Analysis of Biological Development (K. Kalthoff)

Updates to Topic 21: Pattern Formation and Embryonic Fields

Answers to Questions in Text

Antennapedia Gene Expression (p. 534)

  1. Does the Antennapedia allele used in this study represent a loss-of-function or a gain-of-function allele? Answer: gain-of-function
  2. Assume you make Drosophila transgenic for a gene consisting of the coding region of Antennapedia spliced to an inducible promoter. Assuming that the transgenic flies survive to adulthood, what would you expect their antennae to look like? Answer: like complete legs. This experiment has actually been done (Schneuwly et al., 1987, Nature 325: 816-818).

Activin gradient (pp. 543-546)

  1. Why was it necessary in this experiment to label the responding tissue (animal caps)? Answer: In order to recognize the demarcation line between inducing and responding tissue after they had grown together in culture.
  2. Since the investigators wanted to test the inducing effect of activin, why did they supply activin mRNA, rather than protein, to the inducing tissue? Answer: Because mRNAs are generally longer-lived than their cognate proteins.
  3. The authors were concerned that the distance effect might be a deceptive appearance caused by the movement of responding cells away from the inducer. To investigate this possibility, the investigators constructed two layers of potentially responding cells labeled with two different dyes. No movement of the green fluorescent cells, which were located next to the inducer, was observed. Did this test cover the possibility that such cell movement might be mimicked by the induction of rapid cell divisions in the layer immediately adjacent to the inducer, with concomitant loss of Xbra+ gene expression in the daughter cells from this layer? If so, explain. If not, how could this possibility be tested independently? Answer: This possibility should have been covered by the experiment described, because rapid cell division should have led to a distinct reduction in cell size next to the inducer. Nevertheless, the investigators did test this possibility independently by raising the conjugates in the presence of cytochalasin, which inhibits microfilament formation and hence prevents cytokinesis (see Sections 2.3 and 2.4). This treatment caused the appearance of multinucleate cells and diminished the distance effect somewhat, but cells with Xbra mRNA were still separated from the activin source by cells without Xbra mRNA (see Fig. 3e,f in Gurdon et al., 1994, Nature 371: 487-492).


Clarifications and Corrections

p.545, Fig. 21.20. The headline for the second column should read "Blastula dissection", rather than "Blastula (injection)".

New Review Articles

Smith J.C. and Gurdon J.B. (2004) Many ways to make a gradient. BioEssays 26: 705-706

Tabata T. and Takei Y. (2004) Morphogens, their identification and regulation. Development 131: 703-712.

Teleman A.A., Strigini M. and Cohen S.M. (2001) Shaping morphogen gradients Cell 105: 559-562

New Research Articles

Chen Y. and Schier A.F. (2001) The zebrafish Nodal signal Squint functions as a morphogen. Nature 411: 607-610

The authors investigated the morphogen properties of Cyclops and Squint, two transforming growth factor-ß signals required for mesoderm induction and patterning in zebrafish. After injecting mRNA for either protein into a single cell at the 128-256 cell stage, they used in situ hybrization to assay for the expression of known target genes including goosecoid+ (gsc+) and no tail+ (ntl+). They found that high doses of Squint induced the expression of both gsc+ and ntl+ at close range while ntl+ was also activated more distantly (up to 8 cells away from the Squint-producing cell). To test whether Squint acts through a relay mechanism, they used embryos mutant in the one-eyed pinhead (oep) gene, which do not respond to Squint. If squint mRNA was coinjected with oep mRNA into oep- mutants, then the injected cell expressed ntl+ but the adjacent cells did not. However, wild-type cells grafted close to the injected cell did express ntl+. Thus, the Squint signal can spread through unresponsive territory but does not spread by any relay mechanism independently of oep+ gene activity. Together, the results indicate that Squint acts as a morphogen.

Dubrulle J., McGrew M.J. and Pourquié O. (2001) FGF Signaling Controls Somite Boundary Position and Regulates Segmentation Clock Control of Spatiotemporal Hox Gene Activation. Cell 106: 219-232

The authors found that in chicken embryos fgf8 is transcribed only in tail cells, and that by growth and mRNA degradation a moving wave of FGF8 protein is produced. This wave seems to coordinate the segmentation process and spatiotemporal Hox gene activation.

Goetz JA, Suber LM, Zeng X, Robbins DJ. (2002) Sonic Hedgehog as a mediator of long-range signaling. Bioessays 24: 157-165

Gurdon JB, Bourillot PY. (2001) Morphogen gradient interpretation. Nature 413: 797-803.

Dubois L., Lecourtois M., Alexandre C.. Hirst E. and Vincent J.-P. (2001) Regulated endocytotic routing modulates wingless signaling in Drosophila embryos. Cell 105: 613-624

Most gradient models imply that the morphogen is synthesized at a localized source and inactivated as it moves away from the source. The rate of inactivation will then control the steepness of the gradient. This process was studied directly for the gradient of Wingless (Wg) protein in the ectoderm of the Drosophila embryo. The investigators used transgenic embryos producing a horeseraddish peroxidase-Wingless (HRP-Wg) fusion protein. The HRP moiety, which generates a dense stain in electron micrographs, persists in the cellular compartments involved in protein degradation, such as lysosomes, thus allowing the investigators to "see Wingless after it has been degraded". They found that Wg degradation is faster posterior than anterior of the strip of cells that synthesize Wg. Thus, the Wg gradient falls off steeply to the posterior while being more shallow to the anterior. The low concentration of Wg that is required to make denticles is therefore reached further away from the Wg source to the anterior than to the posterior (see Fig. 22.29 of textbook).

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