Expression analysis of Egr-1 ortholog in metamorphic brain of honeybee (Apis mellifera L.): Possible evolutionary conservation of roles of Egr in eye development in vertebrates and insects

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Highlights

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    The honeybee homolog of Egr-1 (AmEgr) was transiently upregulated in the pupal brain.

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    AmEgr was mainly expressed in post-mitotic cells in optic lobes.

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    Unique variants encoding polyglutamine tracts were expressed in the pupal brain.

Abstract

Specific genes quickly transcribed after extracellular stimuli without de novo protein synthesis are known as immediate early genes (IEGs) and are thought to contribute to learning and memory processes in the mature nervous system of vertebrates. A recent study revealed that the homolog of Early growth response protein-1 (Egr-1), which is one of the best-characterized vertebrate IEGs, shared similar properties as a neural activity-dependent gene in the adult brain of insects. With regard to the roles of vertebrate Egr-1 in neural development, the contribution to the development and growth of visual systems has been reported. However, in insects, the expression dynamics of the Egr-1 homologous gene during neural development remains poorly understood. Our expression analysis demonstrated that AmEgr, a honeybee homolog of Egr-1, was transiently upregulated in the developing brain during the early to mid pupal stages. In situ hybridization and 5-bromo-2’-deoxyuridine (BrdU) immunohistochemistry revealed that AmEgr was mainly expressed in post-mitotic cells in optic lobes, the primary visual center of the insect brain. These findings suggest the evolutionarily conserved role of Egr homologs in the development of visual systems in vertebrates and insects.

Introduction

A unique group of genes, which are quickly transcribed in response to extracellular stimuli without de novo protein synthesis and thereby capable of causing rapid, adaptive changes in cellular function, are known as immediate early genes (IEGs) [1]. In mature nervous systems, the expression of IEGs is closely associated with neural activity and reportedly plays an essential role in synaptic plasticity, which is thought to be the neural basis of memory and learning [2]. Over the past three decades, a lot of IEGs have been identified in vertebrates [3]. On the other hand, until recently, there has been no report of a gene exhibiting clear characteristics as an IEG in insects, with the exception of one gene named kakusei, which was only identified in the honeybee and believed to encode a non-coding nuclear RNA [4]. In 2013, we demonstrated that Apis mellifera Early growth response protein (AmEgr), a honeybee homolog of Egr-1, which is one of the best characterized IEGs in vertebrates, encoding a transcription factor with a DNA-binding domain comprising unique three tandem Cys2His2 zinc finger motifs [5], showed transient and prominent increases in expression after neural activation evoked by seizure induction and pharmacological treatment [6]. Also, in our ongoing studies, induced expression of the Egr homolog by neural activity is observed even in hemimetabolous insects (field cricket, Watanabe et al., Unpublished data), suggesting the conserved role of Egr homologs in mature nervous systems in both vertebrates and insects.

Besides the modification of the mature nervous system, various vertebrate IEGs also play an important role in neural development [7]. As for vertebrate Egr-1, previous studies have reported embryonic expression in the subplate and intermediate zones during corticogenesis [8], and contribution to the development and growth of the visual system [9], [10], [11], [12]. In insects, however, the expression pattern and role of the Egr ortholog in the developing neural system remain unknown. In the present study, we performed expression analysis of AmEgr during metamorphic stages of the honeybee, and found that unique splice variants, distinct from transcripts induced in the adult brain, were transiently upregulated in the optic lobes, the primary visual center of the insect brain, suggesting the evolutionarily conserved role of Egr homologs in oculanogenesis.

Section snippets

Bees

European honeybee (Apis mellifera L.) colonies were purchased from a local dealer and maintained at Tamagawa University. Developmental stages of worker pupae were determined by external morphological features (eye pigmentation and cuticle tanning) described in a previous study [13].

Quantification of the AmEgr transcript

The bees were anesthetized in ice-cold water soon after collection from brood cells, and the brains were dissected. The seizure-induction experiment was performed as described previously [6], [14]. Total RNA samples

Expression change of AmEgr in metamorphic brain

The honeybee has five larval instars (L1–5) and then develops into a prepupa (PP), pupa, and finally emerges as an adult bee. The pupal stage of worker bees lasts for nine days (P1–9) when the larval nervous system becomes completely remodeled to adult-specific systems [17]. To examine the expression pattern of AmEgr during metamorphosis, we sampled worker brains of L5, PP, P1, P4 and P7, and performed qRT-PCR using primer sets, which could amplify the zinc finger-coding region (Fig. 1A), the

Discussion

In the present study, we demonstrated that AmEgr, a homolog of vertebrate Egr-1, was upregulated in the optic lobes of the honeybee from the early to mid pupal stages, suggesting the involvement of the Egr homolog in the development of the insect visual system. To the best of our knowledge, this is the first study to demonstrate an expression pattern of IEGs in the context of the neural development of insects.

Since Cajal first observed structural similarities in neural circuits in both

Acknowledgements

We are grateful to Profs. Toru Miyata and Kazuomi Sato for providing the reagents for the BrdU labeling experiment. This work was supported in part by the Grant-in-Aid for Scientific Research (C) Grant Numbers 26450472, Japan Society for the Promotion of Science (JSPS) for TS. AU was a recipient of Research Fellowships of JSPS for Young Scientists (14J12036).

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