By: Niki Striar, Rachel Fyler, and Julie Henry

Cell Signaling Master Genes Early Embryonic Development

Cell Signaling

Cell signaling in developmental cells usually occurs in the form of chemical signals. Cells often send chemical signals to other cells so that they may interact, by a signaling molecule from one cell binding to a receptor on the cell surface of another cell, causing a chain of events that carries the signal to the interior of the cell and amplifies that signal. Cells may integrate information from several different signals they receive simultaneously to create a unified action plan, as seen in development. (1) Two extremely important effects of cell signaling in development is the migration and patterning of cells, as outlined below. (2)
One important mode of signaling, Eph-ephrin signaling, lets migrating cells know where and when to begin moving, and where and when to stop. Cell migration and adhesion are the main forces morphing the cells into critical anatomical structures, and the signaling at the cell membrane is the ultimate controller. During migration, cells must move considerable distances and must reach a precise target or there will be deformities such as cleft palates. Eph-ephrin interaction ensures that repulsion, attraction and adhesion of cells allows the cells to migrate to their correct places in development. In this signaling pathway, the receptors are called Ephs, and their respective ligands are called ephrins. Eph-ephrin signaling is the largest migratory signaling pathway in a cell by both class and expanse of role. (2)

Extracellular interactions between the Eph receptors on the cell membrane and specific binding partners (ephrin ligands) causes intracellular signalling to change the cell’s activity. These signals interact with cytoskeletal structures within the cell to tell the cells which way to move and for what distance. To add an even greater level of specificity to this signaling pathway, there is a number of different Eph cell receptor types, all sensitive to different ephrin ligands, meaning cells have their own navigational system. Eph-ephrin signaling controls the development of many different tissue systems, perhaps most prevalently in nervous system, particularly in the guidance of axons. Eph-ephrin signaling is also important in the development of the vascular system, particularly with arterial vessels (ephrin-B2) and venous vessels (Eph-B4), cell sorting and boundary formation, neural crest cell migration, midline adhesion, and guiding stem cells in the brain and intestine to their proper niches. (2)

The Eph receptor was discovered in the 1980’s during research on tyrosine kinases involved in cancerous cells. Eph stands for erythropoietin-producing hepatocellular (EPH) receptor, beloning to the largest family of receptor tyrosine kinases (RTKs) with 16 different receptors, divided into Eph-A (1-10) and Eph-B (1- 6). These are distinguished by slight structural differences, but for the most part, all Ephs share similar structural features. In the extracellular domain where the Eph receptor interacts with the ephrin ligand there is a short hydrophobic ephrin binding domain, and in the intracellular domain there is an internal cytoplasmic signalling structure composed of a tyrosine kinase domain and protein interaction sites. (2)

There are 9 different types of ephrin ligands - ephrin-A1 through ephrin-A6 and ephrin-B1 through ephrin-B3. Ephrin-As will only bind to Eph-A receptors, and ephrin-Bs will only bind to Eph-B receptors; however, Eph-A4 will interact with both ephrin -A and -B. The ephrin ligands are expressed on the cell surface, thus close contact between cells is required for signaling to occur, allowing for more specific movement of cells. Only ephrin-Bs have a cytoplasmic tail on the inside of the cell, ephrin-As, on the other hand, have a very short tail linking them to the plasma membrane. The extracellular structure of the ephrin binds to the respective Eph receptor, alloing for intercellular signaling. This high-affinity binding between Eph receptors and ephrin ligands leads to signaling on the inside of the cell by the receptor following the receptor tyrosine kinase (RTK) model. (2)

In the absence of ligand bonding, Eph receptors are distorted by unphosphorylated tyrosines interacting tightly with the kinase domain; however, when the ephrin ligand bonds with the Eph receptor, RTKs become activated through phosphorylation and in turn phosphorylate downstream substrates, beginning a signal transduction cascade. Basically, the binding of the ephrin ligand to the Eph receptor transforms the receptor from a dormant state to an active state, causing signal transduction in the cell. This allows the cell to become motile and repulse, attract and adhere to other cells. Signal transduction in Ephs and ephrins does not target the nucleus primarily to regulate transcription and does not transform the cells or make them more proliferative, rather, it alters the cytoskeleton of the cell in a very precise manner. Defining how the context of a signaling event can promote or discourage repulsion or attraction is the largest challenge for biologists, and is a problem still being worked on today. (2)

