Pax6 Homolog Research Articles

Knockout of a single Pax6 gene (toy but not ey) leads to compound eye deficiency and small head in honeybees

The compound eyes are crucial to honeybees, playing pivotal roles in color recognition, orientation, localization, and navigation processes. The development of compound eyes is primarily mastered by an evolutionarily conserved transcription factor Pax6. In honeybees, there are two Pax6 homologs: ey and toy. To gain a deeper understanding of their functions, we knock out both homologs using CRISPR/Cas9 technology. Intriguingly, we observe that toy knockout mutants have smaller heads without compound eyes and exhibit brain atrophy, while ey knockout mutants develop normal compound eyes, most of which die before/during their metamorphosis from pupa to adult. By comparing the head transcriptomes of four stages (larva, prepupa, pupa, and adult) in toy-knockout mutants versus normal controls, we identify significantly perturbed genes related to DNA binding transcription factors, neuron differentiation, and insect visual primordium development. Additionally, we find the interaction network of toy in honeybees differs obviously from that of D. melanogaster. Our findings suggest the two Pax6 genes serve distinct functions in honeybees and toy takes over the central function of ey in master-regulating the development of honeybee compound eyes. This adds new evidence for breaking the simplified view that some of conservative developmental toolkit genes function as all-or-nothing master regulators.

Evolutionary Conservation and Diversification of Five Pax6 Homologs in the Horseshoe Crab Species Cluster

Evolutionary Conservation and Diversification of Five Pax6 Homologs in the Horseshoe Crab Species Cluster

Coming into clear sight at last: Ancestral and derived events during chelicerate visual system development.

Pioneering molecular work on chelicerate visual system development in the horseshoe crab Limulus polyphemus surprised with the possibility that this process may not depend on the deeply conserved retinal determination function of Pax6 transcription factors. Genomic, transcriptomic, and developmental studies in spiders now reveal that the arthropod Pax6 homologs eyeless and twin of eyeless act as ancestral determinants of the ocular head segment in chelicerates, which clarifies deep gene regulatory and structural homologies and recommends more unified terminologies in the comparison of arthropod visual systems. Following this phylotypic stage, chelicerate visual system development differs fundamentally from other arthropods during the compartmentalization of the ocular segment in that eye and optic neuropil primordia originate independently from each other. Comparative analyses of this phase identified further gene regulatory homologies but also major differences, most notably the possibly compensatory replacement of Pax6 by Pax2 in lateral eye specification. Also see the video abstract here: https://youtu.be/Hdfr3z5kEXU.

Characterization and Comparison of Eye Development and Phototransduction Genes in Deep- and Shallow-Water Shrimp Alvinocaris longirostris and Palaemon carinicauda

The investigations of the molecular components of eye development and phototransduction in deep-sea species are important to elucidate the mechanism of their adaptation to dim light. In this study, eye transcriptomes of the shrimp Alvinocaris longirostris from the deep-sea chemosynthetic ecosystem and the shallow-water shrimp Palaemon carinicauda were compared. Two Pax6 homologs with low expression levels were identified in both species, which are essential transcription factors in eye development. This finding implies that the development of the two shrimp eyes at early embryo–larvae stages might be similar. The multiple components of the phototransduction pathway were identified in both species. However, the number of phototransduction components was significantly reduced in A. longirostris, as well as expression level. Particularly, short-wavelength/UV-sensitive (SWS/UVS) opsins were absent in A. longirostris and only one putative middle-wavelength-sensitive (MWS) opsin was identified in this species. The conserved sites and structures of the putative LWS opsins were found between deep-sea and shallow-water decapods, indicating that the opsins in deep-sea crustaceans may also conserve their spectral absorption and signal transduction function. Phylogenetic analyses supported the monophyly of LWS opsins and SWS/UVS opsins in arthropods, while the MWS clade fell outside of the main arthropod LWS clade. The results are expected to provide baseline for study of visual adaptation in deep-sea shrimps.

