ABSTRACT
Tenebrionid beetles have been highly successful in colonising environments where water is scarce, underpinned by their unique osmoregulatory adaptations. These include a cryptonephridial arrangement of their organs, in which part of their renal/Malpighian tubules are bound to the surface of the rectum. Within the cryptonephridial tubules, an unusual cell type, the leptophragmata, plays a key physiological role underpinning water conservation. Nothing was known about the developmental mechanisms or evolution of these unusual renal cells. Here, we investigate mechanisms underpinning leptophragmata development in Tribolium castaneum. We find that leptophragmata express and require the Tiptop transcription factor, similar to secondary renal cells in Drosophila melanogaster, which express Teashirt and Tiptop, despite Drosophila lacking a crypronephridial arrangement. An additional transcription factor, Dachshund, is required to establish leptophragmata identity and to distinguish them from the secondary cells in the non-cryptonephridial region of renal tubule of Tribolium. Dachshund is also expressed in a sub-population of secondary cells in Drosophila. Leptophragmata, which are unique to the beetle lineage, appear to have originated from a specific renal cell type present ancestrally and to be specified by a conserved repertoire of transcription factors.
INTRODUCTION
Tenebrionid beetles have been extremely successful at surviving where water is scarce, and therefore colonising arid regions (Buxton, 1923). Unique osmoregulatory adaptations are likely to underpin this ability, including differences in the physiology and hormonal control of their renal tubules (also known as Malpighian tubules; MpTs) compared with other insects (Cabrero et al., 2020; Halberg et al., 2015; Halberg and Denholm, 2024; Koyama et al., 2021). Another crucial adaptation is the cryptonephridial complex (CNC). In contrast to the ancestral state, where the MpTs are free within the haemolymph, in the CNC arrangement the distal regions of the MpTs are bound to the surface of the rectum (Beaven et al., 2023). This complex is ensheathed in an insulating tissue: the perinephric membrane (Ramsay, 1964). Ion transport by the MpTs generates a high potassium chloride concentration surrounding the rectum (O'Donnell and Machin, 1991), which functions to draw water osmotically out from the faeces within the rectal lumen that can then be returned by the MpTs to the body (Machin and O'Donnell, 1991). This allows tenebrionid beetles, such as Tenebrio molitor, to generate powder dry faeces, thus minimising water loss (Ramsay, 1964; Wigglesworth, 1932). The ability of the CNC to extract water from the rectum is so powerful that they can even use it to extract moisture from the air in conditions above a certain level of humidity (Buxton and Haldane, 1930; Dunbar and Winston, 1975; Hansen et al., 2004; Machin, 1975).
Leptophragmata are a cell type within the CNC, considered to be key to its function. They have unique access to the haemolymph surrounding the CNC, residing beneath regions of the perinephric membrane where it is present as only an extremely thin ‘blister’ of extracellular material (Grimstone et al., 1968; Ramsay, 1964). Leptophragmata have long been considered the likely route by which chloride ions enter the CNC (Grimstone et al., 1968; Lison, 1937; Ramsay, 1964), although other possible routes have been suggested, including from the fluid in the rectal lumen (O'Donnell and Machin, 1991). In our previous paper using the model insect species the red flour beetle (Tribolium castaneum), we shed light on the molecular mechanisms underpinning leptophragmata function. We identified a cation and/or proton antiporter, NHA1, in the leptophragmata, which likely uses a proton gradient to transport potassium into the CNC (Naseem et al., 2023). In turn, chloride is considered to follow along the electrochemical gradient established by potassium transport (Machin and O'Donnell, 1991; O'Donnell and Machin, 1991). Leptophragmata are an unusual, seemingly unique, cell type, with a highly distinctive cell morphology when compared with other Malpighian tubule cells. They are extremely thin flattened disk-shaped cells, a band of thicker material traverses them and their cell body hangs down into the lumen of the Malpighian tubule (Ramsay, 1964). They also form an unusual contact with the inner layer of the perinephric membrane, displaying a region of highly electron-dense ECM (Grimstone et al., 1968). They are also distinct in their position, aligning beneath openings in the perinephric membrane, and there are indications that they are unique in their physiology.
