- Open Access
The flipflop orphan genes are required for limb bud eversion in the Tribolium embryo
© The Author(s). 2017
- Received: 12 June 2017
- Accepted: 3 October 2017
- Published: 19 October 2017
Unlike Drosophila but similar to other arthropod and vertebrate embryos, the flour beetle Tribolium castaneum develops everted limb buds during embryogenesis. However, the molecular processes directing the evagination of epithelia are only poorly understood.
Here we show that the newly discovered genes Tc-flipflop1 and Tc-flipflop2 are involved in regulating the directional budding of appendages. RNAi-knockdown of Tc-flipflop results in a variety of phenotypic traits. Most prominently, embryonic limb buds frequently grow inwards rather than out, leading to the development of inverted appendages inside the larval body. Moreover, affected embryos display dorsal closure defects. The Tc-flipflop genes are evolutionarily non-conserved, and their molecular function is not evident. We further found that Tc-RhoGEF2, a highly-conserved gene known to be involved in actomyosin-dependent cell movement and cell shape changes, shows a Tc-flipflop-like RNAi-phenotype.
The similarity of the inverted appendage phenotype in both the flipflop- and the RhoGEF2 RNAi gene knockdown led us to conclude that the Tc-flipflop orphan genes act in a Rho-dependent pathway that is essential for the early morphogenesis of polarised epithelial movements. Our work describes one of the few examples of an orphan gene playing a crucial role in an important developmental process.
- Appendage formation
- Epithelial morphogenesis
- Orphan flipflop gene
- Tissue folding
- Tribolium castaneum
The general bauplan of the insect leg is highly conserved in evolution and so are the genes controlling appendage growth and patterning . Yet, principal topological differences within the insects exist. In the fruit fly Drosophila, the leg anlagen invaginate from the larval epidermis and internalise to develop inside the body cavity as imaginal discs. During pupation, appendages evert and only become functional in the adult. In contrast, ventral appendages of the flour beetle Tribolium start as everting epidermal bulges that subsequently grow in length during embryogenesis. This mechanism of appendage formation is representative for most arthropods and similar to apical epidermal ridge formation in vertebrates . Eventually Tribolium larvae hatch with fully differentiated, functional appendages .
Bud formation takes place in a restricted area of the epithelium where cells collectively polarise, undergo cell shape changes and, as a consequence, evaginate. Once this crucial decision is made, the bud grows in length and eventually differentiates [19, 54, 60]. The coordinated contractility of a group of cells at their apical or basal cortices provides the cellular basis for this morphogenetic event: apical constriction leads to tissue invagination while basal constriction results in the formation of an external bud. Constriction at one cortex of a cell usually goes along with expansion of the membrane at the opposite side .
To date, morphogenetic processes that involve apical constriction are intensely studied in a variety of developmental contexts. Most prominent examples are the infolding of cell sheets during gastrulation or neurulation, blastopore formation, trachea development, dorsal- and neural tube closure as well as embryonic tissue sealing during wound healing [22, 25, 27, 34, 40, 45].
However, tissue eversion as a consequence of basal constriction is less well understood and has been analysed in only a few cases: the formation of the midbrain-hindbrain boundary constriction and morphogenesis of the optic-cup in vertebrates, and notochord formation in an urochordate [9, 14, 28, 29, 33]. Classical studies in the polyp Hydra describe basally constricted cells within epithelial sheet curvature during reproductive bud initiation [12, 58].
Different cellular mechanisms such as differential growth or compressing forces from neighbouring cells have also been shown to initiate tissue bending and have been described for morphogenetic events like branching of developing epithelia or gut looping . Moreover, all the described processes are likely to synergise with other types of cell behaviour, such as directed cell migration into the region where a bud will form, or changes in adhesive properties once a bud protrudes out of the plane of an epithelium.
In any case, epithelial cell shape changes require the dynamic and spatial reorganisation of the actomyosin network. Its assembly and disassembly is controlled by small GTPases like RhoA (ras homologue family member A). RhoA becomes activated by the guanine nucleotide exchange factor RhoGEF2 which is transported to the apical cell cortex along the polarised microtubule network through association with the plus-end binding protein EB1 at the tips of the growing microtubules. At the apical cortex, active RhoA triggers myosin contraction through the Rho-associated coiled-coil kinase (ROCK) [27, 41].
