Loss of spam de-evolves the fruit fly’s compound eye

One of the main characteristics of insects is the compound eye, a faceted structure consisting of individual, radially-arranged units called ommatidia. The number of ommatidia varies hugely in different types of insects – some types of worker ants have as little as 6, while some dragonflies have in excess of 25,000.

Each ommatidium contributes a spot to the visual image, which consists of the total inputs from all the ommatidia in the eye. Fruit fly vision can be thought of as being pixelated, with an image being produced in much the same way as the pixels on a computer screen. Each faces a slightly different direction, and together they produce a mosaic view of the visual field. The resolution of the image is determined by the number of ommatidia in the eye and the angle between them. Each ommatidium projects an optic nerve fibre to the brain, where visual information from the eye is processed to produce a visual image.

Because the visual image is a mosaic, and also because it has no central lens or retina, the compound eye does not produce a true, focused image of the environment, and the visual acuity of insects is low. There is, however, a very high temporal resolution, because the compound eye has a very high flicker fusion rate – that is, it can register as may as 200 images per second, in comparison to about 30 for humans. As a result, insect eyes are ideal for motion detection. Movements are tracked from one ommatidium to another, and small changes in the visual field are detected very quickly.

Compound eyes can broadly be categorized into two types. Most insects have apposition compound eyes consisting of ommatidia in which the rhabdomeres – the light-sensitive areas containing the photopigments – are fused together to form a closed system. In contrast, fruit fly Drosophila melanogaster and the mosquito have neural superposition compound eyes, in which the unfused rhabdomeres form an open system. An open rhabdomere system provides higher resolution because the image contains more points (or pixels).

In an advance online publication at Nature, Zelhof et al report a mutation which transforms the architecture of the fruit fly’s compound eye from an open rhabdomere system into a closed one. The findings shed some light on how the open rhabdomere structure of the fruit fly’s compound eye – the most complex of arthropod eyes – evolved from the simpler closed structure of its ancestor.

The compound eye of the adult fruit fly consists of a hexagonal array of about 800 ommatidia. It is a highly polarized structure, with mirror symmetry around its equator. Each individual ommatidium is an assembly of 20 precisely arranged cells – 8 photoreceptor neurons and 12 supporting cells and pigment cells. The ommatidium is covered externally by a transparent, crystalline corneal lens, which focuses light onto the light-sensitive structures underneath.

Like the compound eye’s ultrastructure, the cellular structure of the ommatidium is also hexagonal. Each is shaped like a hollow elongated cone, with six pigment cells, 3 cone cells and 3 mechanosensory bristles forming the walls. Inside, photoreceptors 1-6 (R1-6) are arranged in a ring surrounding R7 and R8, which are stacked on top of each other in the centre. This gives ommatidia a characteristic pattern of 7 spots when viewed from the top or in cross-section (above left).

R1-R8 are arranged around a central space. Each receptor cell sends 60,000 finger-like projections, called microvilli, into a central space. The microvilli from the eight cells are collectively called the rhabdomere, and it is this structure that is responsive to light. Each microvillus has a diameter of 1-2 micrometres, and contains millions of photopigment molecules (primarily opsin), packed closely in arrays of microtubules.

Development of the fruit fly eye involves the production of a precisely arranged geometric pattern of cells within a simple sheet of epithelial cells. Beginning around the eye’s equator, epithelial cells begin to differentiate into ommatidium cells. R8 cells are the first to differentiate, and they initiate a cascade of cell-to-cell interactions mediated by diffusible signalling molecules. Each newly differentiated receptor cell signals neighbours, until the ommatidium has a full complement of cells and is assembled. In all types of compound eyes, the membranes of the 8 receptor cells are still fused at the apical surface soon after assembly of the ommatidium. In insects with superposition compound eyes, the interactions between adhesion molecules in the apical membranes are blocked, and the rhabdomeres become separated, creating the inter-rhabdomal space in which the microvilli will be located. In insects with apposition eyes, the rhabdomeres remained fused.

Zelhof and his colleagues screened different lines of chemically-mutagenized fruit flies, using electron microscopy to look for specimens with altered rhabdomere topology. Two of these lines had eyes containing fused rhabdomeres. The genes containing the mutations were identified and then cloned, and named spacemaker (spam) and prominin (prom). Analysis of the DNA sequences suggested that the spam gene product is a secreted protein and that prom is similar to membrane-spanning proteins of unknown function which are associated with microvilli.

The figure below shows rhabdomeres from wild type (left) and a spam loss-of function mutant flies stained green The wild type fly has a clearly defined inter-rhabdomeral space marked by spam expression (magenta) whereas the mutant hasn’t. Zelhof’s team then showed spam expression in the rhabdomeres is necessary for the apical membranes to separate, and that this expression has to occur within a given time window for the rhabdomeres to be fully separated. If the gene is expressed later than it should be, the apical membranes are separated but the rhabdomeres remain fused together at the middle. This phenotype is similar to that of Prom mutants, and the expression patterns of the two genes are similar, so it is suspected that the two gene products act as receptor and ligand pair to generate the inter-rhabdomal space. That overexpression of spam results in an enlarged inter-rhabdomal space is evidence for this hypothesis. Then, Zelhof’s team showed that the spam-prom interaction induces genesis of the inter-rhabdomal space by blocking the actions of a third molecule, called Chaoptin, a cell adhesion molecule which makes rhabdomere membranes adhere to each other.

From the fossil record, it is known that arthropods have had faceted eyes for at least 540 million years. About 100 million years ago, separate rhabdomeres evolved in the diptera (flies, mosquitoes, gnats and midges) as a way of increasing spatial resolution. The notion that spam was recruited during evolution to separate the rhabdomeres is supported by several other pieces of evidence. First, while prom and chaoptin are expressed in the eyes of insects with open rhabdomere systems and in those of insects with closed systems, spam is expressed only in the eyes of insects with open systems. Secondly, ectopic spam expression in an eye with a closed rhabdomer system – the ocelli, simple compound eyes on the top of the fruit fly’s head, which are involved in navigation and consist of less than 100 photoreceptors – resulted in the generation of an inter-rhabdomal space where one normally wouldn’t be found.

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