We have previously demonstrated that Tribolium has a CNC structure very similar to that of T. molitor, in which the CNC has been most studied. Tribolium is an ideal species in which to study CNC developmental mechanisms and their molecular basis (King and Denholm, 2014; Naseem et al., 2023). It has a sequenced, well-annotated genome and established means for manipulating gene expression, including RNAi. We previously identified Tiptop, a transcription factor of the Teashirt gene family, to be expressed in Tribolium leptophragmata (King and Denholm, 2014). Teashirt and its paralogue Tiptop (hereafter, Teashirt/Tiptop) are required for differentiation of MpT secondary cells in Drosophila. Secondary cells are one of two major cell types found in Drosophila Malpighian tubules, and are considered the major route for the movement of chloride and water (Denholm et al., 2013). Tribolium Tiptop is expressed in leptophragmata but also in the free tubule (the region of the tubule not associated with the rectum and exposed to the haemolymph) in a subset of cells that display properties akin to Drosophila secondary cells, including smaller nuclear size (King and Denholm, 2014). Tiptop is the sole member of the Teashirt/Tiptop family of transcription factors in Tribolium (Shippy et al., 2008).
Here, we build on these insights to gain an understanding of the molecular mechanisms by which leptophragmata identity is specified, and provide clues of their evolutionary origins. We draw on the tissue specific transcriptomic database, BeetleAtlas, which has proven to be a powerful resource for guiding molecular studies in Tribolium (Leader et al., 2024; Naseem et al., 2023), and conduct functional studies using RNAi, cell morphology analyses and physiological methods that are established for this species. We find that the transcription factor tiptop, in combination with dachshund, are required for leptophragmata identity. We also find evidence that this combination of genes defines a specific sub-population of secondary cells in Drosophila, which lack a CNC. Therefore, although differentiated leptophragmata appear unique to the beetle lineage at a morphological and molecular level, they are specified by a conserved repertoire of transcription factors, and therefore likely derived from a specialised renal cell type present ancestrally. This work sheds light on the evolution of an unusual cell type within the CNC that underpins the remarkable water conservation ability of tenebrionid beetles.
RESULTS
Leptophragmata require the secondary cell transcription factor Tiptop
To confirm our previous finding that the transcription factor Tiptop is expressed in the leptophragmata of Tribolium, in addition to the secondary cells of the free tubule region (King and Denholm, 2014), we made a detailed characterisation of Tiptop expression. First, we assessed the expression pattern of tiptop during Tribolium embryonic development using fluorescent hybridisation chain reaction (HCR), counterstaining against cut to mark the MpTs (King and Denholm, 2014, and Fig. S1 provide an overview of MpT development in Tribolium in relation to overall embryogenesis).
We found that tiptop is first expressed in all cells of the MpTs as they bud from the developing hindgut (Fig. 1A), a result consistent with a previous report (Shippy et al., 2008). Subsequently, as the MpTs begin to grow and extend, we observed that tiptop expression becomes restricted to their distal region (Fig. 1B). From Drosophila, we know that the early MpTs, which bud out from the developing gut, comprise solely principal cells (Ainsworth et al., 2000; Denholm et al., 2003; Technau and Campos-Ortega, 1985). Later in embryogenesis, as the MpTs extend, the secondary cells insert into the central MpT region, subsequently dispersing among the principal cells (Campbell et al., 2010; Denholm et al., 2003). Assuming that the secondary cells in Tribolium incorporate into the MpT with a similar timing and distribution, then the tiptop expression in the Tribolium tubule observed in early and mid-stage embryos is likely to be in principal cells. In late stages of Tribolium embryogenesis, tiptop expression was no longer seen in the distal MpT, but was observed in a subset of cells regularly scattered along the entire MpT length. This includes both the regions associated with the CNC, which we refer to as perirectal tubules, as well as the free tubule regions (Fig. 1C). This could be explained by tiptop expression being activated in the secondary cells, and downregulated in the distal principal cells. These distinct phases of tiptop expression echo those found for Tiptop in Drosophila tubules during embryogenesis (Denholm et al., 2013).
