Blastocyst Development

Embryology - 5 Dec 2023        Expand to Translate
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Introduction

Human Blastocyst (day 5)

(Greek, blastos = sprout + cystos = cavity) or blastula, the term used to describe the hollow cellular mass that forms in early development.

The blastocyst consists of cells forming an outer trophectoderm (TE, trophoblast) layer, an inner cell mass (ICM, embryo blast) and a blastocoel (fluid-filled cavity). The inner cell mass will form the entire embryo, and is the source of true embryonic stem cells capable of forming all cell types within the embryo. In mammals, the trophectoderm will form key cells (trophoblast) of the fatal component of the placenta.

In humans, blastocyst stage of development occurs during the first and second week following fertilization (GA week 3 and 4) and is described initially as Carnegie stage 3. This stage is followed by blastocyst hatching and implantation.

One of the first key developmental patterning decisions in the morula to blastocyst development is TE or ICM cell fate. In the mouse model, Hippo(TEAD4) and Notch(Cdx2) together appear regulate this early fate decision.

Blastomere genomic divergence

Mouse blastocyst development

Some Recent Findings

Human blastoids model blastocyst development and implantation "One week after fertilization, human embryos implant into the uterus. This event requires that the embryo forms a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form a blastocyst model, which we termed blastoid1. Here we show that naive human pluripotent stem cells (PXGL hPSCs)2 triply inhibited for the Hippo, TGF-β, and ERK pathways efficiently (>70%) form blastoids generating blastocyst-stage analogs of the 3 founding lineages (>97% trophectoderm, epiblast, and primitive endoderm) according to the sequence and timing of blastocyst development. Blastoids spontaneously form the first axis and we observe that the epiblast induces the maturation of the polar trophectoderm that consequently acquires the specific capacity to attach to hormonally-stimulated endometrial cells, as during implantation. Such a human blastoid is a faithful, scalable, and ethical model to explore human implantation and development." Computational analysis of single-cell transcriptomics data elucidates the stabilization of Oct4 expression in the E3.25 mouse preimplantation embryo "Our computational analysis focuses on the 32- to 64-cell mouse embryo transition, Embryonic day (3.25), whose study in literature is concentrated mainly on the search for an early onset of the second cell-fate decision, the specification of the inner cell mass (ICM) to primitive endoderm (PE) and epiblast (EPI). We analysed single-cell (sc) microarray transcriptomics data from E3.25 using Hierarchical Optimal k-Means (HOkM) clustering, and identified two groups of ICM cells: a group of cells from embryos with less than 34 cells (E3.25-LNCs), and another group of cells from embryos with more than 33 cells (E3.25-HNCs), corresponding to two developmental stages. Although we found massive underlying heterogeneity in the ICM cells at E3.25-HNC with over 3,800 genes with transcriptomics bifurcation, many of which are PE and EPI markers, we showed that the E3.25-HNCs are neither PE nor EPI. Importantly, analysing the differently expressed genes between the E3.25-LNCs and E3.25-HNCs, we uncovered a non-autonomous mechanism, based on a minimal number of four inner-cell contacts in the ICM, which activates Oct4 in the preimplantation embryo. Oct4 is highly expressed but unstable at E3.25-LNC, and stabilizes at high level at E3.25-HNC, with Bsg highly expressed, and the chromatin remodelling program initialised to establish an early naïve pluripotent state. Our results indicate that the pluripotent state we found to exist in the ICM at E3.25-HNC is the in vivo counterpart of a new, very early pluripotent state. We compared the transcriptomics profile of this in vivo E3.25-HNC pluripotent state, together with the profiles of E3.25-LNC, E3.5 EPI and E4.5 EPI cells, with the profiles of all embryonic stem cells (ESCs) available in the GEO database from the same platform (over 600 microarrays)." Physiological profile of undifferentiated bovine blastocyst-derived trophoblasts "Trophectoderm of blastocysts mediate early events in fetal-maternal communication, enabling implantation and establishment of a functional placenta. Inadequate or impaired developmental events linked to trophoblasts directly impact early embryo survival and successful implantation during a crucial period that corresponds with high incidence of pregnancy losses in dairy cows. As yet, the molecular basis of bovine trophectoderm development and signaling towards initiation of implantation remains poorly understood. In this study, we developed methods for culturing undifferentiated bovine blastocyst-derived trophoblasts and used both transcriptomics and proteomics in early colonies to categorize and elucidate their functional characteristics. A total of 9270 transcripts and 1418 proteins were identified and analyzed based on absolute abundance. We profiled an extensive list of growth factors, cytokines and other relevant factors that can effectively influence paracrine communication in the uterine microenvironment. Functional categorization and analysis revealed novel information on structural organization, extracellular matrix composition, cell junction and adhesion components, transcription networks, and metabolic preferences. Our data showcase the fundamental physiology of bovine trophectoderm and indicate hallmarks of the self-renewing undifferentiated state akin to trophoblast stem cells described in other species."

