Evolution and multiple roles of the Pancrustacea specific transcription factor <i>zelda</i> in insects

<div><p>Gene regulatory networks (GRNs) evolve as a result of the coevolutionary processes acting on transcription factors (TFs) and the cis-regulatory modules they bind. The zinc-finger TF <i>zelda (zld)</i> is essential for the maternal-to-zygotic transition (MZT) in <i>Drosophila melanogaster</i>, where it directly binds over thousand cis-regulatory modules to regulate chromatin accessibility. <i>D</i>. <i>melanogaster</i> displays a long germ type of embryonic development, where all segments are simultaneously generated along the whole egg. However, it remains unclear if <i>zld</i> is also involved in the MZT of short-germ insects (including those from basal lineages) or in other biological processes. Here we show that <i>zld</i> is an innovation of the Pancrustacea lineage, being absent in more distant arthropods (e.g. chelicerates) and other organisms. To better understand <i>zld´s</i> ancestral function, we thoroughly investigated its roles in a short-germ beetle, <i>Tribolium castaneum</i>, using molecular biology and computational approaches. Our results demonstrate roles for <i>zld</i> not only during the MZT, but also in posterior segmentation and patterning of imaginal disc derived structures. Further, we also demonstrate that <i>zld</i> is critical for posterior segmentation in the hemipteran <i>Rhodnius prolixus</i>, indicating this function predates the origin of holometabolous insects and was subsequently lost in long-germ insects. Our results unveil new roles of <i>zld</i> in different biological contexts and suggest that changes in expression of <i>zld</i> (and probably other major TFs) are critical in the evolution of insect GRNs.</p></div

<i>zld</i> is required for the generation of the posterior region in the hemiptera <i>Rhodnius prolixus</i>.

<p>(A,C,E) <i>R</i>. <i>prolixus</i> control (<i>dsneo</i> embryos). (B,D,F,F’) A representative embryo collected from <i>R</i>. <i>prolixus zld</i> pRNAi. (F,F’) Embryo inside the chorion, ventral and dorsal views. (C,E) Embryo removed from the egg shell. (G,H) Schematic drawings of control and <i>zld</i> pRNAi embryos. (A) DAPI stainings of control (A,E) and <i>zld</i> RNAi (B) embryos. (D) Asterisk denotes an eye which can be observed at the ventral side (F). (D) After dissection, the eye can be identified due to its characteristic red pigmentation and shape. Scale bar corresponds to 500μM.</p

<i>Tc-zld</i> knockdown in larval stages affects elytra and wings in adult stage.

<p>(A) Control elytron extracted from a <i>dsneo</i> adult shows a parallel venation pattern and rigid chitinous structure. (B) <i>Tc-zld</i> RNAi elytron displays a disrupted vein pattern and a less resistant structure. (C,D) Hindwings were dissected and photographed. Although the overall morphological pattern is not affected in both wings, <i>zld</i> dsRNA wings show severe dehydration after ethanol fixation. (E) PD indexes comparison between <i>dsneo</i> and <i>dszld</i> RNAi hindwings and forewings, a PD index reflects the shape of the wing based on its dimensions ratio [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref052" target="_blank">52</a>]. The statistical analysis was carried out by unpaired t-test assuming unequal variances (asterisks refer to P<0.0001 while ns stands for “no significance”), indicating that <i>Tc-zelda</i> RNAi affects hindwing shape. (F) <i>Tc-zld</i> RNAi elytra and hindwing do not show statistically significant differences in length when compared to their respective <i>dsneo</i> RNAi controls.</p

Larval stages and expression dynamics of <i>zld</i> and its putative target genes.

<p>(A-D) Morphology of <i>Tribolium castaneum</i> larvae on 3rd (A-L3), 5th (B-L5), 7th (C-L7) and early pupal stages (D-P1). (E) Relative expression of <i>zld</i>, <i>Ubx</i>, <i>wingless (wg)</i>, <i>distalless (dll)</i>, <i>Iroquois (iro)</i>, <i>Lim-1</i> and <i>Kruppel-homolog-1 (KRH-1)</i> at L3, L5, L7 and P1. Asterisks represent significant differences between stages (P<0.05).</p

<i>Tc-zld</i> knockdown induces phenotypes in antennae and legs of adult <i>T</i>. <i>castaneum</i>.