Rho family GTPases, the effectors of myosin constriction, are also a target of the planar cell polarity (PCP) signalling pathway  that coordinates the behaviour of cells within an epithelium. The aligning of activated myosin through PCP along an axis eventually leads to polarised tissue-bending and -folding in one direction exemplarily seen during neural tube folding . PCP involves the non-canonical Wnt-signaling pathway upstream of Rho .
In order to further understand the crucial aspects of early evagination processes we analysed genes in Tribolium resulting in a hitherto not described larval knockdown phenotype of inverted rather than everted larval appendages.
Here, we focus on the novel Tc-flipflop1 (Tc-ff1) gene that was identified in the genome-wide RNAi (RNA interference) screen iBeetle  and a newly identified flipflop paralogue (Tc-ff2). RNAi-based knockdown of Tc-ff results in the formation of inverted but otherwise fully developed legs inside the larval thorax rather than growing out distally. A similar appendage phenotype was observed in an insertional mutant identified in the GEKU screen  which is located in the RhoGEF2 gene. Furthermore, we found that both Tc-flipflop genes as well as Tc-RhoGEF2 are essential for the integrity of morphogenetic movements of embryonic cells and extraembryonic membranes. We propose that in the limb-field, the very early decision of an epithelium to either invaginate or evaginate depends on Rho associated signalling with the novel Tc-ff genes as essential mediators to secure tissue eversion. Whether restricted only to Tribolium or fast evolving yet present in other animals, the Tc-ff orphan genes highlight the involvement/importance of novel factors in early epithelial morphogenesis and appendage formation.
RNAi mediated knockdown
For gene specific knockdown non-overlapping fragments were ordered from Eupheria Biotech GmbH (1 μg/μl, 3 μg/μl). For parental RNAi young adult females were sedated on ice and fixed on a petri dish using double-sided adhesive tape. dsRNA (Tc-ff, 500 ng/μl; Tc-RhoGEF2, 200 ng/μl) was injected into the abdomen under a stereomicroscope using a glass capillary connected to a manually controlled syringe. Gene specific effects for all results shown were validated with at least two non-overlapping fragments (NOFs) for each gene (Tc-ff1: NOF1 basepairs 1–320 (xx-90,314-2), NOF2 bp 340–659 (xx-90,314-1), Tc-ff2: NOF1 bp 129–416 (xx-90,313-3), NOF2 bp 417–710 (xx-90,313-2), Tc-RhoGEF2 NOF1 bp 2338–2664 (iB_03492), NOF2 bp 7408–7907) (iB_00510)). Eggs were collected and either fixed (in situ hybridisation, antibody staining, morphological analysis) or incubated at 30 °C to develop a cuticle. Tc-Dll dsRNA was injected as a control to validate gene-specific effects.
Molecular biology and expression analysis
For whole mount in situ hybridisation gene-specific primers were used (Metabion) to amplify gene fragments via PCR using cDNA synthesised from total RNA. The amplified fragments were subcloned into the pCR4 vector (TOPO-TA Cloning Kit, Invitrogen). In vitro transcription for synthesis of DIG-labelled RNA probes was performed using the DIG RNA Labelling Kit (Roche Applied Science). Whole mount in situ hybridisation was performed as previously described . Staining was achieved through application of an Anti-Digoxigenin-AP antibody in combination with NBT/BCIP (Roche Applied Science).
Antibody staining was carried out as previously described  using antibodies raised against short peptides corresponding to Flipflop1 (NH2-CPKTTKPKAK-CONH2), Flipflop2 (NH2-CSKNTEHKTK-CONH2) (Pineda Antibody-Service) and Cleaved Dcp-1 (#9578, Cell Signaling Technology), respectively. A Biotin-SP-conjugated AffiniPure Anti-Rabbit lgG antibody was used as secondary antibody (Jackson ImmunoResearch Europe Ltd). Staining was carried out using Vectastain ABC-AP (Vector Laboratories) and NBT/BCIP.
Eggs (nGFP) were dechorionated in diluted bleach, washed, positioned on a microscope slide and covered with halocarbon oil (Voltalef 10S). Injections were performed under an inverted microscope using a micromanipulator. Live imaging was carried out using a Zeiss Z.1 microscope (Zen 2.3 software) equipped with a motorised stage. Pictures were taken every 1–5 min. Images and videos were processed with Adobe Photoshop CS5.
