. Maximal cell increase occurs at about postnatal day (P)5.4, and some 75% of all inhibitory interneurons are generated prior to P7. Conjoint analysis of the developmental accruement of Pax2-GFP-positive cells and their cell cycle distribution reveals that, at least at P0 and P3, the numerical increase of these cells results primarily from proliferation of a Pax2-negative precursor population and suggests that Pax2 expression begins at or around the final mitosis. Following their terminal mitosis, inhibitory cerebellar cortical interneurons go through a protracted quiescent phase in which they maintain expression of the cell cycle marker Ki-67. During this phase, they translocate into the nascent molecular layer, where they stall next to premigratory granule cell precursors without penetrating this population of cells. These observations provide a quantitative description of cerebellar cortical inhibitory interneuron genesis and early differentiation, and define Pax2 as a marker expressed in basket and stellate cells, from around their final mitosis to their incipient histogenetic integration.