Diagram of Eph-ephrin structure (2)
Diagram of Eph-ephrin structure (2)

In 1996 it was discovered that Eph-ephrin signaling is bidirectional, meaning that the binding of the ephrin to the Eph causes signal transduction in ephrin cell as well. The extracellular domain of the Eph receptors will take on a ligand-like role, and the ephrin ligands will take on a receptor-like role. Ephrins have their own intracellular signalling capability when bound to their respective Ephs - tyrosine kinase in the ephrin cells phosphorylates molecules present at the membrane to initiate signaling transduction in that cell. Scientists refer to the signaling in the eph receptor cell as the forward signal, and refer to the signaling in the ephrin ligand cell as the reverse signal. Both the forward signal and the reverse signal may happen at once. (2)

Eph-ephrin bidirectional signaling (2)
Eph-ephrin bidirectional signaling (2)

Ephs and ephrins control most of the development of the nervous system, determining how neurons wire together and axon guidance. Axons must grow great distances across the body sometimes, so the Eph-ephrin signaling pathway must be extremely precise. At the end of developing axonal profusions are growth cones, and below is a video showing the effects of Eph-ephrin signaling on the growth cones. (3) - a video of growth cone collapse and retraction

Patterning is another important aspect of development that is controlled by cell signaling. Sonic Hedgehog signaling controls patterning of the endoderm, mesoderm and ectoderm, and patterning of overlaying nervous tissue. The notochord sends the sonic hedgehog signal in an outwards wave to all of the surrounding cells. Sonic hedgehog signaling tells the midline cells of an organism to be one type of cell, and then various cells extending away from the midline are determined to be different types depending on how much of the hedgehog signal they receive due to their proximity to the signal. This ensures symmetry in an organism as well. When this signaling doesn’t happen, deformities such as cyclopia may occur, as shown in the master genes section of this page. Sonic hedgehog proteins are involved with just about all multi-cellular animals’ patterning, and is most complicated in vertebrates. (4)
Midline of an organism (5)
Midline of an organism (5)

Sonic hedgehog proteins are involved primarily in pattern formation, particularly in differentiating the dorsal side of the organism from the ventral side. Gene expression patterns are changed when hedgehog signaling acts on undifferentiated cell types to turn them into particular cells. The sonic hedgehog gene is used by just about every organ system at some point in development. There are also two other very similar hedgehog signaling pathways - the Indian hedgehog signal and the desert hedgehog signal. Because hedgehog signaling is involved in determining cell types, it acts in the precursors of tissues to differentiate those cells. (4)
Hedgehog signaling begins with a binding of the hedgehog ligand to a protein receptor called the Ptc. The Ptc receptor is an inhibitor of a protein called Smo, downstream of which there is a multi-protein complex called the Hedgehog signaling complex (HSC), which is made of several kinases. When Smo is inactivated, a protein called Ci undergoes cleavage which inhibits transcription, but when Smo is activated, the cleavage of Ci does not occur and transcription of hedgehog genes is induced. In either case, Ci enters the nucleus of the cell, but only when it does not undergo cleavage is transcription induced. In vertebrates there are 3 hedgehog genes, 2 Ptc genes, and 3 Ci genes, called Gli 1-3. Gli-1 and Gli-2 are transcriptional activators, whereas Gli-3 is a transcriptional repressor. (6)
Hedgehog signaling pathway (6)
Hedgehog signaling pathway (6)

(1) __ - a good website for an overview of cell signaling
(2) __ - an in-depth walkthrough of eph-ephrin signaling
(3)__ - discussion of eph-ephrin signaling's effect on the nervous system
(4) - overview of hedgehog signaling
(5) - image of midline in cells