Tribolium castaneum: http://bioinf.uni-greifswald.de/gb2/gbrowse/tcas4/
UCSC Genome Browser: https://genome-euro.ucsc.edu/cgi-bin/hgHubConnect? redirect=manual&source= genome.ucsc.edu
RNAi induced phenotypes:
The novel gene Tc-flipflop reveals a new RNAi knockdown phenotype
There are two Tc-flipflop genes in the Tribolium genome
In addition to the original Tc-flipflop gene, we identified an obvious Tc-ff paralog in the Tribolium genome. Both genes show 47% sequence similarity at the amino acid level and were therefore named Tc-ff1 (TC032552) and Tc-ff2 (TC030881), respectively. Tc-ff1 and Tc-ff2 are short genes (738 and 756 bp) with two exons each (Fig. 1c, d) that lack any conserved domains.
ff1 is also present in other Tribolium species including T. confusum, T. madens and T. freemani. Due to their incomplete annotation, it is not yet clear whether those genomes all contain a true ff2 paralogue (UCSC Genome Browser data, not shown). However, the genes cannot be found in any other sequenced genomes, including more distantly related coleopteran species.
In Tribolium castaneum, both Tc-ff genes are ubiquitously expressed in all embryonic stages at mRNA level (Additional file 1: Fig. S1B-E, F, H). The initially observed outside-in appendage phenotype was validated for both genes using two independent, non-overlapping fragments (NOFs) (Fig. 1c, d).
The “flipflop syndrome”
RNAi mediated knockdown of both Tc-ff genes results in the outside-in appendage phenotype but also reveals additional lesions of the larval cuticle such as the invagination of abdominal segments and the failure of dorsal closure. These alterations from the wildtype collectively comprise the “flipflop syndrome”.
Tc-flipflop determines the directionality of embryonic appendage formation in Tribolium
It has been proposed that epithelial morphogenesis can depend on an elevated level of cell death . To reveal whether this may play a role in the directionality of appendage growth in Tribolium, we analysed Tc-ff RNAi embryos using an antibody detecting Dcp1 . However, the ingression of cells does not appear to be accompanied by an elevated level of cell death in the limb primordium or its immediate neighbourhood (Fig. 5f).
Knockdown of the highly-conserved Tc-RhoGEF2 gene displays a Tc-flipflop-like RNAi phenotype
Tc-flipflop and Tc-RhoGEF2 influence morphogenetic dynamics and polarity of extraembryonic membranes and embryonic tissues
To determine the function of Tc-ff and Tc-RhoGEF2 in early embryogenesis we analysed RNAi embryos both via time-lapse microscopy and in fixed stages. We found that RNAi mediated knockdown of Tc-ff as well as Tc-RhoGEF2 influences the integrity of extraembryonic membranes and interferes with morphogenetic movements of the young germ anlage and the germ band.
Genes that are required for the allocation and patterning of appendages are well-known [2, 59]. However, not much is known about the factors that determine the initial direction of tissue evagination during appendage bud formation. Here, we show that the novel genes Tc-ff1 and Tc-ff2 are required for early appendage eversion during embryogenesis of Tribolium. Knockdown of those genes leads to an outside-in phenotype of inverted appendages that has not been described so far. We further observed this highly specific appendage inversion phenotype after knockdown of RhoGEF2 function. This leads us to the hypothesis that the Tc-ff genes serve as important co-regulators within a Rho-dependent pathway.
Eversion of embryonic limb anlagen requires the novel flipflop genes in Tribolium
In the wildtype Tribolium embryo, limb development starts as a bud that everts and subsequently grows in length. For the first time, we identified genetic components required for this important initial decision within the limb field tissue. Initially, Tc-ff1 was uncovered via its enigmatic inverted leg phenotype during the iBeetle RNAi pre-screen using randomly picked cDNA clones as a source for dsRNA . By sequence homology we identified a second flipflop gene in the Tribolium genome, named Tc-ff2. Both genes are predicted to code for small proteins of 136 and 127 amino acids, respectively, lacking any known functional domains. A web-tool based analysis of their sequences [1, 26] characterises the Tc-ff genes, at least in the absence of a binding partner, as “unstructured” with long stretches of low complexity domains.
