Publishing the impossible paper: it’s the biology my dear fellow!!

“It is not completely apparent why the authors want to investigate this subject in further detail”, or something else in this vein.

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There you go. Again and again, we must patiently endure the animosity. Indeed, why would anyone want to produce yet another paper on the classic “chamber-specific” paradigm so brilliantly revealed by Wang, Nikovits, Stockdale and collaborators? 

In a series of papers in 1996, 1998 and 2001, Wang and colleagues showed that atrial expression in the quail slow myosin heavy chain (SMyHC3) gene was established in a manner not unlike the one described by Michelangelo for his impressive Moses sculpture in the Basilica of San Pietro. Asked how he created the masterpiece, Michelangelo supposedly said: “Well, I just took out of the marble the pieces that did not resemble Moses!” Similarly, the quail SMyHC3 promoter establishes a nearly specific form of atrial chamber expression by working as a master sculptor. It sets up unrestricted expression throughout the heart via a GATA DNA binding site in the SMyHC3 promoter, while simultaneously inhibiting ventricular expression through IRX-4, the latter being a transcriptional repressor recruited to the SMyHC3 promoter by RXR, a nuclear receptor transcription factor bound to the promoter region.

For a novice working in Nadia Rosenthal’s laboratory in 1997 and keen to enter the world of cardiac developmental biology, moving the quail SMyHC3 atrial-specific paradigm to a transgenic mouse was a godsend. What a beautiful mouse phenotype! The wild type quail SMyHC3 promoter produced a wonderful atrial marker in the mouse, which we and the Stockdale laboratory utilized to map the beginning of atrial specification in embryogenesis. 

Surely, the mouse mutants will replicate the quail phenotypes! Famous last words! The GATA mutant that abolished expression in the quail heart was completely ineffective in the mouse. Likewise, the RXR mutant that eliminated atrial specificity in the quail was a dud in mice. To a pragmatist, these were enough reasons to be thankful for the luck we had with the wild-type promoter in the mouse and call it a well-earned day. Perhaps we should have. However, in 1997 that was not an option, but a biological challenge. Instead, we wanted to use the mouse SMyHC3 paradigm to unravel mechanisms through which such powerful DNA regulators could establish atrial specificity in heterologous contexts. 

Twenty-seven years later, this particular saga acquired aspects beyond the scientific. Early on, we lost Ralff Ribeiro, the bright nuclear receptor specialist that tipped us off to a potential novel nuclear receptor binding site beyond the classic enhancer described by Wang and colleagues. Ralff’s hunch opened the way for us to demonstrate that atrial activation was missing from the bag of tricks originally defined for the SMyHC3 promoter. We also lost Wang, our molecular Michelangelo and, more recently, our beloved Ukrainian friend Esfir Slonimsky. There is not a late Friday evening that I don’t recall Esfir bringing me, without any previous warning, some half a dozen more mouse cages with precisely timed pregnancies to work with. Without their brilliant contributions and efforts, we would have never gotten at the bottom of this little mystery.

 One might ask what we achieved with this rather belated contribution. We could say that we created a novel computational method to screen DNA-binding proteins, that we reintroduced some order in our thoughts about the origins of the SMyHC3 as a galliform-specific myosin gene, that we suggested an evolutionary origin for a specific type of atrial specificity mechanism in a viral infection of a Cretaceous avian ancestor, or even that we exploited heterologous transgenic models with some merit, when others have failed. However, publishing this impossibly difficult paper has a much greater impact. It gives us back a little bit of hope that a form of scientific inquiry motivated by naked, perhaps naive, curiosity can still find space among high quality journals. 

Now it is time that we go back to the reasons why we felt the need to suffer such extensive wait. I did make some important mistakes along the way, and these were followed by years of self-imposed pauses, until we could amass the resources to go ahead again. We also had to cope with reiterated requests for more and more transgenic mouse lines, to define the differences between staining and reporter gene expression, to wreck our brains over the differences between necessary and sufficient, to explain in detail why we used multimers rather than single copies and, rather discouragingly, to read that we should “drop” the evolutionary story, as well as many other typical reviewer suggestions. 

In summary, we wanted to understand how a small piece of DNA could essentially  control the same phenotypic characters, but by clearly different means, while working in evolutionary and ontogenetic contexts separated by hundreds of millions of years. This is time enough for major divergence in both DNA and transcription factor aminoacid sequences, so the persistence of atrial specificity in quail, mice and zebrafish demanded some good explanations from the realm of actual biology, not from a fantastic wonderland in which DNA sequences must work the same way in all species. So, there you go. The anwer is the biology, my dear fellow!!

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