PTEN and A. mellifera social development

Honey Bee PTEN – Description, Developmental Knockdown, and Tissue-Specific Expression of Splice-variants Correlated with Alternative Social Phenotypes.

Abstract

This paper characterizes the PTEN gene homologues in Apis mellifera for the first time and explores its potential role is social behavior.  Larvae were fed dsRNA that targeted PTEN products resulting in lethal phenotypes during high doses. The only viable A. mellifera were fed lower dosages mixed into royal jelly, but presented with an intercaste phenotype.  PTEN isoforms were found to be in different concentrations in specific tissues between the two infertile female classes (nurse and forager).  The results suggested that PTEN has a role in adult social development, with Egfr playing a role in modulating the behavior of worker bees.
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Nurse body function compared to forager bodies from http://jeb.biologists.org/content/212/16/iii/F1.medium.gif

Introduction

Female honey bees have two castes, queens and workers.  The worker bees have two subclasses, foragers and nurses.  These two classes have specialized body types that make the bees more efficient at doing their particular job ( e.g. foragers are better equipped to fly than nurses, as demonstrated in the illustration above).  What this paper is trying to determine is the role of PTEN in the social and physical development of female honey bees.

PTEN is a tumor supressor gene that plays an anatagonistic role in the PI3K/Akt signaling pathway by dephosphorylating IIS and Egfr, (Mutti et al, 2011). IIS and Egfr are highly conserved genes through a wide variety of organisms, including humans, (Chu, 2004). In honey bees down regulation of these and TOR lead to the worker bee phenotype in queen destined larvae. Here is a video that shows where PTEN effects the IIS and Egfr cascades in the PI3K/Akt pathway.

Due to the recent findings of Page et al. 2009 it was discovered that PTEN also influences complex behavior in many metazoans, members of the animal kingdom. In this paper they suggested that further research should be done into PTEN‘s influence in the context of social behavior. A better insight into PTEN‘s mechanisms would allow us to better understand how it effects early development and its influence in adult organisms. These mechanisms can also help explain how PTEN mutant alleles are associated with human behavioral disease (Page et al, 2009). Because A. mellifera are social insects with very distinct castes Mutti et al. believed that A. mellifera would then be a great model to explore mechanisms contributing to the variation in social phenotype.

While a PTEN transcript was found in larvae and an ortholog1 had been identified in the Honey Bee Genome Sequencing project the gene structure was still unknown, (Wheeler, 2006). Mutti et al. sequenced the A. mellifera PTEN gene in order to focus on how it expresses itself within the cells. In the process they found three splice variants from alternate splicing, the rearrangement of exons, in PTEN. These splice variants are isoforms A, B, C, shown in the figure below. Isoforms are proteins encoded by different RNA transcript but have similar amino acid sequences and function.

(A) cDNA sequences of three honey bee PTEN isoforms (A, B and C) were compared to the genomic sequences gleaned from the Honey Bee Genome Resources database (http://www.ncbi.nlm.nih.gov/genome/seq/B lastGen/BlastGen.cgi?taxid=7460) to define the exons (including alternative exons) and introns. (B) Three alternate splice forms were cloned and their structure is shown. (Mutti et al, 2011)

From there they tested phenotype expression through knockdown expression2 using double strand RNA (dsRNA), which induces RNA interference, targeted at PTEN. To better understand how RNAi works and what dsRNA you  can watch: RNAi video.  Their final experiment tested the PTEN tissue specific expression and possible relationship to social behavior through a cohort study.

1 Ortholog: a gene passed down to two species from a common ancestor.
2 Knockdown expression: a way to weaken the expression of a gene by blocking the products it encodes for.

Results

Identification of alternate splice variants:  The intron/exon organization of PTEN is not shared between the A. mellifera and other model organisms for this gene, (including humans), (Riehle 2007).  Despite these differences the encoded proteins shows relatively high amino acid sequence homology, or similarity due to a common ancestor, as shown in the figure below, (Mutti et al, 2011).  The A. mellifera PTEN isoforms encode the putative phosphate domain, how it suppresses tumors, and the C2 lipid domain which has an affinity to phospholipid membranes, (Das, 2003).  It was found that isoform A encoded several serine and threonine amino acids, while isoform B contained a PTEN PDZ binding motif.  Isoform C had neither traits and was identified by such, (Mutti et al, 2011).  (The figure above shows the differences between the isoforms).

