Diversity in insect axis formation: two orthodenticle genes and hunchback act in anterior patterning and influence dorsoventral organization in the honeybee (Apis mellifera)

INTRODUCTION

Axis formation is a key step in the developmental process but the genes that are involved in insect axis formation are fast evolving. This is evident in the fact that several genes that control axis formation in Drosophila are missing from other insects. Orthodenticle (otd) are known to be involved in the anterior patterning in vertebrates and invertebrates. Along with Otd1 and Otd2, Hunchback (hb) has also been implicated in anterior patterning in several insects. These genes are common in several species but the interactions between the proteins and their targets differ between species.

Everyone knows that honey bees are studied for their social interactions but honey bees are worth more than their social wealth.  In this article, the honey bee (Apis mellifera) axis formation and gene patterning were examined to analyze the differences and similarities in the fast evolving axis formation of insects.

Apis Mellifera

RESULTS

Identification and Expression of Am-otd1, Am-otd2 and Am-hb

BLAST searches were used to identify Otd and hb orthologs with similarities to Drosphila Otd. After identifying predicted genes, phylogenetic analysis indicated that the genes clustered with clade of perspective gene. GB16866 was designated as Am- orthodenticle-1(Am-otd1), GB11566 as Am-orthodenticle-2 (Am-otd2), and GB19977 as Am-hunchback (Am-hb).

The expression of the genes was examined using situ hybridization to determine if expression is consistent with anterior specification. Am-otd1 becomes enriched in the anterior of the embryo with the highest concentration at the pole (Fig 1 C). Posterior expressed vanishes by stage 6(Fig 1 F) and at stage 9 (Fig 1 G) Am-otd1 is detected in the CNS.

Am-otd2 is expressed by posterior nurse cells. Am-otd2 expression fades in an anterior to posterior sequence after lying of eggs (Fig 1 K,L). At stage 4 (Fig 1 M), Am-otd2 expression appears in cells at the anterior and posterior poles. At stage 9 (Fig 1 P,Q), expression is limited to the developing CNS.

Am-hb is present in maturing oocytes and nurse cells (Fig 1 R). Am-hb becomes enriched in the anterior of the embryo at stage 2 (Fig 1 T). The posterior expression expands to a cap of cells, but Am-hb expression is found throughout the embryo. At stage 9 (Fig 1 W), Am-hb is is expressed in the CNS.

Figure 1. Expression of Am-otd1, Am-otd2, and Am-hb in honeybee queen ovaries and embryos. Embryos and ovaries are orientated with anterior left and dorsal up, unless otherwise stated. (A) Maternal expression of Am-otd1. (A-G) Am-otd1 RNA id associated with energids in stage 1 embryos (A) and is detected in the anterior of stage 2 embryos (C). This expression domain becomes weaker (D) until stage 5 (E) where Am-otd1 is detected in a triangular anterior domain and at the posterior pole. By stage 6 (F) the anterior domain narrows, weak expression occurs at the edges of the gastrulation furrow and posterior expression is not visible. From stage 9 (G) Am-otd1 RNA is present in CNS cells. (H,I,J) Queen ovary expression of Am-otd2. Am-otd2 RNA is present in posterior nurse cells and throughout the oocyte. (K-Q) In stage 1 embryos (K), Am-otd2 RNA is enriched around energids in the posterior, where RNA remains, though loses association with energids by stage 2 (L). By stage 4 (M), Am-otd2 is expressed in an anterior domain and posterior cap that remains through stage 5 (N) and 6 (O). By stage 8 (P) and 9 (Q). Am-otd2 is expressed in the CNS. (R) Queen ovary expression of Am-hb. Am-hb RNA is detected in nurse cells and oocytes. (S-W) In stages 1 (S) and 2 (T) embryos Am-hb RNA is present throughout the embryo. RNA is enriched in the anterior and in a posterior stripe of cells by stage 4 (U). By stage 6 (V), only a posterior cap remains, which fades and is replaced at stage 9 (W) with expression in the CNS.

RNAi Knockdown

RNAi was used to test the theory that Am-otd1, Am-otd2, and Am-hb pattern the anterior by removing there function early to test for defects in anterior patterning.  In situ hybridization for e30 RNA allows better definition of segments. Am-otd1 severely affected knockdown embryos stained for e30 have staining in only the posterior segments, showing loss of pattern from the anterior. Severely affected Am-otd2 knockdown produces loss of all anterior segments and posterior segments appearing twice-normal size indicating fused segments. A double-knockdown of Am-otd1 and Am-otd2 was performed to determine if the genes were working redundantly resulting in extensive loss of anterior structures and fusion of segments. Severely affected knockout of Am-hb resulted in few segments remaining.

Maternal Genes?

The RNAi knockdown of Am-otd1, Am-otd2 and Am-hb indicated that these genes play a role in maternal patterning of anterior regions of the embryo. If these genes do act as maternal anterior patterning genes then they should also regulate the expression of gap genes.  In a previous study by Wilson et al.  (Wilson et al., 2010), Honey bee gap genes were identified by blast searches to find similarities to known Drosophila gap genes. Three gap genes were identified: Am- Krṻppel (Am-Kr), Am-caudal (Am-cad), and Am-giant (Am-gt). The effect of knockdown was observed on the gap gene expression and the results are displayed in the table below. The gap genes are strongly affected by Am-otd1 and Am-hb knockdown, and less by Am-otd2.