Based on the expression pattern of tiptop in the perirectal tubules, and on our earlier observations (Denholm et al., 2013), we considered that these tiptop-positive cells were likely to be leptophragmata (Fig. 1C). To test this, we dissected adult CNCs and used an antibody to detect Tiptop protein, in combination with counterstaining for F-actin, allowing us to identify the leptophragmata by position and cellular morphology. In Tribolium and the related Tenebrionid beetle Tenebrio molitor, the perinephric membrane ensheaths the CNC but is punctuated with small openings under which the leptophragmata sit (Beaven et al., 2023; Grimstone et al., 1968; Naseem et al., 2023; Ramsay, 1964). The outer perinephric membrane, which stains strongly for F-actin, and the openings within it, can readily be identified. The leptophragmata are also apparent, both by their position beneath the opening and by the distinctive bar-shaped F-actin structure contained within them. Using this approach, we identified leptophragmata and confirmed that they express Tiptop (Fig. 1D). Together, these data show that Tiptop expression initiates in Tribolium tubules from embryonic stages and is subsequently maintained in tubule cells (including leptophragmata) into adults, and point towards a likely role in tubule cell identity.
To test for a functional requirement for Tiptop in the development of the leptophragmata, we sought to determine the consequence of tiptop depletion. To do this, we injected dsRNA targeting tiptop into adult females, and observed their offspring at the stage of 1st instar larvae. In control 1st instar larvae, we identified leptophragmata based on their expression of Tiptop, their small nuclear size (relative to surrounding principal cells), their position under the perinephric membrane openings and their F-actin bar structure, which, despite being smaller than in the adult, are still discernible (Fig. 2A). After knockdown, Tiptop expression is abolished (indicating efficient knockdown) and there is no evidence of leptophragmata based on their distinctive morphologies of the F-actin bar structure and small nuclei. (Fig. 2A,B). This suggests that tiptop is required for the specification and/or normal differentiation of leptophragmata in the embryo. This is in line with the role for teashirt/tiptop genes in the differentiation of secondary cells in Drosophila (Denholm et al., 2013).
To test whether Tiptop is required for the maintenance of leptophragmata identity, we performed RNAi-mediated tiptop knockdown by injecting last instar larvae and analysed adult phenotypes after eclosion. We observed severe morphological abnormalities, including deformations in elytra shape and, in the large majority of adults that emerged, incomplete shedding of the pupal case (Fig. S2A,B). The abnormal adults appeared to desiccate rapidly and generally died within a few days of eclosion.
We dissected CNCs from these tiptop-depleted adults, and found that leptophragmata are lost based on morphological criteria, including the absence of the F-actin bar structure and their distinctive staining for microtubules at this stage (Fig. 2C,D). Additionally, we tested for the presence of leptophragmata using an independent physiological assay. Evidence that leptophragmata act as a major site of chloride ion transport was first obtained by incubation with silver nitrate (Lison, 1937). This precipitates as silver chloride in the presence of chloride ions, developing into a dark staining of silver in the presence of light. The darkly stained leptophragmata are completely absent after knockdown of tiptop (Fig. 2E,F). As part of a parallel study, we also found that staining of NHA1, which we demonstrated to be a cation/proton antiporter expressed in the leptophragmata, is not observed after tiptop knockdown (Naseem et al., 2023). Together, these findings suggest that tiptop is required to specify and maintain leptophragmata identity, including their normal structure and physiological functions.
