Injecting <em>Gryllus bimaculatus</em> Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

Bowdoin College

In cultured neurons, the exogenous application of neurotrophins (in homogenous concentrations) alters many features of axonal and dendritic arbors. In vivo, however, release of endogenous neurotrophins from neuronal processes creates spatially heterogeneous neurotrophin distributions. To probe the consequences of such endogenous neurotrophin distribution, we produced 'donor neurons' in ferret cortex brain slices that co-expressed brain-derived neurotrophic factor (BDNF) and red fluorescent protein (RFP). Using two-photon microscopy, we analyzed their effects on 'recipient neurons' that expressed green fluorescent protein (GFP) alone. BDNF released from dendrites and cell bodies acted directly on nearby recipient neurons to increase dendritic branching in a distance-dependent manner. Three-dimensional analysis of donor and recipient dendrites indicated that the BDNF source had to be within 4.5 microm to induce dendritic growth in the recipient neuron. Thus, BDNF released from an individual cell alters the structure of nearby dendrites on an exquisitely local scale.

BDNF release from single cells elicits local dendritic growth in nearby neurons.

The development of dendrites is a crucial step in the formation of cortical circuitry. The morphogen brain-derived neurotrophic factor (BDNF) may mediate the effects of activity on dendritic morphology since its expression and release are thought to be activity-dependent. Using two-photon microscopy, the autocrine and paracrine effects of BDNF on dendritic morphology were assessed. Overexpression of BDNF profoundly altered the form and stability of basal dendritic arbors via an autocrine mechanism. Paracrine BDNF also altered dendritic branching, though in a highly local fashion. BDNF is capable of acting as an intercellular morphogen, and could hypothetically shape dendritic arbors to best fit the developing structure and function of the pre-synaptic circuit.

Local effects of BDNF on dendritic growth.

Neurones that lose their presynaptic partners because of injury usually retract or die. However, when the auditory interneurones of the cricket Gryllus bimaculatus are denervated, dendrites respond by growing across the midline and forming novel synapses with the opposite auditory afferents. Suppression subtractive hybridization was used to detect transcriptional changes 3 days after denervation. This is a stage at which we demonstrate robust compensatory dendritic sprouting. Whereas 49 unique candidates were down-regulated, no sufficiently up-regulated candidates were identified at this time point. Several candidates identified in this study are known to influence the translation and degradation of proteins in other systems. The potential role of these factors in the compensatory sprouting of cricket auditory interneurones in response to denervation is discussed.

Differential gene expression during compensatory sprouting of dendrites in the auditory system of the cricket Gryllus bimaculatus.

The auditory system of the cricket has the unusual ability to respond to deafferentation by compensatory growth and synapse formation. Auditory interneurons such as ascending neuron 2 (AN-2) in the cricket Gryllus bimaculatus possess a dendritic arbor that normally grows up to, but not over, the midline of the prothoracic ganglion. After chronic deafferentation throughout larval development, however, the AN-2 dendritic arbor changes dramatically, and medial dendrites sprout across the midline where they form compensatory synapses with the auditory afferents from the contralateral ear. We quantified the extent of the effects of chronic, unilateral deafferentation by measuring several cellular parameters of 3 different neuronal components of the auditory system: the deafferented AN-2, the contralateral (or nondeafferented) AN-2 and the contralateral auditory afferents. Neuronal tracers and confocal microscopy were used to visualize neurons, and double-label experiments were performed to examine the cellular relationship between pairs of cells. Dendritic complexity was quantified using a modified Sholl analysis, and the length and volume of processes and presynaptic varicosities were assessed under control and deafferented conditions. Chronic deafferentation significantly influenced the morphology of all 3 neuronal components examined. The overall dendritic complexity of the deafferented AN-2 dendritic arbor was reduced, while both the contralateral AN-2 dendritic arbor and the remaining, intact, auditory afferents grew longer. We found no significant changes in the volume or density of varicosities after deafferentation. These complex cellular changes after deafferentation are interpreted in the light of the reported differential regulation of vesicle-associated membrane protein and semaphorin 2a.

Bilateral consequences of chronic unilateral deafferentation in the auditory system of the cricket Gryllus bimaculatus.

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.

Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases.

