the WTF factor

Somethingsaddendum below really wrong here.

French speaking people may follow the discussion here and there, hoping for a focused reply.

page 32, column 2, §2:

However, it has been shown that many growth factors especially that of the FGF family are in fact regulated linearly by pressure [142].

Where [142]: Tracheal occlusion increases the rate of epithelial branching of embryonic mouse lung via the FGF10-FGFR2b-Sprouty2 pathway, Mathieu Unbekandt, Pierre-Marie del Moral, Frederic G Sala, Saverio Bellusci, David Warburton and Vincent Fleury, Mechanisms of Development Volume 125, Issues 3-4, March-April 2008, Pages 314-324 doi:10.1016/j.mod.2007.10.013

From the [142] article’s outline

4. Experimental procedures
4.1. Lung cultures
4.2. Phosphorylated Histone-H3 and phosphorylated ERK immunofluorescence
4.3. Embryonic lung growth development movies
4.4. RT-PCR
4.5. β-galactosidase expression
4.6. Fgf10 Whole mount in situ hybridization
4.7. Fgfr2-IIIb Antisense RNA
4.8. Fgf10 LacZ/− embryonic lungs
4.9. Statistics

No pressure measurement mentioned and you aren’t going to find any in the paper itself.

And you can hear Fleury himself explaining that they failed to do pressure measurements. [french, 47:30-48:02]

In the Unbekandt et al. paper, there is a single measure of Fgf10 mRNA, after 48h of culture. There are three other genes tested in parallel, Shh, Vegf and Sprouty2, none of the of the “FGF family

Regulated linearly by pressure? How do you get this result with a single measurement of one of the variables (gene expression) and none of the other one (pressure)?

Image-14.jpg

How?

Maybe Dr Fleury is able to explain that.
Dr Fleury, make your •


[addendum 2009.05.18 22:01]

I’m really interested by the upregulation of any gene from pressure as I’ll have to discuss it for the next sections of Fleury’s paper. So, while reading the Unbekandt et al. paper I stumbled upon something really weird and that makes for a WTF2 factor.

Figure 4 presents the results of semi-quantitative PCR using as template cDNA from the control and test (occluded trachea) tissues. Panel B is the interesting part. No error bars! Could that be that the assay was performed only once? Well, could be. Error bars are used for other histograms in the study (see fig. 1, 2 or 3 for example). Not for this one.

Neither the method employed for quantification, from the relatively low quality gels, is described.

Let’s go a step further to reach the WTF3 stage.

At the time the paper was submitted [Received 21 March 2007; revised 16 September 2007; accepted 31 October 2007. Available online 7 November 2007] the role of hypoxia in lungs development was known: Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung, Am J Physiol Lung Cell Mol Physiol doi:10.1152/ajplung.00185.2004 First published Sep 17, 2004 288:167-178, 2005, Minke van Tuyl, Jason Liu, Jinxia Wang, Maciek Kuliszewski, Dick Tibboel and Martin Post.

Minke van Tuyl et al. shown that hypoxia is sufficient to enhance epithelial branching morphogenesis:

The rate of branching of both tissue elements [epithelial and endothelial cells] was increased in explants cultured at 3% oxygen compared with 20% oxygen.

During this period,

Expression of two crucial epithelial branching factors, fibroblast growth factor-10 and bone morphogenetic protein-4, were not affected by low oxygen. Epithelial differentiation was maintained at low oxygen as shown by surfactant protein C in situ hybridization. To explore epithelial-vascular interactions, we inhibited vascular development with antisense oligonucleotides targeted against either hypoxia inducible factor-1 or VEGF. Epithelial branching morphogenesis in vitro was dramatically abrogated when pulmonary vascular development was inhibited.

So, without increasing the Fgf10 expression, neither occluding the trachea and thus with internal lung pressure equal to the atmospheric, the branching morphogenesis of the lung is active, in low oxygen cultures. And the effect is mediated by an increased expression of Vegf.

In the absence of measures of internal lung pressure in tissues with an occluded trachea, and the absence of oxygen partial pressure in the same lung, knowing that an increase of Fgf10 expression is not necessary for lung morphogenesis, how can one publish a report expecting to demonstrate that “Tracheal occlusion increases the rate of epithelial branching of embryonic mouse lung via the FGF10-FGFR2b-Sprouty2 pathway” ?

Or didn’t he knew about the Minke van Tuyl et al. paper ?

Well, maybe Fgf10 expression increase due to internal pressure in the occluded lung, but it’s over-expression isn’t necessary for lung development and from the data presented in the Unbekandt et al. paper one can certainly not conclude “that many growth factors especially that of the FGF family are in fact regulated linearly by pressure

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