For the first stage of the experiment, preliminary testing the hypothesis, we would not need a large number of test subjects. A few blood samples from healthy volunteers would be enough to obtain a sufficient amount of neutrophils to use in a chi-square test. Only in the latter phases would we need a wider selection of subjects.

The ideal technique for the previously described experiment would be an in situ hybridisation combined with a nuclear membrane or background staining, but a very strict conservation of nuclear morphology is essential and current hybridisation protocols show a limited conservation of nuclear shape.

All of the experiments described in this paper need a way of combining an in situ hybridisation with nuclear staining and a practically perfect nuclear shape conservation. The deformation is most likely caused by fixation and more efficient fixation techniques can help solving the problem completely or partially (Contreras-Dominguez et al. 2010), but also the high temperatures needed for the separation of DNA strands can play a role in it and we may have to face this problem in order to follow this research line.

The possible increase in nuclear loss of shape caused by denaturalisation temperatures could be avoided either by:

1. Previously fixating the nuclear membrane in a way that protects it from thermal deformation.
2. Achieving the separation of DNA strands by methods less invasive than high temperature.
3. Hybridising with an intronic sequence of a highly transcribed hnRNA instead of DNA, thus eschewing the need to denaturalise the DNA at all.

The less invasive methods mentioned in point 2 might be genetic tools like target-specific transcription factors and other signals used by the cell to separate both strands. Depending on how the transcription of each locus is regulated, it could be less reliable than high temperature. If we want our probe to arrive at the vacant sequence and stay hybridising, we may also need to prevent the RNA polymerase from joining or transcribing the locus or to employ nucleic acids with higher affinity for DNA than the other DNA strand, such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) (Briones and Moreno 2012).

Hybridisation with an intronic sequence of hnRNA, as mentioned in point 3, would solve the problem of having to separate both strands of DNA, but it would be extremely difficult to wash away the excess probe without depleting the nucleus, so we may need to employ FRET or hairpin probes that only emit their signal when they have reached the target.

The main problem in this case would be telling apart two copies of the gene being transcribed in the same lobe from only a single copy being transcribed at all, since the probes would not join a single spot of the strand but rather a region of the nucleus with copies of the RNA strand (Fig. 8). Different, allele-specific probes in different colours for polymorphic genes would help to solve the problem in heterozygosis, but it adds a new complexity layer while also diminishing the number of loci we can study at the same time.

Figure 8.  

When hybridising with intronic hnRNA, we would need an additional probe in order to discard false positives because of a copy of the gene not being transcribed and it would only work in heterozygotes. In this case, using red and green probes for different alleles would show as yellow if both copies are transcribing in the same lobe or as red or green if only one of them is being transcribed.

The reason different loci can be distributed between following a fixed pattern amongst the lobes may be that regulatory factors present in one lobe are absent in other.

Another option, in the case of neutrophils, is that some unused genes for this line (or the unused copy of a gene) are being processed in one lobe to convert them into neutrophil extracellular traps (NETs). DNA in these loci would be treated in such a way as to keep it prepared for quick release, but not available for gene transcription.

This said, the NET would not be enough to explain eosinophil and basophil segmentation if we take it on its own, so it would more likely be either a matter of gene regulation or both. At any rate, it may help to explain potential patterns in which both copies of a gene are present in different lobes in neutrophils (Fig. 9).

Figure 9.  

Distribution between lobes can help both copies of the unused sequence to be modified to become NETs (A), but it can also help to modify only one copy of a used gene (B) by containing the modifying enzymes in one lobe and keeping a functional copy of the gene for normal transcription.

About the mechanisms involved in either or both of these cases:

• It can be as simple as regulatory RNA acting only in the lobes that their genes are located. If this hypothesis is the only true one, then the physical barriers themselves are enough to achieve the desired goal (Fig. 10).
• On the other hand, the process can be as complex as protein transcription factors or DNA-modifying proteins being distributed heterogeneously. In this case, since these factors are synthesised in the cytoplasm, the responsible mechanism would be differential migration, which would point to specific structures in or near nuclear pores amongst the lobes (Fig. 11).

Figure 10.  

Regulatory RNA would function in the same lobe as it was transcribed, affecting only genes distributed along with its own locus.

Figure 11.  

Protein transcription factors would be synthesised in the cytoplasm, so their genes can be in any lobe. Their presence in each lobe would depend on the properties of its nuclear pores. In the image, red pore complexes would reject the factor while the blue ones facilitate their importation into the lobe.

Both of these mechanisms can be present at the same time, so experimentally proving one of them does not mean the other one is neither true nor false.

If this theory is proven, the next step would be a descriptive study in which we take groups of loci to study at the same time, so we learn which genes are grouped together. The different lobes would receive names according to their properties or DNA composition. The nomenclature here proposed uses the Greek alphabet:

• Alpha for the lobe in which the centromere of chromosome 1 is (or the next chromosome in numeric order, if this centromere’s position is distributed at random).
• Beta for the lobe in which is the next centromere in numeric order that is not in lobe Alpha and is not randomly distributed.
• Gamma, delta and so on, following the stated criteria in cells with more than two lobes, including polysegmented cells.

A better, more descriptive nomenclature can be devised once we know more about the eventual specific properties and function of the different lobes.

In this stage, it would be desirable to obtain blood from a wider pool of healthy subjects so we can attest for individual and ethnic variation.

At this point in the research, since we would have proven the existence of a non-random pattern, it might be worth trying to apply chromosome painting instead of short probes, which may have appeared too expensive in the early steps but would be much more practical now.

After all of these experiments are done, assuming we find evidence of a non-random pattern of distribution of DNA, the last foreseeable step (which can be taken at the same time as the clinical approach) is to find out which mechanisms cause this phenomenon. Since the theories mentioned above are not mutually exclusive, each must be tested on its own.

Regulatory RNA can be easily tested, in theory. All we need to do is to detect their presence exclusively in the lobes along with their coding loci.

Protein transcription factors or DNA-modifying enzymes can be also studied by looking for them directly, but the pore complexes in different lobes should also be analysed.

In the case of NETosis, it can additionally be useful to search for DNA modifications in each lobe.

These seemingly easy tasks come with the added challenge of having to combine these techniques with at least one lobe identification. This longer-term problem will no doubt be more easily addressed, if needed and when it presents itself, due to the accrued experience we would have in this field at that point.

If the previous step shows a potential clinical usefulness, the most cost-effective technique for routine use would be using a probe for each involved locus (provided there are not technical issues that prevent us from employing this approach). Developing standard labelling for each lobe can make the work of probe designers simpler, be it through the addition of lobe-specific loci probes or through the detection of specific nuclear proteins, in the nuclear membrane or otherwise.

We are facing an obvious question that has not been raised before. Unfortunately, new experimental techniques are needed in order to properly study it but, should they be developed, we have a clear path for the investigation to follow.

Even though there is a clear possibility for this subject to have repercussions on some diseases, the first step would be to investigate it from a descriptive viewpoint before we can try a clinical approach, which might discourage some clinically orientated groups from investing in this line at first, but the potential here, especially for groups with less direct clinical implication, is large enough that others will surely try.