CRISPR/Cas9-mediated mutation reveals Pax6 is essential for development of the compound eye in Decapoda Exopalaemon carinicauda

The compound eye in crustaceans is a main eye type in the animal kingdom, knowledge about the mechanism to determine the development of compound eye is very limited. Paired box protein 6 (Pax6) is generally regarded as a master regulator for eye development. In the present study, a genome-based analysis of the Pax6 gene in the ridge tail white prawn Exopalaemon carinicauda was performed and two members of Pax6 homologs, named Ec-Eyeless (EcEy) and Ec-Twin of eyeless (EcToy) were identified. To understand the function of these two homologs of Pax6 gene in the prawn, the CRISPR/Cas9 genome editing technique was applied to generate EcEy and EcToy knock-out (KO) prawns and their phenotypes were analyzed. The surviving EcEy-KO embryos and larvae exhibited severe abnormal eye morphology, suggesting that EcEy is necessary for the compound eye development in prawn, while no mutant phenotype was found in EcToy-KO individuals. These findings highlighted the conservative role of Pax6 gene in the compound eye formation, and the functional differentiation between EcEy and EcToy gene may reveal a novel regulating mechanism of Pax6 on the compound eye development in the decapods. These data will provide important information for understanding the regulation mechanism for crustacean compound eye development.

Gonadal, Not Maternal, Acquisition of Duplicated pax6 Orthologs in Megalobrama Amblycephala.

The highly conserved transcription factor Pax6 is involved in the development of the eyes, brain, and pancreas in vertebrates and invertebrates, whereas the additional expression pattern in other organs is still elusive. In this study, we cloned and characterized two pax6 homologs in blunt snout bream (Megalobrama amblycephala), named Mapax6a and Mapax6b. The protein alignment and phylogenetic tree showed that Mapax6a and Mapax6b were highly conserved compared with their counterparts in other species. Genomic information analysis revealed that the synteny conservation of Wilms tumor, Aniridia, genitourinary abnormalities, and mental retardation loci was also maintained in this species. By reverse transcription polymerase chain reaction, the expression of Mapax6a was later than that of Mapax6b which was found in the blastula stage, while the expression of Mapax6a started from the somite stage, and both of them persisted in a subsequent stage during the embryonic development. By RNA and protein detection, Mapax6a and Mapax6b were detected in the eye and brain as canonic patterns, and most importantly, they were also enriched in germ cells of the testis and ovary. Therefore, our findings validate the duplication of pax6 in fish, confirm the classical expression patterns in the brain and eye, and, for the first time, present a new acquisition of Mapax6a and Mapax6b in gonadal germ cells in particular. Therefore, our results enrich the expression pattern and evolutionary relationship of pax6 by suggesting that duplicated Mapax6 is involved in gametogenesis in Megalobrama amblycephala.

Hipk promotes photoreceptor differentiation through the repression of Twin of eyeless and Eyeless expression

Organogenesis is a complex developmental process, which requires tight regulation of selector gene expression to specify individual organ types. The Pax6 homolog Eyeless (Ey) is an example of such a factor and its expression pattern reveals it is dynamically controlled during development. Ey׳s paralog Twin of eyeless (Toy) induces its expression during embryogenesis, and the two genes are expressed in nearly identical patterns during the larval stages of development. While Ey must be expressed to initiate retinal specification, it must subsequently be repressed behind the morphogenetic furrow to allow for neuronal differentiation. Thus far, a few factors have been implicated in this repression including the signaling pathways Hedgehog (Hh) and Decapentaplegic (Dpp), and more recently downstream components of the retinal determination gene network (RDGN) Sine oculis (So), Eyes absent (Eya), and Dachshund (Dac). Homeodomain-interacting protein kinase (Hipk), a conserved serine–threonine kinase, regulates numerous factors during tissue patterning and development, including the Hh pathway. Using genetic analyses we identify Hipk as a repressor of both Toy and Ey and show that it may do so, in part, through Hh signaling. We also provide evidence that Ey repression is a critical step in ectopic eye development and that Hipk plays an important role in this process. Because Ey repression within the retinal field is a critical step in eye development, we propose that Hipk is a key link between eye specification and patterning.