<p>(A) Relative expression of <i>Tc-Dll</i> at pupal stages after <i>neo</i> or <i>zld</i> dsRNA injections. (B-D) Antennae extracted from control injected individuals (B) display 11 segments, including two proximal (S = scape, P = pedicel), six intermediate (Flagellum = dots) and three distal modified segments that form the club (Cl). (C,D) Injection of <i>zld</i> dsRNA caused fusion of the club segments in mild phenotypes (C), while in stronger phenotypes (D) the antennae fail to segment, developing as an antennal rudiment. (E,F) Legs of <i>T</i>. <i>castaneum</i> display six podomeres: Coxa (Cx), Trochanter (Tr), Femur (Fe), Tibia (Ti), Tarsus (T) and pretarsus (pT). (E) Mesothoracic legs of <i>neo</i> dsRNA injected individuals show five tarsal segments, or tarsomeres. (F) <i>zld</i> dsRNA beetles lacks tarsal segments resulting in a reduced tarsus, while other leg structures do not display large morphological defects.</p

<i>Tc-zelda</i> is maternally provided and progressively confined to the posterior growth-zone during embryonic development.

<p>In all embryos, the anterior region points to the left. (A) Relative expression of <i>Tc-zld</i> in ovary and carcass. (B) Relative expression of <i>Tc-zld</i> 0–3, 3–6 and 6–9 hours after egg lay. Expression values were normalized using the constitutive gene <i>rps3</i> in both experiments. (C) Pre-blastoderm stage embryo (0–3 hours) shows <i>Tc-zld</i> transcripts uniformly distributed and its respective DAPI staining in D. (E) At uniform blastoderm stage (3–6 hours) transcripts begin to occupy the germ rudiment, respective DAPI in F. (G) Shortly before posterior invagination, <i>zld</i> expression occurs along the whole germ rudiment, with higher levels at the posterior region where growth zone will form (6–9 hours). (H) Shortly before the beginning of germ band elongation <i>zld</i> expression is highly expressed at the GZ, although lower levels of mRNA can be observed at the along the whole embryonic region. (I) Approx. 13 hours after egg lay (AEL), during the beginning of germ band extension, <i>Tc-zld</i> is expressed at the posterior GZ (arrow). (J) An embryo slightly older than the one in H, highlighting <i>Tc-zld</i> expression at ventral serosal cells during serosal window closure. (K) Approx. 18-21hours AEL <i>zld</i> is still expressed at the posterior most region (arrow), head lobes (arrowheads) and a single gnathal segment (asterisk). (L) Approx. 39–48 hours AEL, during early dorsal closure, expression is observed at the nervous system (arrow).</p

<i>Tc-zelda</i> parental RNAi affects oviposition and cellularization, while embryonic RNAi affects embryonic posterior patterning.

