Developmental biology can be a wild field to study. Through looking into the fundamental genes controlling body structure and the formation of the body as a whole, strange and bizarre effects can be discovered from the smallest of changes. One of the most famous being the creation of fruit flies that grow a pair of legs from their faces due to the reassignment of body position genes.

It doesn’t take a huge change to have major impact when dealing with these genes because of how involved they are in so many aspects of embryonic and later development. And it is only through the manipulation of these genes that we have slowly begun to tease apart how all the interlocking systems work together to make up the organism that is you.

Insects have worked out well for this field of study due to their simpler and modular body plans and the speed at which they reproduce. Among this research has been the study of ectopic development, the abnormal placement of organs, like the previously noted fly legs. The perturbation of developmental genes and the changes this causes in regards to ectopic body part placement helps to directly show how the regulation of said genes changes the body plan schema.

The Why of an Eye

Researchers at Indiana University decided to look into ectopic development involving the eyes of insects, specifically beetle species and a developmental gene called orthodenticle (otd). A previous study from last year had indicated that reducing the activity of this gene results in the formation of extra compound eye-like structures in members of the beetle genus Onthophagus.

This was fairly surprising, as otd had been studied in other, more commonly used in science, beetle species like the Tribolium genus and in Drosophila fruit flies. For the former, it is involved in photoreceptor formation in the eye, but not in placement or overall development. And for the latter it is involved in photoreceptors and several other eye structures, but again, not the eye as a whole. No insects are known to produce extra “distinct” compound eyes in the middorsal region of the head.

So they chose to do their own experiment using RNA interference (RNAi) to see what would result.

Ectopic Observation

Samples of the otd gene were taken from several Onthophagus species and then cloned, before being broken into fragments and amplified to produce a high amount of them. These fragments were transcribed into double stranded RNA (dsRNA) sequences for use in RNAi (whereby cellular systems use dsRNA fragments to silence the original gene sequences in the genome). Incorporation of these sequences into larval beetles resulted in downregulation of the otd gene and its transcripts.

The first results to look into was whether this alteration is isolated to the two particular species investigated in the 2016 study (O. taurus and O. sagittarius) or whether it happens to other species in the genus and other genera in the tribe Onthophagus is in. Some other changes downregulation of otd caused include defects in the head plate, a reduction in size of the horns and other ornamental protrusions, and the formation of smaller ectopic horns elsewhere on the head.

But the meaningful part is that ectopic compound eyes were observed in all of the species tested and thus are not isolated to the Onthophagus genus. Though their level of development varied significantly, with the most complex being in O. sagittarius. Scanning electron microscope imaging showed that these eyes, while smaller than the normal eyes of the beetles, appear to be fully formed with the correct underlying structures.

An experiment using a blinking light in a dark environment confirmed that the ectopic eyes are connected to the beetle nervous system and functional, though they appear to be more nearsighted than normal beetle eyes.

RNAi and Developmental Biology

The regulatory systems and pathways for how lacking the otd gene leads to these ectopic eyes will require further study to discover, but it does indicate that RNAi is a worthwhile method for testing developmental genes. Especially since it can be used across a wide variety of species as compared to other techniques that are more limited.

This result in the downregulation of a key regulatory gene also indicates that the regulatory pathways and body plan systems are able to compensate and turn what would otherwise be a mess of misplaced body parts into a working set of eyes. Such a revelation opens up ectopic eyes as a part of the developmental biology field and a new way to see how life has evolved over time.

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Photo CCs: Insect eye from Wikimedia Commons

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