Aataa

+ FLP

Gene of interest lacZ Z^

Gene of interest ^Z +

Gene of interest ^Z +

M FIGURE 15-11 Gene manipulation in analysis of signaling systems. (a) In the simplest case, a gene is activated with the use of a promoter that is specific to certain cells or, as with "heat shock" promoters, that is inducible in all cells. This procedure has the limitation that the transgene may be detrimental and transgenic animals cannot be isolated. (b) An improvement is to make two lines of flies transgenic, one carrying the gene of interest under the control of an upstream activating sequence (UAS, X-ref) from yeast. The UAS is activated when the yeast transcription factor GAL4 is present, as it is when the UAS-bearing flies are crossed with flies having GAL4 expressed in certain cells. (c) The opposite goal is to remove a gene's function selectively from certain cells. The yeast recombinase FLP acts on FRT sequences that have been inserted near the base of a chromosome. Starting from a fly that is heterozygous for a mutation of interest, mutant/wild-type, one can obtain clones of cells that are homozygous for either the mutant or the wild-type allele. (d) It is difficult to recognize and analyze small clones of cells obtained as in part (c) if the cells are not marked. In this refinement, the recombination removes GAL80, a protein that inhibits GAL4 function, at the same time as the mutant allele is made homozygous. The unleashed GAL4 then activates a UAS that drives the production of a fluorescent protein. A mutant cell, like the neuron shown, can be analyzed to see the effect of the mutation on, for example, wiring the brain. (e) To activate a gene in small, randomly generated clones of cells, FLP is again used but this time to remove an intervening transcriptional termination sequence that prevents the gene of interest from being active. At the same time, a lacZ or other marker gene is removed; so the clone of cells with the gene turned on is identifiable by the lack of marker gene expression.

Inductive Signaling Operates by Gradient and Relay Mechanisms

In some cases, the induction of cell fates includes a binary choice: in the presence of a signal, the cell is directed down one developmental pathway; in the absence of the signal, the cell assumes a different developmental fate or fails to develop at all. Such signals often work in a relay mode. That is, an initial signal induces a cascade of induction in which cells close to the signal source are induced to assume specific fates; they, in turn, produce other signals to organize their neighbors (Figure 15-12a). Alternatively, a signal may induce different cell fates, depending on its concentration. In this gradient mode, the fate of a receiving cell is determined by the amount of the signal that reaches it, which is related to its distance from the signal source (Figure 15-12b). Any substance that can induce different responses depending on its concentration is often referred to as a morphogen.

The concentration at which a signal induces a specific cellular response is called a threshold. A graded signal, or morphogen, exhibits several thresholds, each one corresponding to a specific response in receiving cells. For instance, a low concentration of an inductive signal causes a cell to assume fate A, but a higher signal concentration causes the cell to assume fate B. In the gradient mode of

(a) Relay signaling

(a) Relay signaling

▲ FIGURE 15-12 Two modes of inductive signaling. In the relay mode (a), a short-range signal (red arrow) stimulates the receiving cell to send another signal (purple), and so on for one or more rounds. In the gradient mode (b), a signal produced in localized source cells (red arrows) reaches nearby cells in larger amounts than the amounts reaching distant cells. If the receiving cells respond differently to different concentrations of the signal (indicated by width of the arrows), then a single signal may create multiple cell types.

▲ FIGURE 15-12 Two modes of inductive signaling. In the relay mode (a), a short-range signal (red arrow) stimulates the receiving cell to send another signal (purple), and so on for one or more rounds. In the gradient mode (b), a signal produced in localized source cells (red arrows) reaches nearby cells in larger amounts than the amounts reaching distant cells. If the receiving cells respond differently to different concentrations of the signal (indicated by width of the arrows), then a single signal may create multiple cell types.

signaling, the signal is newly created, and so it has not built up to equal levels everywhere. Alternatively, the signal could be produced at one end of a field of cells and destroyed or inactivated at the other (the "source and sink" idea), so a graded distribution is maintained.

Mesoderm Cell Fates in Xenopus Blastula Studies with ac-tivin, a TGF^-type signaling protein that determines cell fate in early Xenopus embryos, have been sources of insight into how cells determine the concentration of a graded inductive signal. Activin helps organize the mesoderm along the dorsal/ventral (back/front) axis of an animal. The endoderm and ectoderm form first after fertilization of a Xenopus oocyte; the mesoderm forms slightly later. These three distinct cell populations (germ layers) make up the blastula, a hollow ball of cells.

Specific genes are used as indicators of the tissue-creating effects of signals such as activin. For instance, a low concentration of activin induces expression of the Xenopus brachyury (Xbra) gene throughout the early mesoderm. Xbra is a transcription factor necessary for mesoderm development. Higher concentrations of activin induce expression of the Xenopus goosecoid (Xgsc) gene. Xgsc protein is able to transform ventral into dorsal mesoderm; so the local induction of Xgsc by activin causes the formation of dorsal, rather than ventral, mesodermal cells near the activin source. Using 35S-labeled activin, scientists demonstrated that Xenopus blastula cells each express some 5000 type II TGF^-like receptors that bind activin. Findings from additional experiments showed that maximal Xbra expression was achieved when about 100 receptors were occupied. At a concentration of activin at which 300 receptors were occupied, cells began expressing higher levels of Xgsc. Similar results were obtained with blastula cells experimentally manipulated to express sevenfold higher levels of the activin type II receptor. These findings indicate that blastula cells measure the absolute number of ligand-bound receptors rather than the ratio of bound to unbound receptors, and confirm the importance of signal concentration.

Vulva Development in C. elegans An example of cell-fate determination by a combination of graded and relayed signals is the development of the vulva of the nematode worm C. ele-gans. This structure develops from a group of epidermal vulval precursor cells (VPCs) whose fates are controlled by an inductive signal from a nearby cell called the anchor cell. All the VPCs have the potential to become any of three different cell types: 1° and 2°, which refer to different vulval cell types, and 3°, which is a nonvulval type. A set of cells, such as the VPCs, is called an equivalence group if each cell in the set has equal capacity to form more than one cell type. The inductive signal secreted by the anchor cell is LIN-3, which is similar to vertebrate epidermal growth factor (EGF). Like the EGF receptor, the receptor for LIN-3 is a receptor tyrosine kinase, called LET-23, that acts through a Ras-MAP kinase pathway (see Figure 14-21).

The results of early studies suggested that LIN-3 was a graded signal inducing the 1° fate in the nearest VPC (nor-

P3.p

P6.p RELAY

P4.p

Your Heart and Nutrition

Your Heart and Nutrition

Prevention is better than a cure. Learn how to cherish your heart by taking the necessary means to keep it pumping healthily and steadily through your life.

Get My Free Ebook


Post a comment