The honey bee isoforms were aligned with the sequences of D. melanogaster (AF161258), Nasonia vitripennis (NP_001128398), Tribolium castaneum (XP_974994), Aedes aegypti (ABM21568) and Homo sapiens (NP_000305) using ClustalW version 1.82. The PTEN signature motif (HCXXGXXR) is highlighted (grey), the phosphatase domain underlined with a solid black line, the C2 domain underlined with a dashed line, and PDZ binding motif is double underlined. The Genbank accession numbers for three honey bee PTEN isoforms A, B and C are FJ_969918, FJ_969919 and FJ_969920, respectively. (Mutti et al, 2011)

Effects of reducing PTEN gene expression during larval development:  Knock-down expression of PTEN gene was induced by feeding larvae dsRNA against PTEN during the first five instars.  The dsRNA was effective against all PTEN isoforms.  There were four trial diets: a queen and worker bee diet with high amounts (450μg/ml) of dsRNA and a queen and worker bee diets with lower amounts of dsRNA (150 μg/ml).  The control group diets consisted of either a queen or worker diet mixed with the corresponding amounts of dsRNA against green fluorescent protein, (GFP), which does not share a homolgy with A. mellifera genes, (Mutti et al, 2011), and will therefore not block any gene expression.  This additional step to the control group is to rule out any effects that could be caused by the presence of dsRNA.

There was a 100% mortality rate amongst the bees in the higher dose group which displayed body region deformities and did not complete metamorphosis.  While the lower dosage did not affect expression at the whole-body level, larvae that were given a worker diet still did not complete metamorphosis.  Whereas those given a queen diet completed development but had intercaste phenotypes.  This phenotype was characterized by a mixture of queen body type structure with worker body type.

Test of gene knockdown in honey bee larvae fed worker (A) vs. queen (B) diet in each of two separate experiments (n = 24). The larvae were fed with a higher dosage (450 µg/ml) of dsRNA which elicited significant PTEN knockdown (A,B). Bars represent mean ± s.e, different letters (a, b) denote significantly different groups, main effect ANOVA, p<0.05). (Mutti et al, 2011)

Expression profiling of brain, ovary, and fat body in different behavioral groups:  (The statistical data is on the chart below followed by the correlation data)  This experiment was based on two single cohort colonies, focusing on nurses and foragers.  All PTEN isoforms were expressed at significantly higher rates in the brains and ovaries in forager as compared to age-matched nurses, who only showed significantly elevated levels of isoform B, (Mutti et al, 2011).  It was also found that all isoforms had a positive correlation in the brain and ovary but there was no fat body association.

Statistical analysis using main effects ANOVA.

(A) Isoforms A and B, (B) Isoforms A and C (C) Isoforms B and C. Relative levels in brain (red circles), ovary (blue squares) and fat body (green triangles) are shown. Open circles, squares and triangles represent the forager data points while the closed symbols represent the nurse bee data points. As a general pattern, expression of PTEN isoforms is positively correlated in brain and ovary (Pearson's correlation, p<0.05), but not in the fat body of the workers (p>0.05). (Mutti et al, 2011)

Discussion

Knockdown of PTEN during larval development: The study found that PTEN did not specifically play a role is caste differentiation,  because both castes displayed the lethal phenotype due to PTEN suppression.  They reasoned that the intercaste development was due to the down regulation effecting certain tissues and regions more than others.  This then led the to the conclusion that PTEN plays a crucial role in nutrient sensing, and will throw off larval development if disturbed. They did not investigate further into the lethality of PTEN suppression because it has been well documented in other species where the cause is various developmental defects in different tissues and organs, (Goberdhan, 1999).

PTEN expression and correlation to social behavior: PTEN is essential for proper localization of the cytoskeleton in Dictyostelium discoideum permitting the formation of filopodia1 necessary for both locomotion and chemotaxis2, (Wessels, 2007).  Which would make dendritic arborization3 a plausible explanation of why PTEN expression, known for suppressing growth, is so high in the brains of foragers.  This hypothesis is helped by the increase in both locomotion and olfactory learning in forgers due to their need to develop navigational skills.  However measurement of the overall transcription levels in the smaller brain compartments, or mushroom bodies, cannot be explained. The authors hypothesized that the increase in PTEN in forager ovaries contributes to their reduced propensity of reproductive activity, (Hansen, 2004).  The variance in the fat body is much less noticeable and the transcript levels don’t diverge for isoforms A and C, as shown in the figure below.

RT-qPCR was used to determine isoform-specific PTEN transcript levels in (A–C) Brain; (D–F) Ovary; and (G–I) Fat body. Age matched (20-day old bees) nurses and foragers from single-cohort colonies were used for the expression analysis. All RT-qPCR samples were run in triplicate. Brain, ovaries and fat body from 3 individual bees were pooled by tissue to make up one biological sample (n = 6). Bars represent mean ± s.e, different letters (a, b) indicate significant differences as determined with a Fisher LSD post-hoc test, p<0.05.

Honey bees as a model system to study PTEN function: They can provide a model for understanding the molecular mechanisms that regulate complex behavior, which can be affected by physiological feedback between the brain, gonad, and adipose tissue, (Flatt, 2008).  The feedback is partly linked to energy-/nutrient-sensing pathways like IIS.  A. mellifera can use IIS to form a complex regulatory network that influences social behavior, (Corona, 2007).  The results from the study suggest that PTEN may play a role in adult social behavior, which the authors use to suggest the Efgr could effect the regulatory loop in charge of worker bee behavior.  This is because PTEN influences Efgr signal transduction.  A. mellifera are smaller and have shorter life spans as compared to vertebrates and would therefore make studying these complex relationships more accessible to researchers.

