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Opsins and spectral sensitivity
The capacity of an animal to adapt the visual system to its needs and to the environment that surrounds him, is intrinsically related to the spectral sensitivity given by the opsin.
Opsins are members of the G protein coupled receptors, composed of 7 transmembrane helices, which activate internal signal transduction pathways. They are covalently bound to an UV sensitive chromophore (usually a retinal) at lysine residue of the 7th helix. The connection of the opsin to the chromophore will shift its absorbance spectrum towards the red. Fine tuning of the spectral sensitivity is then determined by specific amino acids present in the opsin, at the side chains of the binding pocket.
Based on phylogenetic analysis of opsin sequences, we can distinguish 4 major classes of opsins in the animal kingdom (Porter et al., 2012a):
– C- opsins, present in ciliary photoreceptors
– Cnidopsins, present only in cnidarians and ctenophores
– R-opsins, present in rhabdomeric photoreceptors
– Group 4 opsins, less characterized opsins, including retinal G-protein-coupled receptor neuropsins and peropsins.
The most common types of opsins are the C and R opsins. They differ in their modes of function: when activated by light, C-opsins cause the hyperpolarisation of the cell, followed by a GT signalling cascade, whereas R-opsins depolarize and have a Gq signalling transduction pathway. For 3 of these 4 groups we can find members of all animal taxa, suggesting that multiple lineages of opsins were already present in the last common metazoan.
Opsins are expressed in a wide variety of tissues and cell types, and not all are used for image formation. Examples include the pinopsins and parapinopsin (C-opsins), found in the pineal organ of birds, lizards and lamprey, peropsin, expressed in the bee’s brain, melanopsins (R-opsins), which are present in the vertebrate retina but are responsible for setting of the circadian rhythms rather than image formation (Do et al., 2009) and the R-opsins expressed in the tube feet of sea urchins(Lesser et al., 2011).
Visual opsins can be separated into 3 classes, based on their absorbance spectra: long-wavelength (LWS), middle-wavelength (MWS) and short-wavelength (SWS), corresponding to green-yellow, blue and UV absorbance spectra respectively (Fig. 0-3).
Fig. 0-3 The electro-magnetic spectrum Visible light has a frequency from ~400 to ~750 nm
Vision in arthropods
Arthropods have the widest diversity of eye designs in the animal kingdom making this an exceptional group for studying eye evolution. The panoply of species, their wide range of habitats and diverse modes of living are reflected in the number of eye designs present, revealing the importance of the visuals system to the adaptation of the animals to their habitats.
In extant arthropods, we can find 4 main types of eyes (reviewed in (Nilsson and Kelber, 2007; Strausfeld et al., 2016)):
Compound retinas with fixed number of photoreceptors per ommatidium and lenses formed by crystalline cone cells
o Typical in insects, crustaceans and scutigeromorphs (Myriapoda)
Large corneal eyelets surmounting a varying number of stacked photoreceptors o Found in myriapods (except Scutigeromorpha)
Compound retinas with variable numbers of photoreceptors and corneal lenses o Found in Xiphosuran eyes
Single lens eyes
o Found in Chelicerates (except Xiphosura)
The earliest compound eyes found in the fossil record belonged to radiodontans, a lineage belonging to the arthropod stem group, whose emergence preceded arthropodization. Radiodontans are considered to be the largest predators during the Cambrian. They possessed enormous compound apposition eyes, with up to 16000 facets (Cong et al., 2014; Paterson et al., 2011).
The finding of radiontan compound eyes in the Cambrian, supports the position that high resolution apposition compound eyes, with isomorphic ommatidia and a fixed number of photoreceptor cells, are the ground pattern organization for arthropods (Strausfeld et al., 2016). From that ancestral state, we see significant conservation in crustaceans and insects, and radical divergence in chelicerates (including single lens eyes of arachnids) and myriapods (except scutigeromorphs).
The architecture of the visual system has been extensively used to reconstruct the phylogenetic relationships between arthropods. The first theory that insects and malacostracan crustaceans would share a common ancestor was based on comparisons of their retinal structures by E. Ray Lankester in 1904.
While there is an ongoing debate on the phylogenetic relationships of different arthropod groups, recent studies point clearly towards a shared ancestor of insects and crustaceans, giving rise to the monophyletic group Pancrustacea. The interrelationships within this group are still controversial (Fig. 0-4) (Budd and Telford, 2009; Cong et al., 2014; Legg et al., 2013; Regier et al., 2010).