Most genomic resources available for insects represent the Holometabola, which are insects that undergo complete metamorphosis like beetles and flies. In contrast, the Hemimetabola (direct developing insects), representing the basal branches of the insect tree, have very few genomic resources. We have therefore created a large and publicly available transcriptome for the hemimetabolous insect Gryllus bimaculatus (cricket), a well-developed laboratory model organism whose potential for functional genetic experiments is currently limited by the absence of genomic resources. cDNA was prepared using mRNA obtained from adult ovaries containing all stages of oogenesis, and from embryo samples on each day of embryogenesis. Using 454 Titanium pyrosequencing, we sequenced over four million raw reads, and assembled them into 21,512 isotigs (predicted transcripts) and 120,805 singletons with an average coverage per base pair of 51.3. We annotated the transcriptome manually for over 400 conserved genes involved in embryonic patterning, gametogenesis, and signaling pathways. BLAST comparison of the transcriptome against the NCBI non-redundant protein database (nr) identified significant similarity to nr sequences for 55.5% of transcriptome sequences, and suggested that the transcriptome may contain 19,874 unique transcripts. For predicted transcripts without significant similarity to known sequences, we assessed their similarity to other orthopteran sequences, and determined that these transcripts contain recognizable protein domains, largely of unknown function. We created a searchable, web-based database to allow public access to all raw, assembled and annotated data. This database is to our knowledge the largest de novo assembled and annotated transcriptome resource available for any hemimetabolous insect. We therefore anticipate that these data will contribute significantly to more effective and higher-throughput deployment of molecular analysis tools in Gryllus.

Developmental gene discovery in a hemimetabolous insect: de novo assembly and annotation of a transcriptome for the cricket Gryllus bimaculatus.

Dendrite and axon growth and branching during development are regulated by a complex set of intracellular and external signals. However, the cues that maintain or influence adult neuronal morphology are less well understood. Injury and deafferentation tend to have negative effects on adult nervous systems. An interesting example of injury-induced compensatory growth is seen in the cricket, Gryllus bimaculatus. After unilateral loss of an ear in the adult cricket, auditory neurons within the central nervous system (CNS) sprout to compensate for the injury. Specifically, after being deafferented, ascending neurons (AN-1 and AN-2) send dendrites across the midline of the prothoracic ganglion where they receive input from auditory afferents that project through the contralateral auditory nerve (N5). Deafferentation also triggers contralateral N5 axonal growth. In this study, we quantified AN dendritic and N5 axonal growth at 30 h, as well as at 3, 5, 7, 14, and 20 days after deafferentation in adult crickets. Significant differences in the rates of dendritic growth between males and females were noted. In females, dendritic growth rates were non-linear; a rapid burst of dendritic extension in the first few days was followed by a plateau reached at 3 days after deafferentation. In males, however, dendritic growth rates were linear, with dendrites growing steadily over time and reaching lengths, on average, twice as long as in females. On the other hand, rates of N5 axonal growth showed no significant sexual dimorphism and were linear. Within each animal, the growth rates of dendrites and axons were not correlated, indicating that independent factors likely influence dendritic and axonal growth in response to injury in this system. Our findings provide a basis for future study of the cellular features that allow differing dendrite and axon growth patterns as well as sexually dimorphic dendritic growth in response to deafferentation.

Quantification of dendritic and axonal growth after injury to the auditory system of the adult cricket Gryllus bimaculatus.

The auditory system of the cricket, Gryllus bimaculatus, demonstrates an unusual amount of anatomical plasticity in response to injury, even in adults. Unilateral removal of the ear causes deafferented auditory neurons in the prothoracic ganglion to sprout dendrites across the midline, a boundary they typically respect, and become synaptically connected to the auditory afferents of the contralateral ear. The molecular basis of this sprouting and novel synaptogenesis in the adult is not understood. We hypothesize that well-conserved developmental guidance cues may recapitulate their guidance functions in the adult in order to facilitate this compensatory growth. As a first step in testing this hypothesis, we have generated a de novo assembly of a prothoracic ganglion transcriptome derived from control and deafferented adult individuals. We have mined this transcriptome for orthologues of guidance molecules from four well-conserved signaling families: Slit, Netrin, Ephrin, and Semaphorin. Here we report that transcripts encoding putative orthologues of most of the candidate developmental ligands and receptors from these signaling families were present in the assembly, indicating expression in the adult G. bimaculatus prothoracic ganglion.

De novo assembly of a transcriptome for the cricket Gryllus bimaculatus prothoracic ganglion: An invertebrate model for investigating adult central nervous system compensatory plasticity.

Hadley W. Horch

Department Biology and Department of Neuroscience

Bowdoin College

Injecting Gryllus bimaculatus Eggs