1 Filopodia:  microspikes on cells that are thought to be involved in sensing chemicals in their environment and the direction change because detected chemicals.
2 Chemotaxis:  a phenomenon in which cells use the presence of a chemical in their environment to direct their movements.
3 Dendritic arborization: caused by cytoskeleton changes in the neurons which results in the determination of the nature and extent of innervation of a neuron.

In brain and ovary, PTEN (red circles) isoforms A, B and C, which could potentially down-regulate insulin/insulin-like signaling (IIS), are more abundant in foragers than in nurses. However, foraging behavior is positively associated with IIS via the release of insulin-like peptide 1 (ilp-1, orange ellipse) in the brain (pink) {32}. Ilp-1 may cause juvenile hormone (JH, green ellipse) levels to increase {91}; JH is also positively correlated with foraging behavior {94} and may enhance IIS by feedback suppression of vitellogenin (Vg, violet ellipse), a proposed negative regulator of IIS {34},{86},{92},{93}. These relationships contradict the repression of IIS by elevated PTEN in forager brain tissue. In contrast, suppressed IIS by PTEN in ovary tissue is consistent with the reduced reproductive propensity of foragers {31}. In fat body (yellow), PTEN isoform B, ilp-1 and insulin-like peptide 2 (ilp-2) are elevated in nurses compared to foragers (K. Ihle, unpublished data; and results in this paper), but effects on metabolic biology are currently unclear. The ilp gene products from fat body or brain may also take part in remote signaling to other organs {32}. In this illustration, larger-size circles/ellipses, and thicker arrows (positive)/blocked arrows (negative) denote higher levels of expression, enhancement and suppression, respectively. Dotted arrows indicate the yet unresolved effects on worker phenotypes. (Mutti et al, 2011)

Critique

For the most part this paper was clear and informative about what they were researching and how.  Their experimental techniques were very thorough, though it might have been nice to seem more trails testing different levels of RNAi in the diets.  Possibly with lower levels to see if it was possible to obtain workers that survived through metamorphosis.  For the most part they were very vague about how studying PTEN’s involvement in honey bees could relate to human social behavoir, causing the reader to look elsewhere for the information.  Overall this was a good paper, though it relied heavily on the audience already understanding PTEN’s role in social development and IIS and Efgr and their role in the PI3K/Akt signaling pathway.

Important Takeaways

  • PTEN does not play a role in the differentiation between queens and workers, but plays an important in the developmental process
  • PTEN could possibly play a role in the social differences between nurses and foragers due to the physical changes caused by PTEN
  • Though there is a lot more research that needs to be done into the developmental roles of PTEN and its effects on social behavior this study is the first step in that direction

Sources:

Chu EC, Tarnawski AS: PTEN regulatory functions in tumor suppression and cell biology. Med Sci Monit 2004, 10:RA235-241.

Corona M, Velarde RA, Remolina S, Moran-Lauter A, Wang Y, et al. (2007) Vitellogenin, juvenile hormone, insulin signaling, and queen honey bee longevity. Proc Natl Acad Sci U S A 104: 7128–7133.

Das S, Dixon JE, Cho W (2003) Membrane-binding and activation mechanism of PTEN. Proc Natl Acad Sci U S A 100: 7491–7496.

Flatt T, Min KJ, D’Alterio C, Villa-Cuesta E, Cumbers J, et al. (2008) Drosophila germ-line modulation of insulin signaling and lifespan. Proc Natl Acad Sci U S A 105: 6368–6373.

Goberdhan DC, Paricio N, Goodman EC, Mlodzik M, Wilson C (1999) Drosophila tumor suppressor PTEN controls cell size and number by antagonizing the Chico/PI3-kinase signaling pathway. Genes Dev 13: 3244–3258.

Hansen IA, Attardo GM, Park JH, Peng Q, Raikhel AS (2004) Target of rapamycin-mediated amino acid signaling in mosquito anautogeny. Proc Natl Acad Sci U S A 101: 10626–10631.

Mutti NS, Wang Y, Kaftanoglu O, Amdam GV. Honey bee PTEN – description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes. PLoS ONE. 2011;6:e22195.

Riehle MA, Brown JM (2007) Characterization of phosphatase and tensin homolog expression in the mosquito Aedes aegypti: six splice variants with developmental and tissue specificity. Insect Mol Biol 16: 277–286.

Wessels D, Lusche DF, Kuhl S, Heid P, Soll DR (2007) PTEN plays a role in the suppression of lateral pseudopod formation during Dictyostelium motility and chemotaxis. J Cell Sci 120: 2517–2531.

Wheeler DE, Buck N, Evans JD (2006) Expression of insulin pathway genes during the period of caste determination in the honey bee, Apis mellifera. Insect Mol Biol 15: 597–602.

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