To answer the question, whether the Tc-ff genes are indeed protein-coding or function as long-non-coding RNAs, we raised anti-peptide-antibodies that recognise a single specific band of the expected molecular weight (Additional file 1: Fig. S1A) in a Western blot using embryonic extracts. We take this as indication that the predicted ORFs are indeed translated, however, the antibody does not detect a distinct spatial or subcellular pattern in the embryo.
The Tc-ff genes can be found in the Tribolium lineage (including T. confusum, T. madens and T. freemani. UCSC Genome Browser) but seem absent from any other sequenced genomes. Based on their lack of conservation, small size and apparent disordered structure, we classify Tc-ff1 and Tc-ff2 as orphan genes . Given that orphan genes of small size tend to be evolutionarily more recent , these genes may well be limited to a small subset of colepteran species. However, we also cannot exclude that the Tc-ff genes provide a conserved function and homologs cannot be identified due to small size and a fast-evolving sequence. In any case, the Tc-ff genes represent one of few examples of orphan genes with an essential early embryonic function. In addition, these genes illuminate an early decision in the development of appendages which had been overlooked so far.
With the Tc-ff knockdown we observed a variety of phenotypes in affected larval cuticles ranging from severe to weak. We categorise the inverted growth of one or just a few appendages without additional defects as a weak knockdown phenotype. More severely affected cuticles display many outside-in events at once combined with a dorsal closure defect (Fig. 3h). The strongest effects are represented by cuticle remnants without recognisable structures or the complete lack of a cuticle (“empty eggs”), respectively, underlining that Tc-ff function is required in different tissues. While the Tc-ff2 knockdown results in a somewhat higher penetrance regarding phenotypic defects compared to Tc-ff1, both genes appear to be non-redundant as the single knockdown of either gene is sufficient to disrupt their morphogenetic function. This is also highlighted by the fact that the combined Tc-ff1/ff2 double RNAi knockdown does not increase the overall penetrance compared to the single knockdowns (Additional file 2: Table S1).
We found that the direction of limb budding in Tribolium is determined as early as the beginning of appendage bud formation and is significantly influenced by Tc-ff function. We have no indications that in Tribolium the eversion of the limb epithelium depends on an elevated level of cell death as it has been shown for other cases [32, 44].
The flipflop genes may act in a RhoGEF-dependent cell polarity network
Given that the orphan Tc-ff genes lack evolutionarily conserved characteristics to associate them with any known molecular pathway, we searched for other genes that display the same or a similar RNAi knockdown phenotype. We found that RNAi-mediated (partial) knockdown of the ubiquitously expressed Tribolium homolog of RhoGEF2 did also result in the disruption of appendage eversion, resembling the characteristic Tc-ff RNAi outside-in phenotype (Fig. 6). In contrast to Tc-ff, Tc-RhoGEF2 represents an evolutionarily highly conserved gene. By activating members of the Rho-family GTPase , RhoGEF2 plays an essential role in a number of morphological processes involving cell-cell adhesion, cell polarity, cell migration and cell motility [20, 36, 47]. Furthermore, RhoGEF2 is well known to be a key factor controlling cell shape change and apical constriction through the regulation of actomyosin contractility [3, 15, 24, 38]. In Drosophila an impairment of factors involved in Rho-dependent apical constriction can result in salivary gland formation outside the embryo, instead of forming inside the embryo as in wildtype . Here, we show that both Tc-ff and Tc-RhoGEF2 initially determine the direction of appendage growth. However, the number and localisation of inside-out events seen in different Tc-ff RNAi larvae does not follow a certain pattern. This variability of the phenotype led us to hypothesise that the cell fate decision of the epithelium whether to invaginate or evaginate may be a quantitative local event rather than an all-or-nothing epithelial switch. Depending on the number of cells within the limb field undergoing uniform polarised constriction either at the apical or the basal side provides the physical ground for an epithelium to buckle to one of the two directions. Disruption of the uniformity of this collective cell behaviour through absence of the same polarity cue in all cells increases the likelihood of adjacent cells being forced into a different direction. In Drosophila it has been shown that inhibition of apical constriction in a defined area of the epithelial tissue disrupts ventral furrow formation depending on the number of cells affected as well as the intensity of the inhibitory signal . Tissue invagination does still function when a smaller section of cells is affected as long as there are still enough cells undergoing cell shape change “dragging” adjacent corrupted cells with them.
