Evidence that the limb bud ectoderm is required for survival of the underlying mesoderm.

Figure 1. Healthy male rooster (5)

 

 

 

 

 

 

 

 

 

 

 

Based on paper done by Marian Fernandez-Teran, Maria A. Ros and Francesca V. Mariani

Chicken as Model Organism for Limb Deveopment

Chicken (gallus gallus) has been used as model organism for studying limb development as it is relatively easy to manipulate the developing limb in vivo. From previous studies based on chicken embryology, information such as fate maps and cell-cell interactions specifying limb pattern has been obtained. The principles in chicken limb development can be applied to other vertebrates, such as humans. For instance, during embryonic development, same chemical signals shape human hand and chicken wing. Using chickens as model organism in embryology is important to understanding human embryology.

HumanVsChicken

Figure 2. Comparison of human arm to chicken wing (3)

Chicken Embryo Development

Introduction

Limbs develop from limb buds, which are derived from flank mesenchyme. Limb buds form as a result of interactions between mesoderm and ectoderm and they are active throughout limb development. Signals from the limb bud induce formation of Apical Ectoderal Ridge (AER), which regulates proximo-distal patterning, and the zone of polarizing activity (ZPA), which regulates anterior-posterior polarity.

AER

Figure 3. Scanning electron micrograph of an early chick forelimb bud, with its apical ectodermal ridge in the foreground. (Courtesy of K. W. Tosney; 4)

 

Previous studies have shown that AER plays an important role in limb development by promoting proliferation and survival of the underlying limb mesoderm. Fibroblast growth factor (FGF), which is expressed in AER, has shown to affect limb development. Previous studies showed that inactivation of Fgf8 resulted in a range of alterations in limb pattern and severe cases showing complete loss of the limb (2, 4). Besides AER, non-AER ectoderm also has a role in limb patterning by providing cell signals for dorsal and ventral patterning. In studying the patterns of programmed cell death in AER-FGF mutant, the authors noticed that there was cell death in both proximal mesoderm of the limb bud and in the proximal dorsal ectoderm. In the paper, they explored whether the non-AER ectoderm has a survival function in addition to a role in patterning (2).

mesoderm+ectoderm

Figure 4. Formation of limbs in chicken embryo (3)

Results

1. After removing the dorsal ectoderm of stage 20-21HH wing bud, with AER intact, there was a massive cell death in the underlying dorsal mesoderm. White dots on figures represent DNA fragments from cell death. As seen on figure 5A-D, removal of the dorsal ectoderm resulted in cell death in the underlying dorsal mesoderm. Figures 5G and I shows that the underlying mesoderm becomes independent of influences from the dorsal ectoderm as cell death at later stages results in reduced cell death (2).

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Figure 5. Pattern of cell death after removal of the dorsal ectoderm (2).

2. With the removal of dorsal ectoderm,  the limb skeleton phenotype was affected. The limbs that developed after the dorsal ectoderm was removed were shorter. Fig 6A represents normal limb bud and Figs 6B-D shows abnormal limbs resulting from dorsal ectoderm removal (2).

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Figure 6. Analysis of skeletal patterning after DE removal (2).

3. Wnt7a is expressed in the dorsal ectoderm. Wnt7a is gene that codes for signaling proteins that help guide the development of anterior-posterior axis. Also, it gives rise to dorsal/ventral patterning. Loss of Wnt7a causes limb defects. For example, loss of Wnt7a causes the dorsal side of the limb to be switched to ventral sides. The paper studied whether there was a residual of dorsal ectoderm left by studying the expression of Wnt7a. Immediately after the removal operation, there was absence of Wnt7a expression (Fig. 7A). Wnt7a expression was not recovered after the operation (Fig. 7B-C). Then, the paper studied whether ectoderm would heal over the “denuded” mesoderm. Figure 7E shows that after 20 hours after the operation, the ectoderm has covered most of the dorsal surface. Compared to normal lib bud, the recovered ectoderm was morphologically different (Fig. 7F) (2).

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Figure 7. Wnt7a is not expressed but an ectoderm-like layer of cells covers the operated dorsal side (2).

4. Shhwhich depends of Wnt7a signal from the dorsal ectoderm, became down-regulated (Fig. 8A-C). Shh (sonic hedgehog) codes for a morphogen involved in a variety of patterning systems. It is also important in limb development as it helps create ZPA. 16 hours after the removal operation of the dorsal ectoderm, Shh expression was detected, but its level was lower level and in a reduced domain compared to the normal limb bud (Fig. 8D-G). Simultaneous detection for both Shh and Wnt7a showed that later expression of Shh expression after the operation showed that Wnt7a was absent (Fig. 8H-J). This shows that later expression of Shh is not caused by recovery of Wnt7a expression. Expression of Lmx1b was reduced, but not to the extreme of reduction in Shh expression, showing the sensitivity of Shh expression to Wnt7a signals (Fig. 8K and L). Lmx1b is “a homeobox-containing transcription factor responsible for establishing dorsal identity in the subjacent mesoderm” (2).