Retinal differentiation in Drosophila

Drosophila eye development has been extensively studied, due to the ease of genetic screens for mutations disrupting this process. The eye imaginal disc is specified during embryonic and larval development by the Pax6 homolog Eyeless and a network of downstream transcription factors. Expression of these factors is regulated by signaling molecules and also indirectly by growth of the eye disc. Differentiation of photoreceptor clusters initiates in the third larval instar at the posterior of the eye disc and progresses anteriorly, driven by the secreted protein Hedgehog. Within each cluster, the combined activities of Hedgehog signaling and Notch-mediated lateral inhibition induce and refine the expression of the transcription factor Atonal, which specifies the founding R8 photoreceptor of each ommatidium. Seven additional photoreceptors, followed by cone and pigment cells, are successively recruited by the signaling molecules Spitz, Delta, and Bride of sevenless. Combinations of these signals and of intrinsic transcription factors give each ommatidial cell its specific identity. During the pupal stages, rhodopsins are expressed, and the photoreceptors and accessory cells take on their final positions and morphologies to form the adult retina. Over the past few decades, the genetic analysis of this small number of cell types arranged in a repetitive structure has allowed a remarkably detailed understanding of the basic mechanisms controlling cell differentiation and morphological rearrangement.

Analysis of the transcriptomes downstream of Eyeless and the Hedgehog, Decapentaplegic and Notch signaling pathways in Drosophila melanogaster.

Tissue-specific transcription factors are thought to cooperate with signaling pathways to promote patterned tissue specification, in part by co-regulating transcription. The Drosophila melanogaster Pax6 homolog Eyeless forms a complex, incompletely understood regulatory network with the Hedgehog, Decapentaplegic and Notch signaling pathways to control eye-specific gene expression. We report a combinatorial approach, including mRNAseq and microarray analyses, to identify targets co-regulated by Eyeless and Hedgehog, Decapentaplegic or Notch. Multiple analyses suggest that the transcriptomes resulting from co-misexpression of Eyeless+signaling factors provide a more complete picture of eye development compared to previous efforts involving Eyeless alone: (1) Principal components analysis and two-way hierarchical clustering revealed that the Eyeless+signaling factor transcriptomes are closer to the eye control transcriptome than when Eyeless is misexpressed alone; (2) more genes are upregulated at least three-fold in response to Eyeless+signaling factors compared to Eyeless alone; (3) based on gene ontology analysis, the genes upregulated in response to Eyeless+signaling factors had a greater diversity of functions compared to Eyeless alone. Through a secondary screen that utilized RNA interference, we show that the predicted gene CG4721 has a role in eye development. CG4721 encodes a neprilysin family metalloprotease that is highly up-regulated in response to Eyeless+Notch, confirming the validity of our approach. Given the similarity between D. melanogaster and vertebrate eye development, the large number of novel genes identified as potential targets of Ey+signaling factors will provide novel insights to our understanding of eye development in D. melanogaster and humans.

Conserved role for the Dachshund protein with Drosophila Pax6 homolog Eyeless in insulin expression

Members of the insulin family peptides have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. The Drosophila genome encodes seven insulin-like peptide genes, dilp1-7, and the most prominent dilps (dilp2, dilp3, and dilp5) are expressed in brain neurosecretory cells known as "insulin-producing cells" (IPCs). Although these dilps are expressed in the same cells, the expression of each dilp is regulated independently. However, the molecular mechanisms that regulate the expression of individual dilps in the IPCs remain largely unknown. Here, we show that Dachshund (Dac), which is a highly conserved nuclear protein, is a critical transcription factor that specifically regulates dilp5 expression. Dac was strongly expressed in IPCs throughout development. dac loss-of-function analyses revealed a severely reduced dilp5 expression level in young larvae. Dac interacted physically with the Drosophila Pax6 homolog Eyeless (Ey), and these proteins synergistically promoted dilp5 expression. In addition, the mammalian homolog of Dac, Dach1/2, facilitated the promoting action of Pax6 on the expression of islet hormone genes in cultured mammalian cells. These observations indicate the conserved role of Dac/Dach in controlling insulin expression in conjunction with Ey/Pax6.

