LifeMap Discovery provides easy access to a wealth of information on mammalian development and integrates it with stem cell biology and regenerative medicine information.

The Tri-leveled Structure

"System": A collection of organs, which share similar functions. The system tag provides a physiological context to organs and tissues. For example, the reproductive system, which includes the ovary and testis organs.

The Organ/Tissue Level

The Anatomical Compartment Level

Each anatomical compartment has a corresponding card that provides a brief description, accompanied by images describing its structure, anatomy and development, as well as links to its populating cells. In addition, the card provides lists of genes specifically expressed in the anatomical compartment or in its populating cells, as annotated from large scale in situ hybridization databases, and relevant high throughput experiments. The card also provides links to related stem, progenitor and primary cells, as well as details of associated diseases and cell therapy applications related to the anatomical compartment or to its populating cells.

The Cell Level

The information provided in the cell cards aims to assist stem cell researchers to match and identify stem cell derivatives, obtained from differentiation protocols, with embryonic in vivo cells by means of gene expression profiles and selective gene markers. The information provided may also assist researchers in enhancing differentiation protocols, based on available signaling information.

The Ontology Tree Structure

The interactive ontology viewer in LifeMap Discovery is designed to enable researchers to learn about the hierarchical order of development, within the spatiotemporal context of each developing tissue/anatomical compartment/cell. Each entity is connected, via a navigable link, to its ancestor(s) ("developed from") and its descendants ("develops to"). All compartments and cells are also linked to their related organ/tissue, where all the information is aggregated.

Developmental Time

LifeMap Discovery provides annotations in the cell cards for the developmental stage of cells in the following fashion:

DPC (days post-coitum) – Embryonic days (e.g., E13.5 for 13.5 dpc). Human gestational weeks are sometimes added as a note in the cell card of a mouse cell, and mouse developmental times are sometimes annotated in the notes field of human cells.

Stage - numbers representing TS (Theiler Stage), when mouse development is being described, CS (Carnegie stage), when human development is being described, HH (Hamburger-Hamilton) when chick development is being described.

When the cells are present postnatal, the exact days are also annotated (e.g., P10). When the exact developmental time is not available, the timing of first appearance of the cells is generally annotated as either prenatal/postnatal. When information for the developmental time is completely absent, the N/A (not available) annotation is presented. Thus, the developmental time annotations enable us to describe complex multi-staged developmental processes without losing context.

Species Selection

LifeMap Discovery aims to present a carefully compiled and comprehensive view of mammalian development, which is a complex and well-orchestrated process. While some developmental stages are conserved among most mammalian species, significant interspecies variations in developmental processes require a species-specific description of development.

The organ/tissue and anatomical compartment levels do not have a species annotation since information from multiple species can be aggregated to them. However, all the cells in the database have a species annotation in the form of an icon depicting the species in which the provided information was observed.

Due to the wealth of information available on early human development, and the clinical relevance of human embryonic stem cells (obtained from human blastocysts) the "early development" stages presented in the database describe human stages, defined as the stages from the zygote to the three germ layers (endoderm, mesoderm and ectoderm). Due to ethical and practical limitations, detailed developmental information for later stages of the human embryo development is scarce. Therefore, the later stages of embryonic development, from the formation of the three germ layers to birth, are described mostly for the mouse embryo, which has been extensively studied by developmental biologists and stem cell researchers worldwide. In some rare cases, information from chick species is provided.

Species type is also annotated for gene expression, and appears as an icon near the gene name. The logic behind presentation of species-diverse gene expression information is:

• A lot of data on gene expression during development belongs to species whose development is not described in our database, thus the information is not lost and is included in the relevant mouse/human cells.
• Often, information from the literature is partial and the ortholog is not known.

Signal Modeling

Incoming, Internal and Outgoing Signals

Signals that are activated by secreted proteins are defined in LifeMap Discovery as "incoming signals" and are detailed in the cell cards relating to cells that are affected by the signal. When the cells that secrete the signal are known, they are mentioned in the signal details as the source cells. In such cases, the signal is presented in the source cell card as an "outgoing signal". Often, the actual secreted molecule that activates the signal is unknown, while its effect, e.g., induction or inhibition of certain genes, such as transcription factors, is known. Such signals are defined in the database as "internal signals" and their name depicts the downstream genes affected by the signal, followed by their expression effect (as 'induced' or 'inhibited').