Regulation of Am-zen

In flour beetles, Tribolium, Tc-otd1 regulates the expression of Tc-zen and Tc-sog (Kotkamp et al., 2010). The researchers wanted to determine whether a similar process occurs in the honey bee so they examined the expression of honey bee zen in Am-otd1 RNAi embryos. In Am-otd1 RNAi embryos, Am-zen RNA expression is lost from only the anterior and posterior poles. They also examined the Am-zen expression in Am-hb RNAi embryos. Knockdown of Am-hb results in disorganization of Am-zen expression. In Am-hb RNAi embryos, Am-zen mRNA is absent from the anterior but is expressed across central regions and expands dorsally. Therefore it was concluded that both Am-otd1 and Am-hb contribute to the regulation of Am-zen.

The phenotype of Am-zen knockdown was examined, to determine if, like Tribolium, loss of Am-zen causes anteroposterior patterning defects. If responsible then knockdown will cause anterior segmentation defects and changes in the expression of gap and pair-rule genes. Am-zen RNAi embryos survived to hatching but had deformed head in dorsal regions and extended embryo flanks. DAPI staining at stage 9 shows that the dorsal side of the embryo collapses in mild cases and in severe cases, the germ rudiment extends all the way to the dorsal side of the embryo. All segments are present in Am-zen RNAi embryos and pair-rule gene expression is normal. Loss of Am-zen does not affect segmentation and does not have a significant effect on gap gene expression.

Regulation of Am-gt expression

Based on previous implications of Am-otd1, Am-otd2, and Am-hb regulating Am-gt through direct regulation and plays a role in regulating of gap genes, the researchers searched for cis-regulatory motifs (CRMs) that regulate the expression of Am-gt. A region indicated from searching for regulators of giant upstream was cloned upstream of a lacZ reporter gene and used to produce transgenic Drosophilalines. lacZ expression driven by the Am-gt was examined by in situ hybridization.

Figure 8. Expression and regulation of Am-gt CRM in Drosophila embryos. (A-D) Am-gt CRM embryos stained for lacZ RNA. Embryos are oriented anterior left, dorsal up. (A) Weak background expression is seen in stage 1 embyros. (B,C) Stage 5 embryos display an anterior domain of expression. (D) Dorsal view of a stage 6 embryo showing expression in a dorsal domain. (E) Expression of Am-gt CRM in a homozygous otd -/- embryo. Expression in the anterior domain is reduced. (F) Expression in a hb -/- embryo showing loss of expression. (G) lacZ reporter expression in a zen -/- embryo showing loss of dorsal expression. (H) Expression in a cad -/- mutant embryo with ubiquitous ectopic expression.

At stage 1 (Fig 8A), embryos, lacZ expression was weak with significant expression appearing by stage 4 (Fig 8B,C) in an anterior domain. At stage 5 (Fig 8D), lacZ is expressed along the dorsal midline in the developing aminoserosa. The Am-gt CRM reporter was crossed into mutants homozygous for transcription factors that have the possibility to regulate binding to this CRM. In Otd mutants, lacZ is absent in the anterior but remains in the dorsal midline (Fig 8E). In hb mutants (8F) lacZ expression is reduced through the embryo.  In zen mutants lacZ expression is lost from the aminoserosa (Fig 8G). It was also tested to determine if the same interactions that exist in cad mutants  in Drosophila are present in the honey bee. RNAi knockdown of Am-cad (Fig 8H) resulted in a posterior shift of the posterior border of the anterior Am-gt domain. This indicates that Am-cad plays a role in repressing Am-gt expression.

CONCLUSION

Am-otd1, Am-otd2, and AM-hb have anterior patterning roles in honeybee embryos.  These genes also probably directly regulate the expression of the gap gene giant. The RNAi knockdown of these genes affect the expression domain of the gap genes studies therefore it has been proposed that Am-otd1 and Am-otd2 together with Am-hb act as maternal anterior patterning genes in the honeybee embryo. The effects of knockdown of Am-otd1 and Am-hb are greater than any other insect reported leading to the conclusion that definition of the anterior seems to be a crucial event in patterning in the entire body of the embryos.

STRENGTHS AND WEAKNESSES

The experimental design was really well planned and executed. Presentation of results was clear and the use of tables and figures greatly added to the overall understanding of the article. The researchers used a several methods to ensure that their results were accurate including the use of double knockdowns of genes. The only improvement that could be made in a better conclusion in the paper, demonstrating how the finding in the research adds to or takes away from the overall diversity of in insect axis formation. Also the presentation of the comparisons in the findings of previous studies on different insects could be more clearly stated with possibly using a table.

REFERENCES

Kotkamp, K., Klingler, M. and Schoppmeier, M. (2010). Apparent role of Tribolium orthodenticle in anteroposterior blastoderm patterning largely reflectsnovel functions in dorsoventral axis formation and cell survival. Development137, 1853-1862.

Wilson, M., & Dearden, P. (2011). Diversity in insect axis formation: two orthodenticle genes and hunchback act in anterior patterning and influence dorsoventral organization in the honeybee (apis mellifera). Development, 138, 3497-3507.

Wilson, M. J., Havler, M. and Dearden, P. K. (2010). Giant, Krüppel, and caudalact as gap genes with extensive roles in patterning the honeybee embryo. Dev.Biol. 339, 200-211

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