Drosophila CtBP regulates proliferation and differentiation of eye precursors and complexes with Eyeless, Dachshund, Dan, and Danr during eye and antennal development

Specification factors regulate cell fate in part by interacting with transcriptional co-regulators like CtBP to regulate gene expression. Here, we demonstrate that CtBP forms a complex or complexes with the Drosophila melanogaster Pax6 homolog Eyeless (Ey), and with Distal antenna (Dan), Distal antenna related (Danr), and Dachshund to promote eye and antennal specification. Phenotypic analysis together with molecular data indicate that CtBP interacts with Ey to prevent overproliferation of eye precursors. In contrast, CtBP,dan,danr triple mutant adult eyes have significantly fewer ommatidia than CtBP single or dan,danr double mutants, suggesting that the CtBP/Dan/Danr complex functions to recruit ommatidia from the eye precursor pool. Furthermore, CtBP single and to a greater extent CtBP,dan,danr triple mutants affect the establishment and maintenance of the R8 precursor, which is the founding ommatidial cell. Thus, CtBP interacts with different eye specification factors to regulate gene expression appropriate for proliferative vs. differentiative stages of eye development.

Mab-31 and the TGF-β pathway act in the ray lineage to pattern C. elegansmale sensory rays

BackgroundC. elegans TGF-β-like Sma/Mab signaling pathway regulates both body size and sensory ray patterning. Most of the components in this pathway were initially identified by genetic screens based on the small body phenotype, and many of these mutants display sensory ray patterning defect. At the cellular level, little is known about how and where these components work although ray structural cell has been implicated as one of the targets. Based on the specific ray patterning abnormality, we aim to identify by RNAi approach additional components that function specifically in the ray lineage to elucidate the regulatory role of TGF-β signaling in ray differentiation.ResultWe report here the characterization of a new member of the Sma/Mab pathway, mab-31, recovered from a genome-wide RNAi screen. mab-31 mutants showed ray cell cluster patterning defect and mis-specification of the ray identity. mab-31 encodes a nuclear protein expressed in descendants of ray precursor cells impacting on the ray cell's clustering properties, orientation of cell division plane, and fusion of structural cells. Genetic experiments also establish its relationship with other Sma/Mab pathway components and transcription factors acting upstream and downstream of the signaling event.Conclusionmab-31 function is indispensable in Sma/Mab signal recipient cells during sensory rays specification. Both mab-31 and sma-6 are required in ray lineage at the late larval stages. They act upstream of C. elegans Pax-6 homolog and repress its function. These findings suggested mab-31 is a key factor that can integrate TFG-β signals in male sensory ray lineage to define organ identity.

Mutational analysis of the eyeless gene and phenotypic rescue reveal that an intact Eyeless protein is necessary for normal eye and brain development in Drosophila

Pax6 genes encode evolutionarily highly conserved transcription factors that are required for eye and brain development. Despite the characterization of mutations in Pax6 homologs in a range of organisms, and despite functional studies, it remains unclear what the relative importance is of the various parts of the Pax6 protein. To address this, we have studied the Drosophila Pax6 homolog eyeless. Specifically, we have generated new eyeless alleles, each with single missense mutations in one of the four domains of the protein. We show that these alleles result in abnormal eye and brain development while maintaining the OK107 eyeless GAL4 activity from which they were derived. We performed in vivo functional rescue experiments by expressing in an eyeless-specific pattern Eyeless proteins in which either the paired domain, the homeodomain, or the C-terminal domain was deleted. Rescue of the eye and brain phenotypes was only observed when full-length Eyeless was expressed, while all deletion constructs failed to rescue. These data, along with the phenotypes observed in the four newly characterized eyeless alleles, demonstrate the requirement for an intact Eyeless protein for normal Drosophila eye and brain development. They also suggest that some endogenous functions may be obscured in ectopic expression experiments.

