Evidence favoring the biophysical model for Hox gene collinearity

On March 29, 2011 Patrick Tschopp wrote:

Dear Spyros,

thank you for your message. I have read already your recent review, in fact Denis gave me the print-out you sent him. However, I also stand by the conclusions and the model we deduced from our series of experiments.

The Evx3-Hoxd13 region has been investigated in quite some details, with the recent inversion as well as deletions encompassing Hoxd13. If only this stretch of DNA was deleted, no premature expression was observed, only when fragments more centromeric to Evx2 were affected (see Kondo and Duboule 1999; Tschopp and Duboule 2011).

A clean deletion of this intergenic region would require the generation of two new loxP sites, thus tedious ES cell work and at least 2 -3 years of follow-up. As I will be leaving the lab in less than two months, I will not be able to consider either one of the experiments you suggested!

Thanks anyway for your continued interest in our work.

Sincerely,

Patrick

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The above email gave me the opportunity to read carefully the Kondo and Duboule paper (Cell 97, 407-417 (1999)) and this was a gratifying revelation.

In this paper, among other experiments, K and D describe the following genetic deletions in the Hoxd cluster:

Deletion del0: the posterior region of the cluster is deleted 

                                          ↑---Hoxd13------ Hoxd12------- Hoxd11---↑

Deletion delII: together with del0, the exterior region --Evx2----------- is deleted:

                       ↑--Evx2----------------Hoxd13------ Hoxd12------- Hoxd11---↑

K and D examine the expressions of Hoxd4 and Hoxd10 after deletions del0 and delII for transgenic mice embryos at stage E7.5 to E8.5. Their results are shown in Fig.6 of their 1999 Cell paper and they conclude (p. 412) “ …that both  Hoxd4 and Hoxd10 were activated prematurely in the delII  configuration. Unexpectedly again, analysis of Hoxd4 and Hoxd10 expression in older del0 and delII fetuses did not show any deviation from their normal profiles (not shown), suggesting that the early deregulation was not maintained subsequently”.

Let us make an integrated analysis of the above unexpected results in the framework of the biophysical model:

1. The wild type expression of Hoxd4 at stage E8 is observed posteriorily in the embryo while this expression is missing (at the same stage) in mutant embryos with a posterior Hoxd deletion (see Fig. 6D in P.Tschopp et al. (2009) PloS Genet. 5  (3) ).

This observation was explained recently (S. Papageorgiou (2011) Develop. Growth Differ. 53, 1-8). [Let me repeat in short the explanation: in a posterior deletion, the total negative charge N of the cluster is reduced. In order to compensate this reduction, the positive charge P of the surroundings must increase and this causes a retarded posteriorization of the expression. Hence, at the stage E8 the mutant expression of Hoxd4 has not appeared yet]. At E7.5 to E8.5 and for del0, the Hoxd4 expression is absent –in contrast to the wt Hoxd4 expression.

2. A basic hypothesis of the biophysical model is the representation of the Hox cluster by an elastic spring whose fixed end is located inside the chromosome territory and its free end can be pulled toward the interchromosome domain. The fixed end lies between the 5’end of the cluster and Evx2. When the fixed end of the spring is cut-off, the spring becomes loose and can be pulled and expanded with smaller than normal forces –which means prematurely.

In  delII, besides del0, an extra region is deleted (---Evx2-------) which lies outside the Hoxd cluster. This is the region of the fixed end of the spring. Therefore in delII as compared to del0 a premature gene expression is expected. This is confirmed and depicted in Fig.6 of the K and D (1999) paper. This ‘unexpected’ premature activation is naturally derived from the biophysical model. To my knowledge, only the biophysical model predicts this behavior of the Hoxd transgenic expressions.

3. In the experimental proposals (see my blog entry March11, 2011), the aim of Experiment A is to test whether the Hoxd expressions are prematurely expressed after the deletion of the   --Evx2---------    region. It is ironic that this model expectation of premature activation was already confirmed in retrospect by Kondo and Duboule in 1999! Therefore, Experiment A is not necessary anymore. The proposition for Experiment B still holds.

Spyros Papageorgiou

Email:spapage@bio.demokritos.gr