Cellular and Molecular Biology Topics
Mammalian Cell Determination
Mammalian development is highly regulative. Cells in the very early embryo are totipotent, even so that if one of the cells at the 2-cell stage is destroyed, a normal embryo will still develop from the remaining cell. If two 8-cell morulas are combined, they will still develop into one normal embryo. When changes occur at the totipotent stage, cells adjust their behavior to generate animals that are normal in pattern and size.
Determination is a cellular commitment to a specific developmental pathway without an overt phenotypic change. Differentiation is overt cell specialization. For example, neural crest cells become committed to form neural crest cells before they leave the neural tube, but they do not differentiate into specific cell types until they complete their migration. Determined cells can be non-equivalent. For example, cells from the leg bud are determined to form leg structures but local cues determine appropriate structures within the limb. If a block of a leg bud from a chicken is transplanted to the tip of the wing bud, the result is a wing with toes instead of fingers.
Developing cells may be regulated by unidirectional, reciprocal and lateral inductive interactions. Unidirectional inductive interactions are signals produced by one type of cell that affect the development of another type of cell. When reciprocal inductive interactions occur, two different types of cells (or more) produce different signals that induce each other.
In lateral interactions, cells that are initially identical assume different fates as a consequence of interactions between them. Epithelial-mesenchymal reciprocal interactions regulate development of many organs such as the gut, lungs, gonads and kidneys. For example, in kidney development, the embryonic epithelium known as the uretheric bud and the mesenchyme that givse rise to the kidney are both mesoderm derivatives. The uretheric bud epithelium is induced by the mesenchyme to branch, forming the collecting ducts. Reciprocal induction drives the mesenchyme to form the epithelium that eventually will form the proximal and distal tubules and the glomeruli.
Some lateral interactions are governed by the notch-delta pathway. Notch is the receptor, delta the ligand, and both are transmembrane proteins that interact with each other to transduce a signal to the cytosol of the notch cell. After activation of notch, proteolitic cleavage releases an intracellular domain which then translocates to the nucleus and increases transcription of notch, but decreases transcription of delta. In this manner, random differences in expression of the receptor or ligand are amplified such that any initial asymmetry leads to commitment of cells to different fates.
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Signaling and Cell Migration
Key signaling molecules in vertebrate development include retinoic acid (regulates Hox gene expression), fibroblast growth factor (FGF), sonic hedgehog, the TGFb superfamily, and transcription factors like Hox (?) and T-box (?).
Fibroblast Growth Factors (FGFs) are bound by 4 different cell membrane receptors (FGFR1-4) that belong to the tyrosine kinase receptor family.
Sonic Hedgehog (Shh, a segment polarity gene product) is used in differentiating subpopulations of cells throughout the embryo. Depending on where the signal is being secreted, how far away the responsive cell population is and how Shh is proteolytically cleaved, will determine Shh function. Shh binds to the membrane receptor patched .
The TGFb superfamily controls development of the dorsoventral axis, and includes activin and BMP. These are conserved between vertebrates and invertebrates, but their expression patterns are reversed (?). The patterning of the mesoderm may be accomplished by activin. Bone morphogenic protein (BMP) functions to induce cartilage and bone formation
T-box (Tbx) genes are a family of transcription factors that are differentially expressed within the limb buds of the embryo. Tbx5 is also thought to have a role in heart development
Pax (paired box) factors are a highly conserved family of transcription factors belonging to the helix turn helix class. The expression of Pax genes is temporally and spatially restricted during development of CNS and various other organs.
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The fate of each embryonic cell is governed by interactions with its neighbors. Cell adhesion molecules are important for controlling cell migration and sorting. Examples of such molecules are the cadherins (Ca2+-dependent cell-cell adhesion glycoproteins), N-CAMs (Ca2+-independent cell-cell adhesion glycoproteins) and integrins (transmembrane proteins that bind extracellular matrix proteins).
Because of these interactions, mixed cells will sort out into the correct pattern. This is important in processes like the migration of limb skeletal muscle cells. Even different species will have similar migration patterns, such that if somites are transplanted to a chick embryo from a quail embryo, a limb will develop at the appropriate site, though the bone and muscle cells will be of a quail.
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Neuronal Development
Neural crest cells originate from the lateral margins of the neural plate. Head region (i.e. cranial neural crest) cells migrate before neural tube closure. Trunk neural crest cells leave after neural tube closure.
The pathway of cell migration depends on the interactions between adhesive and motile influences. As they undergo transformation from epithelial to mesenchymal cells, neural crest cells loose N-CAM expression. Fibronectin promotes migration while chondroitin sulfate inhibits migration.