More recent papers

This table allows an automated computer search of the external PubMed database using the listed "Search term" text link. This search now requires a manual link as the original PubMed extension has been disabled. The displayed list of references do not reflect any editorial selection of material based on content or relevance. References also appear on this list based upon the date of the actual page viewing. References listed on the rest of the content page and the associated discussion page (listed under the publication year sub-headings) do include some editorial selection based upon both relevance and availability. More? References | Discussion Page | Journal Searches | 2019 References | 2020 References Search term: Blastocyst Development | Blastocyst | Blastocoel | Inner Cell Mass | | Trophectoderm

Older papers

These papers originally appeared in the Some Recent Findings table, but as that list grew in length have now been shuffled down to this collapsible table. See also the Discussion Page for other references listed by year and References on this current page. Four simple rules that are sufficient to generate the mammalian blastocyst "We implemented experimentally reported mechanisms for polarity, cell-cell signaling, adhesion, and apoptosis as a set of developmental rules in an agent-based in silico model of physically interacting cells. We find that this model quantitatively reproduces specific mutant phenotypes and provides an explanation for the emergence of heterogeneity without requiring any initial transcriptional variation. It also suggests that a fixed time point for the cells' competence of fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) sets an embryonic clock that enables certain scaling phenomena, a concept that we evaluate quantitatively by manipulating embryos in vitro. Based on these observations, we conclude that the minimal set of rules enables the embryo to experiment with stochastic gene expression and could provide the robustness necessary for the evolutionary diversification of the preimplantation gene regulatory network." Asymmetric division of contractile domains couples cell positioning and fate specification "During pre-implantation development, the mammalian embryo self-organizes into the blastocyst, which consists of an epithelial layer encapsulating the inner-cell mass (ICM) giving rise to all embryonic tissues. In mice, oriented cell division, apicobasal polarity and actomyosin contractility are thought to contribute to the formation of the ICM. However, how these processes work together remains unclear. Here we show that asymmetric segregation of the apical domain generates blastomeres with different contractilities, which triggers their sorting into inner and outer positions." Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst "The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established." Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage "We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA)." Blastocyst gene expression correlates with implantation potential "Compared with blastocysts that resulted in healthy fetal development, blastocysts that failed to implant (negative) showed decreased B3gnt5 and Eomes gene expression, while blastocysts that resulted in spontaneous pregnancy loss (absorption) displayed decreased Wnt3a and Eomes gene expression." FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst "Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. ... In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner."

Movies

Human Blastocyst

Human Blastocyst (day 3 to 6)

‎‎Day 3 to 6 Page | Play

‎‎Contractions Page | Play

‎‎Hatching Page | Play

Model Development

‎‎Morula Model Page | Play

‎‎Blastocyst Model Page | Play

Movies

A recent review{{#pmid28681376|PMID28681376}} of the initial morula to blastocyst formation, based upon animal models, identifies important mechanical steps:

1. Compaction - morula blastomeres packing tightly (microfilament cytoskeleton)
2. Cleavage planes - spindle direction of dividing cells (mitosis).
3. Polarisation - blastomeres apico-basal (Hippo pathway, Yes-associated protein (Yap)
4. Cavitation - blastocoel formation with cycles of cavity expansion and collapse.
   1. epiblast cells - contact the polar trophectoderm
   2. primitive endoderm - facing the cavity

Human Blastocyst Formation

The table below shows human blastocyst in vitro development changes during week 1.

Labeled Blastocyst

Blastocyst Hatching

Mouse Blastocyst hatching

Blastocyst hatching from zona pellucida (mouse)	Blastocyst hatching from zona pellucida (human)

Model Human Blastocyst Development

The following figure is from a recent study using video and genetic analysis of in vitro human development during week 1 following fertilization.

• EGA - embryonic genome activation
• ESSP - embryonic stage–specific pattern, four unique embryonic stage–specific patterns (1-4)

Links: Figure with legend

Mouse Blastocyst Gene Expression

General gene expression patterns are indicated from genomic profiling.