<p>(A) Normalized number of <i>dsneo</i> and <i>dszld</i> RNAi collected eggs. <i>zld</i> pRNAi reduces oviposition. (B) Percentage of hatched larvae obtained from eggs after <i>zld</i> pRNAi in comparison to the control (<i>dsneo</i> RNAi). <i>zld</i> pRNAi leads to lethality during embryogenesis. (C) Relative expression of <i>zld</i> in <i>dsneo</i> RNAi (control) and <i>dszld</i> RNAi during the first two egg lays. <i>zld</i> pRNAi almost completely abolishes <i>zld</i> expression. (D) Phospho-tyrosine (red) and DAPI (blue) staining in <i>dsneo</i> and <i>zld</i> RNAi eggs during cellularization. <i>zld</i> pRNAi impairs cellularization. (E,F) <i>Tc-zen</i> expression in the serosa [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref036" target="_blank">36</a>] is reduced after <i>Tc-zld</i> pRNAi (arrowhead) when compared to the <i>dsneo</i> control. (G,H) Expression of the gap gene <i>Tc-mlpt</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref037" target="_blank">37</a>] in control (G) and <i>zld</i> RNAi (H). <i>Tc-mlpt</i> loses its anterior expression domain in <i>zld</i> RNAi when compared to control. (I,J) The expression of the dorsoventral gene <i>Tc-cactus</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref038" target="_blank">38</a>] was reduced after <i>Tc-zld</i> pRNAi (J-arrow) when compared to the (I) control.(K,L) The expression of the dorsoventral gene <i>Tc-sog</i> was reduced after <i>Tc-zld</i> pRNAi (J-arrow) when compared to the (I) control. (M,N) Expression of <i>Tc-Toll7</i> was affected in the posterior region of <i>Tc-zld</i> RNAi embryos (N) when compared to <i>dsneo</i> (M). (O,P) Expression of the segmentation gene <i>Tc-eve</i> in control (O) and <i>zld</i> RNAi (P). <i>Tc-eve</i> expression is essential for segmentation via a pair rule circuit in <i>T</i>. <i>castaneum</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref039" target="_blank">39</a>]. After <i>zld</i> RNAi the characteristic <i>Tc-eve</i> stripe expression in the growth zone is lost. (Q,R) Segment generation from the growth-zone (GZ) is disrupted, as judged by the analysis of <i>Tc-gsb</i> expression after <i>Tc-zld</i> pRNAi (R) and <i>dsneo</i> (Q). (S,T) <i>Tc-zld</i> dsRNA embryonic injection (eRNAi) into nuclear GFP transgenic line affects segment generation from the posterior GZ (L-asterisk), when compared to nGFP embryo injected with <i>dsneo</i> dsRNA.</p

The onset of Maternal Zygotic Transition (MZT) in <i>Tribolium castaneum</i>.

<p>(A-C) Nuclear DAPI staining of <i>T</i>. <i>castaneum</i> embryos between 0–1 hours (A), 3–6 hours (B) and 6–9 hours after oviposition (C). (D,H) Western-blots of embryonic extracts from 0–1, 0–3, 3–6 and 6–9 hours after oviposition using an antibody against the transcriptionally active form of RNA pol II, as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref033" target="_blank">33</a>]. (D) or an antibody against the α-tubulin protein as a loading control (H). (E,F,G) Immunostaining showing nuclear RNA pol II at 3–6 hours (F) and 6–9 hours (G), but not at 0–1 hour (E) after oviposition. Coupling RNA pol II staining with nuclear DAPI staining shows that <i>T</i>. <i>castaneum</i> zygotic transcription starts between 3–6 hours after egg laying, when the energids reaches the periphery (Fig 2A-C-DAPI and E-G-RNA pol II). Similar results were obtained by western-blots using the same antibody (D).</p

Computational identification of <i>Tc-zelda</i> target genes.

<p>(A) TOMTOM comparison of <i>D</i>. <i>melanogaster</i> motif similar to the TAGteam [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref007" target="_blank">7</a>] obtained by DREME and putative <i>T</i>. <i>castaneum zld</i> DREME motif. (B) Venn diagram of the <i>D</i>. <i>melanogaster</i> ChIP-Seq MZT regulated genes (green) from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref008" target="_blank">8</a>] and <i>D</i>. <i>melanogaster</i> genes predicted by FIMO analysis with the putative DREME motif (yellow). (C) Venn diagram of the <i>D</i>. <i>melanogaster</i> Dm-Zld MZT targets (green) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref008" target="_blank">8</a>] and <i>D</i>. <i>melanogaster</i> one-to-one orthologs of putative <i>Tc-zld</i> targets.</p

Tc-Zld proteins from insects and crustaceans.

<p>(A) Multiple sequence alignment of Zelda proteins, representing major groups of arthropods. (B) Conserved protein architecture features of Zelda proteins. Asterisks and x marks represent presence and absence, respectively. Question marks denote that the feature is either partially preserved or could be flagged as absent due to sequencing or assembly errors (e.g. wrong start codons). The cladogram was organized according to a previously reported phylogenetic study [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006868#pgen.1006868.ref079" target="_blank">79</a>]. Two outgroups without <i>zld</i> orthologs were also included.</p