Initially, neural crest cells are specified by inductive signals from non-neural ectoderm, mainly bone morphogenic protein (BMP). The induced cells express the transcription factor known as slug. BMP-4 and BMP-7 from non-neural ectoderm induce slug expression in future neural crest cells. In the neural plate, pax3 and pax7 are uniformly expressed. Shh inhibits pax3 and pax7 in the ventral region, facilitating development of the floor plate. Therefore, the development of the early nervous system depends on two gradients: BMP from the roof plate and Shh from the floor plate (?). Cranial neural crest cells are patterned with level-specific instructions in the head, similar to homeotic transformations, although trunk neural cells are not patterned (?).
Cell proliferation and survival are under the control of cKit produced by migratory cells and steel factor produced by cells in the migratory pathway. Mutations in the cKit gene affect survival of melanocytes during migration.
Early neural crest cells segregate into intermediate lineages that can differentiate into some but not all neural crest derived cell types. For example, trunk neural crest cells cannot give rise to skeletal structures derived from cranial neural crest. Interactions between neural crest cells and the tissues they encounter during migration determine their pattern of differentiation. For example, sympathetic neurons that innervate sweat glands are adrenergic until their axons touch the glands, then they become cholinergic. Another example is how BMP-4 and BMP-2 cause the neural crest cells to differentiate into autonomic nerves.
Neurocrestophaties include trunk neural disease, cranial neural crest disease or both trunk and cranial crest diseases. Trunk neural crest diseases include Hirschsprung’s disease and albinism. Hirschsprung’s disease is the formation of an aganglionic megacolon due to aberrant migration and death of cells that form enteric ganglia. Albinism is a lack of pigmented cells in the skin.
Cranial neural crest diseases include aorticopulmonary septation, heart defects, defects of the anterior chamber of the eye, cleft lip/palate and Digeorge syndrome. Digeorge syndrome is a deletion on chromosome 22, with a similar phenotype as in HoxA3 mutant mice, including hypoplasia of the thyroid and parathyroid, and abnormalities in cardiac outflow tract. Exposure of human embryos to excess retinoic acid can cause similar defects.
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Asymmetry and Apoptosis
In vertebrates, left/right asymmetry occurs in many organs: heart, lungs, visceral organs, vascular system and brain. Proteins involved in left/right asymmetry include nodal, lefty, Pitx2 and microtubule proteins. Nodal and lefty are members of the TGFb family. Pitx2 is a Hox gene.
Spiral beating of cilia drive a current to the left side of the primitive node. This establishes a gradient of signaling molecules that regulates left/right asymmetry. Once the primitive node is established, it will maintain the asymmetry. If the node is rotated, the normal asymmetry axis will also rotate.
As nodal accumulates on the left side by the action of the cilia, it induces expression of lefty1 and Pitx2. Lefty1 is important in maintaining the midline barrier between left and right sides. Pitx2 induces expression of other morphogens that determine “leftness”. Another factor induced by nodal, lefty2, is a feedback inhibitor of nodal.
Mutations in proteins involved in left/right asymmetry cause diseases like Kartagener’s syndrome, Reiger syndrome and ivMice (?). Kartagener’s syndrome is a randomized situs invertus due to mutated axonemal dyneins (i.e. altered microtubules). Reiger syndrome outcomes include dental and eye hyperplasia, umbilical protrusion and misshaped heart, and is due to a mutated Pitx2. ivMice (?) is also due to mutated axonemal dyneins and results in heterotaxia, discordant reversals of heart and visceral organs in situ.
Programmed cell death (apoptosis) is an important part of pattern formation. This is evident in limb development, were the interdigital spaces form by apoptosis. If cell death does not occur, a soft tissue web will connect the digits (syndactyly).
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Skeltal Muscle
Origins
Following gastrulation and nerulation, a broad expanse of mesoderm known as the paraxial mesoderm lies on either side of the neural tube. This paraxial mesoderm is first divided into segments called somitomeres. As development proceeds, somitomeres are replaced by somites in an anterior to posterior direction. The first 7 pairs of cranial (anterior) somitomeres are not replaced by somites but instead give rise to the muscles of the head. Somites 1 thru 7 give rise to the muscles of the tongue, larynx and neck. Trunk somites give rise to muscles of the trunk, diaphragm and limbs, and to the vertebrae.
Somites form as segmented blocks of cells with mesenchymal morphology transform into spheres of epithelial cells within the paraxial mesoderm. This process is driven by unknown inductive signals from the overlying ectoderm, which stimulate expression of paraxis, a basic helix-loop-helix (bHLH) transcription factor.
After formation of the somite, cells of the ventromedial wall are induced by sonic hedgehog (Shh) from the notochord and ventral wall of the neural tube. This results in expression of Pax1 and Pax9 (Hox genes of the paired family) in the ventral half of the somite, which is now called the sclerotome.
Sclerotomes
form vertebrae. They loose N-cadherin, transform back to mesenchymal morphology,
migrate medially and separate from the rest of the somite. Sclerotomes then
beguin to express chondroitin sulfate and other molecules characteristic of
cartilage, as they aggregate around the notochord and the neural tube.