Fig. 0-4 Arthropod phylogeny – Two of the current views on arthropod phylogeny based in Legg et al. 2013 (A) and Regier et al. 2010 (B)
Despite the long-lasting interest in the study of arthropod eyes, most of our knowledge on the development and neural architecture of the visual system comes from few model organisms, usually hexapods. The most profound knowledge we have comes from Drosophila melanogaster, where genetic/molecular tools allow for a careful study the architecture of the visual system and on the molecular players that give rise to it.
The visual systems of crustaceans have been studied largely in the context of ecology and neuro-physiology, but not in a scale comparable to hexapods. Contributions on the development of crustacean eyes are still scarce and largely descriptive. Part of the reason for this is the lack of model organisms suitable for genetic manipulation. This scenario has been changing recently with the adaptation of the small crustacean Parhyale hawaiensis to the lab life.
Parhyale hawaiensis
Life cycle
Parhyale is a small malacostracan crustacean of the order Amphipoda.
The generation time of Parhyale is approximately 2 to 3 months at 26ºC and animals will continue to grow throughout their lifetime (from ~1 to ~10mm in lenght). Reproduction is continuous throughout the year, as long as the conditions are favourable. For reproduction, the male grabs the female, forming a couple, until oviposition and fertilisation of the eggs. The fertilized eggs are carried by the female in a brood pouch, situated ventrally between the thoracic appendages. Hundreds of eggs at 1-cell stage can be obtained daily from anesthetized females for injections. Once the embryos hatch, they are released from the brood pouch and sexual maturation will be reached after ~7 weeks.
Parhyale has a direct development; the duration of embryogenesis is 10-12 days at 26ºC and developmental stages have been described(Browne et al., 2005). Early cell lineage is stereotypical (a common feature of malacostracan embryos (Dohle and Scholtz, 1988; Dohle et al., 2004)): the first cleavage separates left from right side for most of the ectodermal and mesodermal tissues and at the 8-cell stage each blastomere will contribute to a single germ cell layer (Gerberding, 2002; Wolff and Gerberding, 2015). This characteristic allowed for studies on germ layer specification and compensation during development (Alwes et al., 2011; Gerberding, 2002; Price et al., 2010) and limb regeneration (Alwes et al., 2016; Konstantinides and Averof, 2014). Cell divisions and migration during gastrulation are also described (Alwes et al., 2011; Chaw and Patel, 2012).
Fig. 0-5 Parhyale hawaiensis life cycle – Adult Parhyale reach sexual maturation at around 2-3 months. Embryogenesis lasts 10 days at 26ºC. Embryos at one cell stage can be retrieved from dormant females and cultured in sea water. 8 hours after fertilization the egg underwent a total of three cleavages, giving rise to 4 micromeres and 4 macromeres with restricted cell fates: El, Er and Ep give rise to left, right and posterior ectoderm, respectively; Mav gives rise to the anterior and visceral mesoderm; ml, mr originate the left and right mesoderm; en gives rise to the endoderm and g to the germline. After 9 days, at stage 28, the eyes present a red pigmentation. All scale bars are 200 µm except in the adult female that is 1000 µm. Adapted from (Stamataki and Pavlopoulos 2016). Stages after (Browne et al., 2005), early cell lineage from (Gerberding 2002).
Habitat
The colonies that inhabit the labs around the world today, have all come from a single population, found in the filtration system of the John G. Shedd Aquarium in Chicago in 1997. The original source of that population is unknown.
In nature, Parhyale is distributed worldwide in tropical areas, in intertidal and shallow waters such as mangroves or rocky shores. Sighting records include the Lizard Islands (Australia), the Canary Islands, Trinidad, south-eastern Brazil, Fiji Islands. Frequent changes in salinity, temperature and turbidity in these habitats have produced a robust species that can be easily kept in the lab.
Behaviour studies on circadian clocks show some evidence for an increased activity of Parhyale during the night (B. Hunt PhD thesis), peaking at sunrise and sunset hours.
Working with Parhyale
Parhyale was introduced to the lab by Brown and Patel in late 1990s. It has been used as a research model for almost 20 years, with a community of researchers engaged in developing new experimental tools in this species. Transgenesis (Pavlopoulos and Averof, 2005), gene misexpression (Pavlopoulos et al., 2009), gene knockdown(Liubicich et al., 2009; Özhan-Kizil et al., 2009), CRISPR/Cas9-mediated gene editing(Martin et al., 2016) , a sequenced genome and other genomic and transcriptomic resources (Blythe et al., 2012; Kao et al., 2016; Nestorov et al., 2013; Parchem et al., 2010; Zeng et al., 2011) are available in this species.
These tools and the fact that crustaceans are a sister group to hexapods, make Parhyale an attractive organism to compare with Drosophila and to make inferences about the evolution of developmental, morphological and physiological traits.