Distinct Temporal Requirements for the Homeobox Gene Gsx2 in Specifying Striatal and Olfactory Bulb Neuronal Fates

The homeobox gene Gsx2 (formerly Gsh2) is known to be required for striatal and olfactory bulb neurogenesis; however, its specific role in the specification of these two neuronal subtypes remains unclear. To address this, we have employed a temporally regulated gain-of-function approach in transgenic mice and found that misexpression of Gsx2 at early stages of telencephalic neurogenesis favors the specification of striatal projection neuron identity over that of olfactory bulb interneurons. In contrast, delayed activation of the Gsx2 transgene until later stages exclusively promotes olfactory bulb interneuron identity. In a complementary approach, we have conditionally inactivated Gsx2 in a temporally progressive manner. Unlike germline Gsx2 mutants, which exhibit severe alterations in both striatal and olfactory bulb neurogenesis at birth, the conditional mutants exhibited defects restricted to olfactory bulb interneurons. These results demonstrate that Gsx2 specifies striatal projection neuron and olfactory bulb interneuron identity at distinct time points during telencephalic neurogenesis.

Transcriptional activities of the Pax6 gene eyeless regulate tissue specificity of ectopic eye formation in Drosophila

Pax genes encode DNA binding proteins that play pivotal roles in the determination of complex tissues. Members of one subclass, Pax6, function as selector genes and play key roles in the retinal development of all seeing animals. Mutations within the Pax6 homologs including fly eyeless, mouse Small eye and human Pax6 lead to severe retinal defects in their respective systems. In Drosophila eyeless and twin of eyeless, play non-redundant roles in the developing retina. One particularly interesting characteristic of these genes is that, although expression of either gene can induce ectopic eye formation in non-retinal tissues, there are differences in the location and frequencies at which the eyes develop. eyeless induces much larger ectopic eyes, at higher frequencies, and in a broader range of tissues than twin of eyeless. In this report we describe a series of experiments conducted in both yeast and flies that has identified protein modules that are responsible for the differences in tissue transformation. These domains appear to contain transcriptional activator and repressor activity of distinct strengths. We propose a model in which the selective presence of these activities and their relative strengths accounts, in part, for the disparity to which ectopic eyes are induced in response to the forced expression of eyeless and twin of eyeless. The identification of both transcriptional activator and repressor activity within the Pax6 protein furthers our understanding of how this gene family regulates tissue determination.

Conserved role for the Drosophila Pax6 homolog Eyeless in differentiation and function of insulin-producing neurons

Insulin/insulin-like growth factor (IGF) signaling constitutes an evolutionarily conserved pathway that controls growth, energy homeostasis, and longevity. In Drosophila melanogaster, key components of this pathway are the insulin-like peptides (Dilps). The major source of Dilps is a cluster of large neurons in the brain, the insulin-producing cells (IPCs). The genetic control of IPC development and function is poorly understood. Here, we demonstrate that the Pax6 homolog Eyeless is required in the IPCs to control their differentiation and function. Loss of eyeless results in phenotypes associated with loss of insulin signaling, including decreased animal size and increased carbohydrate levels in larval hemolymph. We show that mutations in eyeless lead to defective differentiation and morphologically abnormal IPCs. We also demonstrate that Eyeless controls IPC function by the direct transcriptional control of one of the major Dilps, dilp5. We propose that Eyeless has an evolutionarily conserved role in IPCs with remarkable similarities to the role of vertebrate Pax6 in beta cells of the pancreas.

