An axon often has to navigate great distance to reach its target cell (ie, another neuron or muscle fiber) and establish the synaptic connection. This process is axonal pathfinding. The axon does not navigate its natural pathway due to any single cue, but via combinations of signals. This redundancy makes axonal pathfinding extremely complex. Axonal pathfinding is extremely specific: a particular axon always contacts the same (or at least very similar) set of neurons or muscles.
Neurons have an intrinsic polarity that is always the same for a specific type of neuron. At one end of this polarity, a localized increase in cell membrane activity occurs (with ruffles, aka lamellipodium often visible) and fine filopodia of the cell cytoplasm extend and withdraw. The structure of filopodia is maintained by microfilaments, while axonal strucure is maintained by microtubules. Eventually, a filopodium forms a growth cone, an actively motile region at the far end.
The growth cone travels based on cues from a substrate called the extracellular matrix (ECM). The ECM contains laminin, collagen and fibronectin (the first being the most important) that adhere to cadherins and integrins in the growth cone membrane. Also, the ECM contains unique molecules (ie, nerve growth factor and retinoic acid) that guide the growth cone, sometimes across a gradient and other times along a narrow path. These unique molecules are deposited: by tissues over which the axons grow (ie, epithelia, somites and blood vessels); by cells at crucial migratory points; and even by the target tissue itself.
Once the growth cone reaches its specific target (ie, muscle fibers or another neuron) it must stop growing and establish a synaptic connection. The high specificity of axonal pathfinding is determined by any of three ways:
| Method | Overview |
|---|---|
| Timing | There may a single cell (or group of cells) mature enough to form a synapse with the incoming axon. Therefore, specificity is determined by the timetable of donor and receiver neuronal maturation. |
| Chemoaffinity | The growth cone and target cell may express matching recognition molecules that restrict interactions with other cells. Once in the target region, the growth cone locates its target cell by unique molecular tags that bind to recognition molecules on the target cell. |
| Pruning | Axons may bind target cells in a somewhat non-specific manner; improper connections are recognized via neuronal activity or trophic factors produced by the target cell. Incorrect connections are eliminated via cell death or selective retraction of certain axonal branches. |
Commisural neurons are interneurons in the (usually dorsal) spinal cord that extend their axons across the midline. Commisural projects project ventrally toward the floor plate, and then cross the midline ventral to the floorplate of the spinal cord. Recombination experiments have shown that the floor plate secretes netrin, an attractive signal critical for commisural axons to migrate to the floor plate and cross the midline. In netrin-/- mutants, commisural axons fail to grow to the floor plate.
Ephrins are membrane-bound ligands that bind to membrane-bound ephrin-receptors called ephs. When an ephrin and an eph bind, a signal is generated in both cells — this means the signal is bi-directional. Ephins are repulsive signals in axon migration, with ephrin/eph signaling responsible for setting the retino-tectal topographic map. In a topographic map, the spatial organization of neurons in one region (retina) is replicated in a connected region (tectum). The retina and tectum contain inverse gradients of ephs and ephrins; axons extend from the retina to the tectum until reaching a signaling threshold (inversely proportionate to their starting point) that inhibits further navigation. Thus, axons from a retinal region with low levels eph will target a tectal region with high levels of eph. In the figure below, axons from a given retinal quadrant will target the tectal quadrant of matching color.
All in all, ephrin/eph signaling is critical for:
| Step | Overview |
| 1st Induction | Specific positions along the anterior-posterior axis of the archenteron (cells arising from the organizer to form dorsal mesoderm and endoderm) induce specific regions of the brain to form. For the developing eye, the diencephalon is induced along with its outpatching, the optic vesicle. |
| 2nd Induction | The optic vesicle induces the lens placode in the overlying head ectoderm. This is evidenced by: surgical or genetic removal of the optic vesicle cases failure of lens formation; optic vesicle transplantation causes formation of a new, ectopic lens. The optic vesicle then folds back on itself to form the optic cup: the outer optic cup layer becomes the pigmented retina; the inner optic cup layer becomes the neural retina. The lens placode also invaginates to form the lens vesicle. Axons from the neural retina travel down the optic stalk; once travelled by axons, the optic stalk becomes the optic nerve. Next, the lens cells differentiate. |
| 3rd Induction | The lens vesicle induces the overlying ectoderm to differentiate as cornea. |
A critical gene that is expressed throughout the developing eye is Pax6. Mutants of Pax6 have aniridia (no iris), small eyes and even no eyes. Pax6-/- homozygotes lack eyes and nasal cavities. Pax6 is required in the ectoderm, as shown by tissue recombination studies using Pax6+/+ and Pax6-/- ectoderm and optic cups. Also, ectopic expression of eyeless (the Drosophila homolog of Pax6) in Drosophila larvae causes ectopic eyes to develop on the legs and wings; murine Pax6 has a similar effect, showing its strong conservation.
