Cell Proliferation and Limb Regeneration

On Mouse Limbs With Reference To The Progress Zone Model of the Chick.

This is my summary of research done on the topic of BMP signaling on the mouse limb and pattern formation in the chick limb based on the research done by: Udayan Guha William A. Gomes, Tatsuya Kobayashi, Richard G. Pestell, and John A. Kessler, In Vivo Evidence that BMP signaling is necessary for apoptosis in the mouse limb, Developmental Biology 249, 108-120 (2002).

Project for the course BIO 480, Developmental Biology, Dr. Grant Mastick, Biology Department, University of Nevada, Reno. http://med.unr.edu/homepage/gmastick/BIO480 page/index.html

Introduction:

Researchers were attempting to solve the question of the importance of the BMP (Bone morphogenic protein) signaling in the development of limb formation in the mouse. Further research had been done proving that proteins BMP₂and BMP₄ cause lethality in the early development in the mouse. So, their role in limb apoptosis, which is determined cell death of the mesenchymal cells, has not been studied on mice. Apoptosis and the regression of inter-digital tissue are the processes of sculpting the limb from anterior to posterior regions of the limb bud. The primary goal of this research article, done by Guha et al, is to prove with in vivo evidence that the BMP signaling from the cell is required for proper cell apoptosis in the limb of the mouse. On the topic of pattern formation we can also look at the Review article written by Lee Niswander “Pattern Formation: Old Models out on a limb” it as concluded that in the chick limb, signals from the BMP create the AER (apical ectodermal ridge). Without this signal the AER is not formed and thus there is no differentiation of the posterior, anterior and inter-digital necrotic zones in the chick. This lead to the discovery of the Progress Zone Model, which is the mesenchyme region that explains patterning of the limb along the proximal-distal axis. This is what the AER influences to create the three regions of the chick limb (Stylopod, Zeugopod, and Autopod).

Experimental System:

Western Blot Analysis to determine the transgenic expression in the adult mouse limb, which was prepared by the adult back skin or embryonic limb homogenates with a rat monoclonal.

Cell death was detected by vital dye staining using the Nile Blue Sulfate.

The apoptotic cells were detected by using a TUNNEL assay.

Inhibition of the BMP signals was the outcome of miss-expressing the BMP antagonist Noggin in the ectoderm before the onset of apoptosis in the limb.

Advantages:

The researchers main advantages would have been the ability to use the X-Ray analysis and also by using the Scanning Electron Microscope. Also using the In Situ Hybridization along with the Immunochemistry helped out in visualizing the apoptotic cells in the experiment.

This is the transgene constructed that was injected to promote in vivo and the miss-expression of Noggin by using Keratin (K4) produced by BMP. Provided by Guha et al.

K-14 promoter

Noggin or Bmp4

Results:

The most significant advances that were accomplished were that the researchers were able to determine that the function of inhibiting the signals of BMP indeed induced abnormalities, along with the absence of regression of cells between the inter digits. They induced transgenic mice, by in vivo, the miss-expression of Noggin by using keratin (K14) promoter to drive expression of the Noggin. It reduced the signals that were usually produced by BMP. The Noggin prevented the BMP’s signals from binding to their sites. There were decreased amount of cell proliferation (cell death) in the necrotic zones. Once abnormalities form from inhibition of BMP signaling researchers were able to rescue some BMP signaling in the hind limb and partially in the forelimb by creating a double transgenic. This would miss-express the Noggin and the BMP4 under the K14 promoter thus hypothesizing that the lack of BMP can be a direct consequence of limb abnormalities.

In order for the researchers to determine whether the inhibition of BMP had an effect on the “Noggin Transgenic mice” they analyzed “phosphorylation of the BMP responsive proteins of the Smad family in sections of the E14.5 limbs by using an antibody that recognizes only phosphorylated forms of Smad1 and Smad5.” Smads are the proteins, which are initiated when signals from type II phosphorylates type I receptors and then the signals cascade and Smad proteins become involved. What made these discoveries possible were the increased technological advances such as the Scanning Electron Microscope, which enabled them to see how the digits of their test experiment of the mouse were separated. And by following this process all in vivo they are able to manipulate the production and expression of the protein in cells.

Conclusions:

The data that they concluded was that Noggin mediated inhibition of the signaling from the BMP might be involved in fate changing in the posterior and anterior necrotic zones. Lack of BMP signaling prevents cell proliferation in the mouse limb. Also, conclusions of rescue of the K14- Noggin-BMP4 double transgenic, show that BMP signal is required for regression of the inter-digital tissue and determination of fate in the limb.

As for the chick limb just to reference to the mouse it was also discovered that the AER is needed for the formation of the regions of the limb and that the signals from the BMP influence them and without the BMP there is abnormalities and in the distal tips of the limb. Mainly they realized that BMP signaling is important for a number of responsibilities in formation of growth and sculpting in the limb of the mouse and the chick limb.

Abnormal limb development

Block the BMP signal

Future Directions:

Things to look for in the future of experiment growth factors, is the research of the extensive roles that the BMP’s play in the “development of hair follicles and the peripheral nerve innervations of developing skin.” Research will be able to determine the “biological role” of these highly expressive proteins. I think that by experimenting with the function and the inhibition of these BMP’s and other growth factors is evolutionary important and can help introduce new techniques in the human development of cells. The more research that is done with these growth factors and the more manipulation done with them the more we are able to find out what has imperative influence on them and what doesn’t. Thus the more information gained the more we are able to prevent diseases and mutations or abnormalities early in the cell formation. Also, we with the information we obtain we might be able to detect and “catch” these so called abnormalities early on and change the fate of them.

Significance:

The significance of this research is that Biologists are more knowledgeable about the signals that affect the cells and their fate determination. This is an advance it to research and helps to identify potential answers to questions that were previously unanswered on what determines what in cell formation. And what processes take place and how they take place. Now there is sufficient evidence in the mouse and the chick that identifies the importance of BMP signaling.

References:

1. Udayan Guha William A. Gomes, Tatsuya Kobayashi, Richard G. Pestell, and John A. Kessler, In Vivo Evidence that BMP signaling is necessary for apoptosis in the mouse limb, Developmental Biology 249, 108-120 (2002).

Summary: This research article was about the importance of BMP signaling in the mouse limb and how the signaling is responsible for the sculpting of the limbs and without it there is sufficient abnormalities and deficiencies in the limb.

2. Niswnader, Lee, Pattern Formation: Old Models Out On A Limb, Nature. Genetics; Vol 4. February p.133-143 (2003).

3. Wolpert Lewis, Beddington, R, Jessell, T, Lawrence, P, Meyerowitz, E and Smith, Jim. Principles of Development; second edition. P345-349, (2002).

4. Galis I, Wagner, Joukuschel. Why is limb regeneration possible in amphibians but not in reptiles, birds, and mammals. Evol Dev 2003 Mar-Apr;5(2):208-20 hhtp://ww.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12622738&dopt=Abstract

This page was constructed by Catherine Connel cconnel@unr.nevada.edu 4-13-03.