Gradient Signal

Genes in Downstream Cascade

Each signal affects genes or gene products. When the affected genes are known, the signal cascade is presented, where the effect is defined as: induced, inhibited or involved. A gene that is labeled as "Involved" in a signal means that its product is involved in molecular interactions with other genes products within the cascade, although its own gene expression (or activity) remains unaffected by the signal.

For each signal, the genes known to participate in the downstream cascade, and the relationship between them, can be viewed under "see details" within the signal scheme.

Signal Outcome

Several types of signal outcomes are described:

• • Differentiation:
  • A process in which a less specialized cell becomes more specialized or takes on a more determined fate.
• • Epithelial-to-Mesenchymal Transition (EMT):
  • A process in which epithelial cells lose their cell polarity and cell-cell contacts, resulting in increased migratory and invasive potential and generation of mesenchymal cells.
• • Mesenchymal-to-Epithelial Transition (MET):
  • A process in which motile mesenchymal cells gain cell polarity and cell-cell adhesion properties and eventually become stationary epithelial cells.
• • Proliferation:
  • A process in which cells multiply and increase in number.
• • Migration:
  • A process in which cells move from one region to another.
• • Apoptosis:
  • A cellular death process in which cells undergo a programmed sequence of events, marked by the fragmentation of nuclear DNA, without releasing harmful substances into the surrounding area.
• • Inhibition of Differentiation:
  • A process in which the signal prevents the less specialized cell to differentiate to a specific fate.
• • Cell Cycle Arrest:
  • A process by which the mitotic cycle is halted.
• • Survival:
  • A process enabling the continued existence of a cell. This type of signal effect is common to neurons.
• • Maintenance:
  • A process in which cells retain their cell identity.

If the signal affects the cell by inducing differentiation or another processes which result in a change in cell identity (EMT, MET, Migration, or Inhibition of Differentiation), a descendent cell ('derived cell') will be linked to the signal.

Matching to Stem Cells

Stem cells serve as a platform for extensive research largely aimed toward clinical applications, such as cell replacement therapeutics, trophic support and in vitro disease models. Each stem cell procedure normally follows specific protocols which describe the growth conditions and cell markers for these cells. Often, the stem cells used as source cells for the protocols are difficult to characterize, and as the cells differentiate, cell identity is difficult to establish and is frequently based on few cell surface markers, fragilely linking them to known lineages (and occasionally, uncharacterized side populations of cells remain after the differentiation process, contaminating the otherwise homogenous culture).

The ability to match differentiating stem cells to their counterparts in live, developing organism, offers the researcher powerful tools for characterizing and understanding the differentiation process.

Understanding the molecular signals and gene expression patterns that regulate cellular differentiation and maturation, can further enhance design of differentiation protocols that better mimic in vivo processes.

LifeMap Discovery exploits the wealth of data accumulated on gene expression in specific cells during development, to match in vitro cells to one or more in vivo cells or anatomical compartments of the embryo or mature mammal. The matching is performed either manually or computationally, and is primarily based on gene expression profiles, but can also be based on function and morphology observations described in the literature.

All mammalian embryos begin their development through the same basic process. The zygotes and early embryos of mammals are virtually indistinguishable from each other until the embryo begins to assume its species-specific characteristics as it develops specialized organs, tissues and cells

The Zygote

The zygote is the first stage of the early embryo, and is formed upon fusion of the haploid (n) male and female pronucleus, wherein the chromosome number in the fertilized ovum becomes diploid (2n). One or two polar bodies are often visible in the perivitelline space, the area between the embryo and the zona pellucida (a glycoprotein membrane that surrounds the plasma membrane of an oocyte).

Fertilization, which normally takes place in the ampulla of the uterine tube, requires contact of spermatozoa with the zona pellucida of the secondary oocyte, penetration of one or more spermatozoa through the zona pellucida and formation of the male and female pronuclei. Completion of the second meiotic division (the first meiotic division occurs prior to fertilization and the first polar body is a result of this division) marks the commencement of the first mitotic division, or cleavage, of the zygote.