• red - loss of maternal mRNAs
• green - activation of embryonic genome (EGA)
• purple - maternal gene activation (MGA)
• orange - continuous expression

Inner Cell Mass

Human Blastocyst (day 5)

The inner cell mass forms an inner layer of larger cells is also called the "embryoblast" is a cluster of cells located and attached on one wall of the outer trophoblast layer. In week 2 this mass will differentiate into two distinct layers the epiblast and hypoblast.

The hypoblast (or primitive endoderm) is a transient epithelial layer facing towards the blastoceol, it is replaced in week three by the gastrulation migrating endoderm cells.

The epiblast layer will form the entire embryo and undergoes gastrulation in week three to form the 3 germ layers. It also forms an epithelial layer lining the amniotic cavity.

Trophectoderm

The trophectoderm (TE) outer layer of smaller cells is also called the "trophoblast" epithelium, that will later form a key component of the placenta. A key early function is for the transport of sodium (Na+) and chloride (Cl-) ions through this layer into the blastocoel. Later in week 2 this layer will differentiate into two distinct trophoblast layers the syncytiotrophoblast and cytotrophoblast cells and are key to implantation and early placentation.

Differentiation of the early layer has been shown to be regulated by the transcription factors Tead4 and then Caudal-related homeobox 2 (Cdx2).

Links: trophoblast | OMIM -Tead4 | OMIM - Cdx2

Blastocoel

Mouse - blastocoel formation

• trophectoderm transports of Na+ and Cl- ions through this layer into the blastocoel
• generates an osmotic gradient driving fluid across this epithelium
• distinct apical and basolateral membrane domains specific for transport
• facilitates transepithelial Na+ and fluid transport for blastocoel formation
• transport is driven by Na, K-adenosine triphosphatase (ATPase) in basolateral membranes of the trophectoderm

Blastocyst Metabolism

Mouse blastocyst GLUT8 expression.

At the blastocyst stage, mammalian development metabolism switches on anaerobic glycolysis metabolism to satisfy metabolic demands of growing blastocyst and formation of the blastocoel. This is thought to be driven by the integral membrane protein family of facilitative glucose transporters (GLUT or SLC2A).

• aerobic - oxidation of lactate and pyruvate via the citric acid cycle (Krebs cycle) and oxidative phosphorylation
• glycolysis- converts glucose into pyruvate
• GLUT - GLUcose Transporter (divided into 3 classes I-III)
• SLC2 - Solute Carrier Family 2

Glucose Transporter Expression

• GLUT1 - from zygote to blastocyst. (all mammalian tissues, basal glucose uptake)
• GLUT2 and GLUT3 - from late eight cell stage to blastocyst. (GLUT2, liver and pancreatic beta cells; GLUT3, all mammalian tissues, basal glucose uptake)
• GLUT4 - not expressed. (muscle and adipose tissue)
• GLUT8 - up-regulated at blastocyst stage. (central nervous system and heart)

(Data mainly from mouse development, adult tissue expression shown in brackets)

A mouse study, has shown GLUT8 is up-regulated following insulin stimulation, though a more recent GLUT8 knockout mouse shows normal early embryonic development in the absence of this transporter.

Links: Biochemistry - glucose transporters | GLUT1 | GLUT2 | GLUT8

Blastula Cell Communication

Two types of cell junctions have been identified located at different regions in the developing blastocyst.

Tight junctions Located close to outer surface create a seal, isolates interior of embryo from external medium.	Gap junctions Allow electrically coupling of the cells of epithelium surrounding the fluid-filled cavity.

Adhesion EM Images: GIT epithelia EM1 | GIT epithelia EM2 | GIT epithelia EM3 | Desmosome EM

Adhesion Cartoons: Tight junction | Adherens Junction | Desmosome | Gap Junction

Blastocyst Hatching - zona pellucida lost, ZP has sperm entry site, and entire ZP broken down by uterine secretions and possibly blastula secretions.