One of the most impressive features of Parhyale is its amenability for live imaging. The transparency of the embryos and of the adult cuticle allows imaging and cell tracking in embryonic, juvenile and adult stages for several days (examples in Fig. 0-6). Stunning examples are the reconstruction of the cell lineages underlying limb outgrowth using light-sheet microscopy (Wolff et al., 2017) and in the study of cell dynamics during limb regeneration (Alwes et al., 2016). Combining this characteristic with the possibility of transgenesis makes Parhyale a powerful organism for studying development, regeneration and cell behaviour in real time.
Fig. 0-6 Live imaging in Parhyale – A) Head of a Parhyale embryo seen from the dorsal side, showing dsRed expression driven by the 3xP3 regulatory sequence (white arrows) B) Trunk of a Parhyale juvenile, showing dsRed expression driven by the Ph-MuscleSpecific regulatory regions. From (Pavlopoulos 2005)
Despite the established genetic tools, many others which are routinely used in other model organisms (such as zebrafish and Drosophila) have failed to work in Parhyale. Namely we are still missing a constitutive/ubiquitous promoter despite several trials with endogenous and viral promoters (N. Konstantinides PhD thesis and A. Pavlopoulos personal communication). Also using the Cre/lox and Flp/FRT recombination systems, often employed to generate cell mosaics, proved unfruitful (N. Konstantinides PhD thesis and M. Grillo personal communication).
Cell specific markers are also still missing. One of the reasons for this is the difficulty in exploring the Parhyale’s genome for regulatory regions, due to the large intergenic regions. A gene-trapping approach has been established in Parhyale (Kontarakis et al., 2011), and few gene-trap screens have been conducted in the lab, yielding a few gene-trap lines. However, more often than expected, many of these lines proved to be unstable, both in maintenance of the trap and survival rates.
Purposes of the project
The diversity seen in arthropod visual systems is achieved by modifications on the optical properties of the eye and neuroanatomy. Processing of information is dependent on the connections established between the eye and the brain and within the optic neuropils.
In arthropods, the knowledge on visual information processing has been largely driven by studies in a few model organisms, and principally Drosophila. The development of molecular, genetic and imaging tools in this model organism has provided great insight on the development, the neuroanatomy and sensory processing in the visual system. Outside the diptera clade, most of the studies on arthropod visual systems have relied on the usage of more classical techniques, such as transmitted electron microscopy, electrophysiology and unspecific/stochastic labelling of neuronal cells (e.g. Golgi staining). This is due, in part, on the difficulty applying modern molecular, genetic and imaging tools to non-model organisms.
Studies in Drosophila have been giving us an enormous amount of knowledge on the function of the arthropod visual system, however the lack of other organisms where genetic and imaging tools can be applied leads to a lack of knowledge on the diversity of how the visual system develops and functions, and, consequently, on its evolution. The need for new arthropod models to study the visual system has, therefore, become very important.
Parhyale has proven to be a reliable model organism where genetic and imaging tools can be applied. This provides the opportunity to compare crustacean and dipteran visual systems in greater depth, and gain insights on the diversity and evolution of the visual system development, structure and function.
I started this project with two main (related) objectives: 1) To explore the visual system of Parhyale, focusing on a description of the eye structure, neuroarchitecture and function; 2) to develop genetic tools that allow us to label different neuronal cell types, helping to identify (and possibly manipulate in the future) different components of the visual system, starting from the primary visual sensors, the photoreceptors cells.
Table of contents :
Preamble
1. General introduction to visual systems
2. Vision in arthropods
3. Parhyale hawaiensis
4. Purposes of the project
Part 1 – A description of the Parhyale visual system
I. Introduction
I.1 – The compound eye
I.1.A – Three types of compound eyes
I.2 – Photoreceptors and ommatidia structure
I.2.A – Visual pigments of compound eyes
I.3 – Visual information processing – the optic lobe
I.3.A – Visual information processing – lessons from the fly
I.3.B – Optic lobe evolution in Arthropods
I.4 – Purposes of Part 1
II. Results
II.1 – Structure and growth of Parhyale eyes
II.2 – Photoreceptor types
II.3 – Optic lobe structure
III. Discussion
Part 2 – Eye adaptations to the environment in Parhyale
I. Introduction
I.1 – Light intensity adaptations – pigment cells and the arthropod pupil
I.2 – Polarisation vision
I.3 – Polarisation–related behaviours
I.4 – Purposes of Part 2
II. Results
II.1 – Pupil adaptation in Parhyale
II.2 – Polarisation sensitivity in Parhyale
III. Discussion
Conclusions and future perspectives
Materials and Methods
References