Based on the findings in Drosophila and the highly conserved RhoGEF2 function, we hypothesise that in Tribolium ff and RhoGEF2 play an essential role in the decision where cellular constriction - either at the apical or basal side - takes place, so that in the absence of this apical-basal polarity cue the buckling direction becomes random. However, the genes are not required for the formation of the appendage primordium itself. Thus, we propose that Tc-ff is involved in a Rho GTPase-dependent pathway that regulates the apico-basal polarity of a cell. However, a detailed analysis of the cellular dynamics including suitable markers localising proteins that are involved in cell shape change events will be required to validate our hypothesis.
Tc-flipflop and Tc-RhoGEF2 are also required for the morphogenetic dynamics and polarity of extraembryonic membranes
In contrast to the single reduced amnioserosa of Drosophila, Tribolium has two extraembryonic membranes, amnion and serosa, that actively contribute to the morphogenesis of Tribolium during gastrulation, germ band extension and dorsal closure [18, 39]. We have seen that knockdown of either Tc-ff or Tc-RhoGEF2 affects the directed morphogenetic movements and the cellular integrity of extraembryonic tissues. This aspect of Tc-ff and Tc-RhoGEF2 function is seen in the early embryo where the extraembryonic membranes fold prematurely and fail to fully cover the embryo. As a consequence, a misshaped germband forms on top of the yolk (Fig. 7d). This phenotype clearly illustrates that the dorso-posterior translocation of the extraembryonic tissues and the enwrapping of the embryo require coordinated tissue elongation within a plane of cells, a process likely to involve planar cell polarity (PCP) . As described for other systems, Rho-dependent cell shape changes can be regulated through the PCP pathway that involves non-canonical Wnt signalling. Thus, we propose that the polarised maintenance of extraembryonic tissue dynamics during Tribolium embryogenesis depends on PCP and that Tc-RhoGEF2 and the Tc-ff genes are downstream targets of this important signalling pathway in Tribolium.
Additionally, we have observed premature ruptures of extraembryonic membranes in RNAi embryos (Fig. 8) as well as dorsal closure defects. In Drosophila, dorsal closure depends on actomyosin contractility at the apical cortex of the amnioserosa cells while the actin cable in cells of the leading edge seems dispensable for this process [11, 40]. It is conceivable that the polarity of the extraembryonic cells along the apico-basal axis might also be disturbed in Tc-ff RNAi embryos and, as a consequence, dorsal closure is impaired. An unstructured actomyosin network also may contribute to weaken the integrity of the extraembryonic membranes epithelia so that they cannot withstand strong mechanical tension during the dorsal closure process. Once cell polarity markers become available for Tribolium it will be feasible to evaluate the involvement of PCP in the process of dorsal closure.
Based on our observations we hypothesise that Tc-ff and Tc-RhoGEF2 contribute to the polarisation of cell movement early in development but do not influence the differentiation of epithelial structures per se. Later, they are involved in stabilising the cellular components required for the pulling forces of tissues during dorsal closure.
Assuming that the Tc-ff genes act in the same pathways as Tc-RhoGEF2, we propose that they may be downstream targets of non-canonical Wnt/PCP signalling via the regulation of or in parallel to Rho proteins.
Here, we showed Tc-ff as one of the few examples of an orphan gene playing a crucial role in a developmental process such as in morphogenetic cell movements. This is one more example for an additional novel, species-specific or fast evolving factor that functions in an otherwise conserved pathway. Previously, an orphan gene within the BMP-pathway involved in digit formation and –outgrowth in the limb has been described in a vertebrate . It will be interesting to see if functional equivalents of the Tc-ff orphan genes will be found in other organisms.
We thank the following people for their help: Andrej Lupas for discussing with us the Flipflop protein structure; Verena Hofer-Pretz and Nicole Gehring for excellent technical assistance and beetle care. Lizzy Ge-rischer and Mario Stanke for providing Tribolium genome annotation tools. Christian Schmitt-Engel for the initial discovery of flipflop1, and Jianwei Li for additional flipflop sequence information.
German Research Community DFG; Grant to AB (BE 4850/1–1).
MK: initial identification of the Tc-ff and KT221 phenotypes; ST, AB performing experiments; ST, AB, RS initiation and conception of the experiments; ST, RS writing the manuscript; AB, MK corrections. All authors read and approved the final manuscript.
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