Uterine Glands - secretions required for blastocyst motility and nutrition

Links: MBoC Figure 21-69. The blastula

Blastocyst Hatching

At about day 5 the human blastocyst "hatches" out of the protective zona pellucida. This hatching allows increased growth, access to uterine nutrient secretions and blastocyst adhesion to the uterine lining. Associated with this hatching process are a series of physical contractions. In the blastocyst, repeated contractions occur after blastocoel formation and the frequency of contractions is greater during the hatching period than in the periods both before and after hatching. Interestingly, the same researchers in this mouse study suggest that the weaker contractions (less than 20% volume reduction) seen have a role in hatching, in contrast to strong contractions (20% or more volume reduction) have the opposite effect of inhibiting hatching. In the mouse model, the identified sites of zona pellucida shedding varied (24% mural site, 24% inner cell mass site, 17% equatorial site, and 35% other sites). Links: Blastocyst Day 5-6 Movie

<mediaplayer width='500' height='450' image="http://php.med.unsw.edu.au/embryology/images/a/af/Human_blastocyst_day_5-6.jpg">File:Human_blastocyst_day_5-6.mp4</mediaplayer> Human blastocyst contractions (day 5-6)

Molecular Factors

• TEA DNA- binding domain, these factors bind to the consensus TEA/ATTS cognate binding site
  • TEF-3 - renamed Tead1 and Tead4
  • Tead3 - is expressed in the placental syncytiotrophoblasts
• E-cadherin - Calcium ion-dependent cell adhesion molecule, a cell membrane adhesive protein required for morula compaction
• epithin - A type II transmembrane serine protease, identified in mouse for compaction of the morula during preimplantation embryonic development. Expressed from 8-cell stage at blastomere contacts and co-localises in the morula with E-cadherin.
• Na, K-adenosine triphosphatase - A sodium potassium pump that generates an osmotic gradient for fluid flow into the blastocoel
• Zonula occludens-1 - (ZO-1) Tight junction protein involved in morula to blastocyst transformation in the mouse

Blastocyst in Other Species

Mouse Blastocyst

• 

  Sox2 expression

• 

  Early gene expression

• 

  Early gene expression

• 

  Early gene expression

• 

  Early gene expression

Early mouse development model

Links: Mouse Development

Bovine Blastocyst

• 

  Bovine blastocyst KRT18, FN1 and MYL6 expression

• 

  Bovine blastocyst KRT18 and MYL6 expression

Links: Bovine Development

References

Reviews

Maître JL. (2017). Mechanics of blastocyst morphogenesis. Biol. Cell , 109, 323-338. PMID: 28681376 DOI.

Rasmussen TP & Corry GN. (2010). Epigenetic pre-patterning and dynamics during initial stages of mammalian preimplantation development. J. Cell. Physiol. , 225, 333-6. PMID: 20607796 DOI.

Cockburn K & Rossant J. (2010). Making the blastocyst: lessons from the mouse. J. Clin. Invest. , 120, 995-1003. PMID: 20364097 DOI.

Rossant J. (2007). Stem cells and lineage development in the mammalian blastocyst. Reprod. Fertil. Dev. , 19, 111-8. PMID: 17389140

Articles

Smith HL, Stevens A, Minogue B, Sneddon S, Shaw L, Wood L, Adeniyi T, Xiao H, Lio P, Kimber SJ & Brison DR. (2019). Systems based analysis of human embryos and gene networks involved in cell lineage allocation. BMC Genomics , 20, 171. PMID: 30836937 DOI.

Santos J, Pereira CF, Di-Gregorio A, Spruce T, Alder O, Rodriguez T, Azuara V, Merkenschlager M & Fisher AG. (2010). Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo. Epigenetics Chromatin , 3, 1. PMID: 20157423 DOI.

Dzamba BJ, Jakab KR, Marsden M, Schwartz MA & DeSimone DW. (2009). Cadherin adhesion, tissue tension, and noncanonical Wnt signaling regulate fibronectin matrix organization. Dev. Cell , 16, 421-32. PMID: 19289087 DOI.

Sheth B, Nowak RL, Anderson R, Kwong WY, Papenbrock T & Fleming TP. (2008). Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation. Exp. Cell Res. , 314, 3356-68. PMID: 18817772 DOI.

Nishioka N, Yamamoto S, Kiyonari H, Sato H, Sawada A, Ota M, Nakao K & Sasaki H. (2008). Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos. Mech. Dev. , 125, 270-83. PMID: 18083014 DOI.

Yamanaka Y, Ralston A, Stephenson RO & Rossant J. (2006). Cell and molecular regulation of the mouse blastocyst. Dev. Dyn. , 235, 2301-14. PMID: 16773657 DOI.

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Cite this page: Hill, M.A. (2023, December 5) Embryology Blastocyst Development. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Blastocyst_Development

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© Dr Mark Hill 2023, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G