Cleavage and Blastocyst Formation

While the zygote is transported through the Fallopian tubes to the uterus, its cells undergo subsequent mitotic divisions, called cleavages, wherein the daughter cells are smaller than the parent, thus, the embryo increases its cell numbers without increasing its total mass or size. The 2, 4 and 8-cell embryo (also known as the early morula), is formed and at this stage the embryonic cells undergo compaction, forming the morula. The mammalian embryo obtains the majority of its nourishment through the placenta and umbilical cord, and therefore, the mammalian cleavage is known as holoblastic cleavage, as opposed to meroblastic cleavage in embryos that receive the majority of their nourishment through an egg yolk. The cells in cleavage-stage embryos are known as blastomeres. As they continue to divide, the cells segregate to the inner and outer cells, forming a cavity (the blastocoele cavity), thereby defining the blastocyst-stage embryo. The inner group of cells (called the inner cell mass, ICM) will become the embryo, while the outer group of cells, called the trophoblast, will become specialized extraembryonic membranes, adapted for intrauterine mammalian development ( chorion, amnion, placenta, yolk sac, umbilical cord). The blastocyst stage is therefore the first time that two distinctive tissues are present, as a result of a segregation that is often described as the first lineage-choice of the embryonic cells.

Trophoblast and the Inner Cell Mass

The blastocyst stage includes dramatic events such as, hatching of the blastocyst from the zona pellucida, and implantation of the embryo in the uterine wall. The zona pellucida prevents adherence of the blastocyst to the oviduct walls (a condition which in humans is called an ectopic ortubal pregnancy). When the embryo reaches the uterus, zona hatching enables implantation, a complex series of events that involves the formation of the enveloping syncytiotrophoblast layer and cytotrophoblast layers of the placenta. Both layers originate in the embryonic trophoblast, and invade the maternal decidua (the maternalpart of the placenta), creating an extensive interface with the maternal tissues.

The epiblast is formed as the ICM segregates into a bilaminar embryonic disc (bilaminar blastoderm), which consists of two epithelial layers: the external (dorsal) epiblast and the internal (ventral) hypoblast. The hypoblast cells delaminate from the inner cell mass to line the blastocoel cavity, where they give rise to the extraembryonic endoderm, which forms the yolk sac.

Germ Layer Formation

Gastrulation is the process in which the ICM is converted into the three germ layers: ectoderm, mesoderm and endoderm. Cells begin to migrate into the center of the blastula and to form primitive germ layers, transforming the blastula into a gastrula. Formation of the primitive streak marks the first event of gastrulation. The primitive streak originates from the anterior epiblast, and appears as an elongating groove (primitive groove) on the dorsal midsagittal surface of the epiblast, along the anterior-posterior axis of the embryo. The rostro-caudal and medial-lateral axes of the embryo are defined by the primitive streak. The rounded primitive node, or Hensen's node, is situated at the cranial tip of the primitive streak, and contains a depression called the primitive pit, which is continuous with the primitive groove. Cells from the epiblast migrate into the interior of the embryo, via the primitive streak, in a process termed ingression, which involves a cellular epithelial-to-mesenchymal transition (EMT). The initial wave of migrating cells streams through the primitive streak, displacing the hypoblast cells to become the definitive endoderm, which ultimately produces the future gut derivatives and gut linings.

The second wave of migrating cells populates a layer between the epiblast and the definitive endoderm, thereby forming the mesoderm layer. The intraembryonic mesoderm cells later give rise to five subpopulations of cells: paraxial mesoderm, intermediate mesoderm, lateral plate mesoderm, cardiogenic mesoderm and a population that forms a midline tube called the notochordal process. The notochordal process originates in the primitive node and is the precursor of the flat-shaped notochordal plate, which after detaching from the endoderm, fuses its free rims together to form a rod that is known as the notochord. Once the mesoderm has formed, the remaining epiblast cells cease to ingress and form the ectoderm. The ectoderm gives rise to two distinct lineages, namely, the surface ectoderm and the neural crest.