(6) - explanation of hedgehog signaling pathway

Master Genes

Homeobox genes are genes that contain homeoboxes, which are short sequences of DNA that contain a protein homeodomain (binds to DNA) and help regulate the expression of other genes. Homeobox genes may be homeotic, which generally control the identity of body parts.
Hox genes are a subgroup of homeotic homeobox genes. Hox genes help determine positional cell differentiation and development, as well as aid in laying out the head to tail body plan of the organism (4). They make sure cells know what to develop into and that body segments are ordered correctly. When mutated, Hox genes cause the transformation of one part of the body into another part of the body. The common example of this is in Drosophila (aka the fruit fly), in which an extra pair of legs replaces the antennae. Hox genes are temporally co-linear, meaning they are arranged on the chromosome in the order that they will be expressed during development and are turned on in such order (2).
The Hedgehog Signalling Pathway, or Hedgehog genes, are involved in patterning and pattern formation within the body. Vertebrates have three different hedgehog genes; Sonic Hedgehog, Desert Hedgehog, and Indian Hedgehog.
Sonic Hedgehog works to establish the front/back and dorsal/ventral pattern of the nervous system. These genes act on different cells in order to turn them into various types of neurons based on their bodily position (1). This signaling protein is secreted from the notochord of a developing embryo, forming a concentration gradient that determines which cells will form. The embryo is able to establish a sense of which end is front, back, head, tail, etc. based on the concentration of the protein and turns on certain genes in its cells based on their placement. Sonic Hedgehog plays a big role in the determination of the placement of limbs and organs (5). It is also involved in the separation of a single eye field into two bilateral fields. A mutation of this gene can result in the condition known as cyclopia, in which there is a single eye in the center of the face (6).

Hedgehod Signaling Video

Hox genes help determine positional cell differentiation and development, as well as aid in laying out the head to tail body plan of the organism. They make sure cells know what to develop into and that body segments are ordered correctly.

1) http:/






Early Embryonic Development

HHMI Embryonic Developement Video


Early embryonic development occurs during the first 1 to 12 days after fertilization. The steps of early embryonic development can be broken down into 6 stages (1): fertilization, cleavage, early blastocyst, beginning of implantation, completion of implantation, and primitive streak. Fertilization begins with the initial ejaculation of male gamete, sperm into the receptacle of the female, and ends with a single celled, diploid organism (1). Cleavage of the zygote begins as a single cell division into two cells and commences the start of many other divisions. Every 20 hours new blastomeres, newly divided cells, are formed. The zygote becomes a morula once around 16 blastomeres have been formed. When the embryo changes its shape from a round shape to a mulberry shape, the process is called compaction (3) During the early blastocyst stage, the morula travels to the uterus. This occurs after approximately 4 days. Once in the uterus, a blastocele cavity forms in the middle of the morula, caused by continued cell division. Within the cavity, embryoblast cells, the cells that will in time form the animal (4), begin to form into a flattened and compact structure. Outside of the cavity, trophoblast cells form, which will in time form the placenta. The embryo is now considered a blastocyst. During the beginning of implantation the blastocyst outgrows its zona pellucida shell, leaving the trophoblast cells exposed and able to secrete an enzyme that will break down the uterine lining enough for the blastocyst to implant itself. To help maintain health of the uterus, human chorionic gonadotrophin is secreted by the trophoblast, helping to keep the uterine lining rich with blood. The uterus is full of newly formed blood capillaries, stimulated by the egg about six days prior, and allow continuous blood circulation between the blastocyst and mother (4). Towards the completion of implantation, the placenta around the blastocyst begins to form when the trophoblast cells have broken down enough of the uterine lining to create pools of blood and induce new capillary growth. At this point in time, the inner cell mass begins to separate into two cell layers: epiblast and the hypoblast. The epiblast will become the embryo and the hypoblast will become the yolk sac (4). During the primitive streak phase, the blastocyst becomes fully engulfed by the placenta, which further secures the embryo to the uterine wall. Blood vessels appear in the placenta, connecting to the uterus. The embryo attaches to the developing placenta via a stock that will eventually become the umbilical cord. A “primitive streak” forms between the epiblast and hypoblast layers in the embryonic disk, showing the gasturation, or separation and migration of cells from the outer layer, towards the embryonic disk, ultimately forming a third layer. These layers are called the endoderm, mesoderm, and ectoderm layers (4). The ectoderm is the top layer of the embryonic disk. It will become skin, hair, ear, nose, mouth, anus, tooth enamel, pituitary and mammary glands, as well as the nervous system. The mesoderm (middle) will become the muscles, bones, lymphatic system, blood cells, lungs, heart and reproductive system, as well as the spleen and excretory system. The endoderm will become the lining of lungs, tongue, tonsils, urethra, bladder, and digestive tract (4).
a depiction of the 3 embryo layers

1) - A great site that gives pictures for each stage of embryonic development
2) - a good site for detailed information on the ‘players’ in the reproduction process
3) - this site is the home to a good video that summarizes in a simplistic manner the early stages of embryonic growth