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Figure 8. Shh and Lmx1b expression after removal of the dorsal ectoderm (2).

5. The paper also showed that ventral mesoderm is dependent on the overlying ectoderm for survival signals. Although the amount of cell death was less than that of dorsal mesoderm after dorsal ectoderm removal, removing ventral ectoderm at HH20-21 showed cell death in the ventral mesoderm (Fig. 9A’-A”). Both skeletal and muscle patterning were normal (Fig. 9B-D). The reduction of Shh expression was also less than that of dorsal ectoderm removal (Fig. 9E-I’). They also checked for the effect of the size of dorsal ectoderm removal on dorsal mesoderm survival. When they removed half of the dorsal ectoderm, either the proximal or distal half, at 20-21HH, there was less severe level of cell death (Fig. 9A). Removal of distal half of dorsal ectoderm resulted in full survival of dorsal mesoderm, suggesting that survival signals are coming from the AER or those remaining in proximal dorsal ectoderm (Fig. 9B). There were mild skeletal defects with the removal of proximal and distal half of the ectoderm (2).

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Figure 9. Removal of the ventral ectoderm results in cell death (2).

6. In order to test the whether the dorsal ectoderm provides a specific signal to the dorsal mesoderm, recombinant limbs were created. In these recombinant limbs, the limb ectoderm was removed and the limb mesoderm were surround either in a limb ectoderm (‘sham control’) or wrapped in ectoderm from the back of the embryo. These recombinant limbs were grafted to the somite area of the host embryos. The sham control showed little cell death while the limb mesoderm wrapped in ectoderm from the back of the embryo showed massive cell death (Fig. 10C-D). The paper suspected that the cell death from the limb mesoderm wrapped in back ectoderm was caused by lack of AER, which is known to produce survival signals to mesoderm. In another recombinant test, limb buds that had the dorsal ectoderm removed but had AER intact was used. The recombinant limbs wrapped with limb ectoderm showed no cell death (Fig . 10E-F). However, the recombinant limbs wrapped with back ectoderm produced cell death (Fig. 10D-G). The recombinant study showed that the cause of cell death in the mesoderm is due to the absence of ectoderm alone (2).

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Figure 10. Partial removals and ectoderm substitution experiments show that the dorsal ectoderm emits specific survival signals (2).

 

Conclusion and Future Studies

The results showed that the non-AER limb ectoderm, dorsal and ventral, is important in the survival of adjacent mesoderm, as the removal of the ectoderm results in cell death in the mesoderm. Cell death and damage to the mesoderm have been shown to affect the phenotype of skeleton and muscles of the limb. Just like AER, non-AER has been shown to produce specific survival signals to the underlying mesoderm. In order to expand on the results of the experiment, the specific survival signal from the non-AER limb ectoderm should be explored. Also, there needs to be a study done to determine whether the skeletal patterning defects observed after removal of the ectoderm is due to massive cell death or  due to lack of limb development inducing signals.

Strength and Weaknesses

The study was very thorough in showing that removal of the ectoderm lead to cell death in the mesoderm, which is critical to normal limb development. It also conducted a recombinant study to show that there the limb ectoderm directly produces survival signals to the limb mesoderm. The authors did a good job illustrating this point as AER had been shown to be important to the survival of the underlying limb mesoderm in earlier stages of limb development. The weakness in the paper is that they were not able to identify any specific signals or group of signals from the limb ectoderm that directly influences the proliferation and survival of the adjacent mesoderm. Further study could be done in identifying these signals in the limb ectoderm.

References 

1. Tickle, C. (2004) The contribution of chicken embryology to the understanding of vertebrate limb development, Mechanisms of development 121, 1019-1029.

2. Fernandez-Teran, M., Ros, M. A., and Mariani, F. V. (2013) Evidence that the limb bud ectoderm is required for survival of the underlying mesoderm, Developmental Biology 381, 341-352.

3. Riddle, R.D and Tabin, C.J. How Limbs Develop. http://genepath.med.harvard.edu/~tabin/Pdfs/Riddle.pdf (accessed 4/02/14).

4.Gilbert SF. Developmental Biology. 6th edition. Sunderland (MA): Sinauer Associates; 2000. Formation of the Limb Bud. Available from: http://www.ncbi.nlm.nih.gov/books/NBK10003/

5. “Get ready Hot Chicken Nation.” Photo. 2014 Music City Hot Chicken Festival. 13/04/14. <http://mchcf.blogspot.com >

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