Analysis of twin of eyeless regulation during early embryogenesis in Drosophila melanogaster

The Drosophila Pax6 homolog twin of eyeless ( toy) is so far the first zygotically expressed gene involved in eye morphogenesis in Drosophila. The study of its expression during embryogenesis is therefore informative of the initial events of eye development in Drosophila. We have analyzed how the initial expression domain of toy at cellular blastoderm is regulated. We show that the three maternal patterning systems active in the cephalic region (the anterior, terminal and dorsal–ventral systems) cooperate with zygotically activated gap genes to shape the initial expression domain of toy. Whereas Bicoid, Dorsal and Torso signaling synergistically act as activators, Hunchback, Knirps and Decapentaplegic act as repressors.

Cis-regulatory organization of the Pax6 gene in the ascidian Ciona intestinalis

The Pax6 gene has attracted intense research interest due to its apparently important role in the development of eyes and the central nervous system (CNS) in many animal groups. Pax6 is also of interest for comparative genomics since it has not been duplicated in tetrapods, making for a direct orthology between the Ciona intestinalis gene CiPax6 and Pax6 in mammals. CiPax6 has been shown to be expressed in the anterior brain, caudal nerve cord, and in parts of the brain associated with the photoreceptive ocellus. This information was extended here using in-situ hybridization, and shows that CiPax6 transcripts mark the lateral regions of the nerve cord, remarkably similar to Pax6 expression in the mouse. As a means of dissecting the cis-regulation of CiPax6 we tested 8 kb of sequence using transient reporter transgene assays. Three separate regions were found that work together to drive the overall CiPax6 expression pattern. A 211 bp sequence 2 kb upstream of the first exon was found to be a major enhancer driving expression in the sensory vesicle (the anterior portion of the ascidian brain). Other upstream sequences were shown to work with the sensory vesicle enhancer to drive expression in the remainder of the CNS. An “eye enhancer” was localized to the first intron, which controls specific expression in the central portion of the sensory vesicle, including photoreceptor cells. The fourth intron was found to repress ectopic expression of the reporter gene in middle portions of the embryonic brain. Aspects of this overall regulatory organization are similar to the organization of the Pax6 homologs in mice and Drosophila, particularly the presence of intronic elements driving expression in the eye, brain and nerve cord.

Differential requirements for the Pax6(5a) genes eyegone and twin of eyegone during eye development in Drosophila

In eye development the tasks of tissue specification and cell proliferation are regulated, in part, by the Pax6 and Pax6(5a) proteins respectively. In vertebrates, Pax6(5a) is generated as an alternately spliced isoform of Pax6. This stands in contrast to the fruit fly, Drosophila melanogaster, which has two Pax6(5a) homologs that are encoded by the eyegone and twin of eyegone genes. In this report we set out to determine the respective contributions that each gene makes to the development of the fly retina. Here we demonstrate that both eyg and toe encode transcriptional repressors, are expressed in identical patterns but at significantly different levels. We further show, through a molecular dissection of both proteins, that Eyg makes differential use of several domains when compared to Toe and that the number of repressor domains also differs between the two Pax6(5a) homologs. We predict that these results will have implications for elucidating the functional differences between closely related members of other Pax subclasses.

Growth and specification: fly Pax6 homologs eyegone and eyeless have distinct functions.

Development requires not only the correct specification of organs and cell types in the right places (pattern), but also the control of their size and shape (growth). Many signaling pathways control both pattern and growth and how these two are distinguished has been something of a mystery. In the fly eye, a Pax6 homolog (eyeless) controls eye specification together with several other genes. Now Dominguez et al.1 show that Notch signaling controls eye growth through a second Pax6 protein (Eyegone). In mice and humans the single Pax6 gene appears to encode both specification and growth controlling proteins through alternative mRNA splicing.