Further Development

Central Signaling Pathways

The Wnt Pathway

WNT signaling outcomes can be contradictory, as manifested during embryonic skeletal muscle formation. While canonical WNTs (WNT1 and WNT3a) are important in paraxial mesoderm (i.e., somite) induction and differentiation towards muscle cells in the trunk region (axial muscles), the same signals were shown to be inhibitory for head myogenesis.

The SHH Pathway

This pathway is conserved among species and, when aberrant, causes severe malformations and growth arrest. Sonic Hedghog is one of three mammalian orthologues of the drosophila Hedghog (the other two are Indian Hedghog and Desert Hedghog) and acts in the pathway is involved in regulation of cell fate and embryonic patterning during early vertebrate development and in the formation of multiple organs, such as the heart, lungs, eye, brain, bone, pancreas and spinal cord, among others. The Shh pathway remains active in the adult organism, where it plays a role in maintaining populations of multipotent stem cells.

The Notch Pathway

In mammalian Notch pathways, members of the Delta-like Jagged (JAG1, JAG2) and the DLL (DLL1, DLL3, DLL4) families serve as ligands for the Notch receptors. The ligand-receptor complex formation induces a three-series cleavage event, which facilitates downstream effects on transcription via the activation of the canonical Notch target genes: Myc, p21, and the HES-family members

Notch signaling plays a key role in hair follicle cells differentiation, by defining the outer root sheath (ORS) cells. Mutations of Notch family members cause a switch from ORS to epidermal differentiation, resulting in replacement of hair follicle by an epidermal cyst. In addition, during epidermal stratification, Notch signaling plays a key role in inducing differentiation of suprabasal layers. Differentiation of spinous keratinocytes into granular keratinocytes is Notch-dependent and is defined in two-step signaling process: first, Notch1 inhibits differentiation, allowing emergence of a sufficient number of cells by proliferation. When the appropriate number of cells is accumulated, Notch induces cell cycle exit and progress into the differentiation stage.

The BMP Pathway

BMPs induce the formation of both cartilage and bone. During bone formation, BMPs facilitate the development of functional bone marrow. In addition, BMPs play a role in a number of non-osteogenic developmental processes. BMP-2 induces the neuronal phenotype of neural crest cells, while BMP-4 directs somite development by inhibiting myogenesis. BMP-4 and BMP-7 specifically induce a sympathetic adrenergic phenotype. In the limb bud, and as part of the FGF-4 and SHH interaction, BMP-2 inhibits limb bud expansion and induces the formation of chondrocyte and osteoblast precursor cells. BMPs are also involved in dorsal/ventral patterning. BMP-4 specifies the development of ventral structures (skin from ectoderm and connective tissue/ blood from mesoderm). Competitive inhibition of BMP4 is responsible for the development of dorsal structures (nervous system and muscle). Recent reports indicate that a heterodimer composed of BMP-4 and BMP-7 is a potent inducer of mesoderm.

Types I and II TGF-beta receptors play significant roles in BMP binding and signaling. Upon ligand binding, heterotetramerization occurs, as the type II receptor activates the type I receptor kinase domain, thereby initiating phosphorylation of cytoplasmic Smad proteins and downstream signal transduction.

The FGF Pathway

To date, the FGF superfamily consists of 23 members, all of which contain a conserved 120-amino-acid (aa)-long core region which is comprised of six identical, interspersed amino acids. The signaling pathway is executed via one of the four tyrosine kinase FGF receptors (FGFRs). In vertebrates, the extracellular domains of the receptors undergo alternative splicing to generate a vast variety of receptors, with variable affinities for their respective FGF ligands. FGF ligands bind the extracellular domain of the FGFR, in combination with heparan sulphate, to form a complex resulting in the transphosphorylation of specific intracellular tyrosine residues, thereby triggering the downstream activation of cytoplasmic signal transduction pathways. Well known signals activated as a result of FGFR activation are the Ras/ERK pathway (affects differentiation), the Akt pathway (affects cell survival) and the protein kinase C (PKC) pathway (involved in cell morphology and migration).