Wnt secreted by the dorsal neural tube counteracts or modulates the effects of Shh at the dorsal half of the somite, allowing its transformation into the dermomyotome. Wnt action leads to expression of Pax3, Pax4 and paraxis.
Cells from the lateral edge of the dermomyotome migrate into the limbs to form limb muscle. Cells from the dorsomedial border of the dermomyotome migrate beneath the epithelial somite to form the myotome, which gives rise to skeletal muscle of the trunk. The remaining somite epithelia is the dermotome and will give rise to the dermis.
(All figures in this section were taken form the same web site: http://www.med.unc.edu/embryo_images/unit-mslimb/mslimb_htms/mslimb002.htm, which also have many cool animations)
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Limb Development
Limb development is highly regulative. If part of the limb primordia is removed, the reminder reorganizes to form a complete limb. If the limb primordia is split in two and prevented from combining back again, two complete limbs will develop. If two equivalent halfs of the limb primordia are combined, a single limb will develop.
Limb skeletal muscle migrates from the somites. Pax3 induces cMet, which is required for migration. cMet is the ligand for HGF/SF, which is expressed in the limb bud. Developing limbs are organized relative to three body axes: anterior/posterior (1st to last digit), dorsoventral (back of hand to palm), and proximodistal (base of limb to the digits).
Shortly after its initial establishment, the limb primordial begins to bulge from the body wall. At this stage, the limb bud consists of mesodermal cells covered by a larger ectoderm. The limb primordial is intrinsically determined as it can guide its own development when transplanted.
Axial control in the developing limb is determined by three structures: the apical ectodermal ridge, the zone of polarizing activity (posterior mesoderm) and the dorsal and ventral ectoderm. The apical ectodermal ridge produces FGF-2, FGF-4 and FGF-8 which control the proximodistal axis. The zone of polarizing activity generates sonic hedgehog, which controls the anterior/posterior axis. The dorsal and ventral ectoderm create Wnt7a gradients that control the dorsoventral axis.
The apical ectodermal ridge is a thickening of the ectoderm along the anterior/posterior
plane of the apex of the limb bud. It forms at the junction of the dorsal and
ventral ectoderm and interacts with the underlying mesoderm to promote limb
outgrow. Mutation of the ectodermal ridge results in arrested limb development.
The apical ectodermal ridge initially produces FGF-8 as a signal. As the limb bud grows out, it also produces FGF-2 throughout its length and FGF-4 in its posterior half. The FGFs stimulate proliferation of the underlying mesodermal cells. As development proceeds, the ridge begins to break up, regressing by apoptosis between the digits to sculpt the interdigital spaces.
The posterior region of the limb mesodermal cells cells contain a zone of polarizing activity that acts as a signaling center and determines the organization of the limb along its anterior/posterior axis. The main signal is Shh, which maintains the apical ectodermal ridge. This zone of polarizing activity establishes a morphogen gradient that detertmines digital patterning. The precise morphogen concentrations in a given region determines which digit will form.
The dorsal ectoderm produces Wnt7a, which induces the Hox gene LMX1. The ventral ectoderm produces En1, which inhibit Wnt7 expression in ventral cells.
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Regulation of Myogenesis
Myogenic regulatory factors include the myogenic bHLH proteins and the Hox gene (?) Pax3. Pax3 induces cMet, which is a ligand required for cell migration.
The myogenic bHLH proteins include MyoD, myogenin, Myf5 and MRF4 (Myf6). These are all transcription factors with a high degree of homology in the bHLH region. They are very highly conserved through evolution, much so that the sea urchin and nematode forms can activate myogenesis in mammalian cells. All the myogenic bHLH proteins can induce myogenesis of embryonic fibroblasts. They act as heterodimers with ubiquitously expressed HLH proteins orE-proteins, which bind to the E box in genes with the sequence CANNTG. The myogenic bHLH are exclusively expressed in skeletal muscle.
The myogenic factors MyoD and Myf5 are activated during determination of myoblasts, although not all cell lines express both. Myogenin is only expressed following growth arrest, and is always found when muscle cells differentiate (?). MRF4 is expressed later in mature myotubes. Myogenin is required for myoblast differentiation, while MyoD is required for myoblast identity (?). In mouse, Myf5 is the only myogenic regulator expressed before muscle formation. Activation of Myf5 may therefore trigger a cascade, but is not required for MyoD expression. Positive signals for muscle cell commitment and myogenic induction include Wnts from the neural tube, Shh frm the notochord and Wnts from the ectoderm. BMP4 is a negative signal from the lateral plate mesoderm. Wnts and Shh induce Pax3 and Myf5 production. Myf5 and Pax3 induce MyoD.
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