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Reciprocal signaling between the lung endoderm and surrounding mesenchyme controls the pattern of bronchial (lung) branching. Mesenchymal cells close to budding region produce Fgf10, which promotes proliferation of bronchioli. The tracheal bud does not branch in Fgf-/- mice, thus confirming that FGF induces cell proliferation. However, if FGF were the only signal then the bronchioli would continually grow without branching. Several mechanisms define branch points, one of which involves Shh.
Shh inhibits FGF expression, and is secreted from the tip of the bronchiolus when branching is about to occur. This leads to a high FGF concentration along a growing bronchiolus, but a low FGF concentration at its tip. As a result, the bronchiolus splits at the tip into two branches; each new branch grows toward the high FGF concentration. Shh-/- knockout mice have primitive sacs instead of lungs due to complete failure of branching.
Drosophila has a signaling mechanism similar to the aforementioned FGF/Shh system. The Drosophila respiratory system begins as a series of invaginating placodes which later future to form multiple branches. Growth of tracheal primordia is induced by binding of its FGF receptor by an FGF homolog called branchless. The tip of the tracheal primordium expresses sprouty, which diffuses and inhibits FGF-stimulated proliferation in basal cells. This causes a T-shaped expansion of the tracheal primordium tip.
Similar to the tracheal and pancreatic buds and their branches, the hepatic (liver) bud is an evagination of the endoderm which branches profusely to form the liver.
Similar to the tracheal and hepatic buds and their branches, the peancreatic bud is an evagination of the endoderm which branches profusely to form the liver. This branched endodermal endothelium differentiates into two types of glandular tissue: exocrine glands, which produce essential digestive enzymes that are secreted into the small intestine; and endocrine glands (aka islet of Langerhans or islets) that produce the hormones insulin and glucagon.
The brain develops from the neural tube. Before even fully developing at the posterior, the neural tube (initially straight) begins to form bulges at the anterior that will give rise to the brain. First, three primary vesicles are formed; starting at the most anterior, these are the forebrain (prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon). Many transcription factors confer identity upon the mammalian brain, including Hox genes.
| Primary Vesicle | Overview |
| Rhombencephalon |
The anterior of the rhombencephalon is the metencephalon, which gives rise to the cerebellum and pons; the posterior is the myelencephalon, which gives rise to the medulla oblongata. Unlike the rest of the brain, the rhombencephalon segments into totally separate and morphologically distinct rhombomeres. There are seven rhombomeres, and also seven Hox genes whose regions of expression end at the anterior end of each rhombomere.
The rhombencephalon of a knockout for any of these seven Hox genes develops normally anterior to the range of expression of that Hox gene; however, rhombomeres within the range of expression of that Hox gene develop abnormally. This suggests that Hox genes play a role in rhombomere identity, just as they control vertebral identity in the trunk. |
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| Mesencephalon | The mesencephalon does not subdivide. |
| Prosencephalon |
The prosencephalon subdivides to form the telencephalon (at the anterior) and the diencephalon (at the posterior). The telencephacon gives rise to the cerebral hemispheres; the diencephalon evaginates to form much of the eye in addition to the thalamic and hypothalamic brain regions. Subdivision of the prosencephalon is managed by spatially localized expression of transcription factors. However, the mechanism of subdivision is still vague due to difficulty knocking out relevant genes, and because the segments are not clearly distinct. Outgrowing neurons mark off groups of cells that do not intermix, suggesting there are three or four subdivision (neuromeres or prosomeres) in the telencephalon and diencephalon. There are at least 25 homeobox genes expressed in restricted regions of the forebrain, also implying that the forebrain is divided into functional units. These genes (ie, Emx, Otx and Dlx) often have overlapping expression regions and are homologous to genes (ie, ems, otd, Dll) expressed in the embryonic fly head semental patterns. |
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