Testing for additional transcription factors required for leptophragmata identity
As both leptophragmata and free tubule secondary cells express Tiptop, we hypothesised that additional transcription factor(s) are involved in regulating leptophragmata versus free tubule secondary cell identity, and further hypothesised these additional transcription factor(s) are also likely to continue to be expressed in the mature leptophragmata, playing a maintenance function, as seen for tiptop. We therefore decided to carry out a targeted screen to identify such factors, using expression data to guide the selection of candidates.
We first identified over 600 transcription factor genes in the genome of Tribolium (Table S1). These are either orthologues of Drosophila melanogaster genes annotated as transcription factors or genes from the Regulator database of metazoan transcription factors (Wang and Nishida, 2015; http://www.bioinformatics.org/regulator/). From this list, we then identified genes for which expression was enriched in the CNC relative to other tissues (Table S1). To do so, we used BeetleAtlas: a comprehensive bulk RNA-Seq expression database from dissected adult and larval tissues (Leader et al., 2024; Naseem et al., 2023) (BeetleAtlas.org). We selected the genes that are most highly enriched in the CNC relative to other tissues, and excluded those where expression is also high in the non-CNC region of the hindgut, or the free section of the tubule. This allowed us to identify 10 transcription factors with expression most specific to the CNC (Fig. 3).
We injected dsRNA targeting each gene into final instar larvae and analysed adult CNCs from these animals morphologically, as well as physiologically using the silver staining assay. From this, we identified several phenotypes after gene knockdown (Figs S3, S4 and Table S2).
Knockdown of twist, SoxD and disco resulted in weak silver staining in leptophragmata (Fig. S3A,E,I,J and Table S2), suggesting an impaired ability to transport or retain chloride ions. Depletion of twist and SoxD also resulted in an abnormal structure of the inner layer of the perinephric membrane, which is rich in microtubules. Anti-tubulin staining reveals a region less dense in microtubules surrounding each leptophragmata (Fig. S4A), but this feature appeared to be lost in twist and SoxD knockdown animals (Fig. S4E,I and Table S2). Fluorescent in situ hybridisation-hybridisation chain reaction (FISH-HCR) shows these three transcription factors (twist, SoxD and disco) are expressed in the perinephric membrane but not in leptophragmata (Fig. S5), and therefore their effect on the leptophragmata is likely to be indirect. We also found that the leptophragmata appear less well aligned with the openings in the outer layer of the perinephric membrane upon knockdown of Pph13 (Fig. S4A,B and Table S2), and that the outer layer of the perinephric membrane had a more fragmented appearance after knockdown of eve (Fig. S4A,D and Table S2). Although these factors do not appear to be involved in specifying leptophragmata identity, they are candidates for further studies of CNC development and function. For dachshund [a transcription factor with well-known developmental roles, including retinal specification and limb patterning (Morata, 2001; Tavsanli et al., 2004)], we identified phenotypes that appear to directly relate to a role in the leptophragmata (see following sections).
Identification of a requirement for Dachshund in leptophragmata identity
Leptophragmata were still clearly discernible upon knockdown of dachshund, which is in contrast to what we found for tiptop knockdown; however, their cytoskeletal morphology is dramatically altered: the F-actin bar structure is severely reduced and the microtubules form a knot within the cell that is not present in mock-injected animals (Fig. 4A,B and Table S2). Again, unlike tiptop knockdown, sliver staining revealed that leptophragmata are present and are physiologically functional, at least in their capacity to transport and/or accumulate chloride ions. However, the darker bar like staining across the leptophragmata, which can be seen in the controls and mock-injected animals, was absent (Fig. 4C,D), consistent with the altered cell morphology observed when staining for F-actin (Fig. 4A,B).
The majority of dachshund knockdown animals survived into adulthood, allowing us to assess both whole-organism physiology and, more specifically, CNC function upon Dachshund depletion. We found clear indications that osmoregulation is abnormal. To assess the ability of the CNC to cope under conditions of water scarcity, we raised dachshund knockdown and mock-injected controls under low humidity conditions (∼5% relative humidity). We saw a small but significant increase in mortality in the dachshund knockdown group (Fig. 4E). In non-desiccating conditions, we also identified a reduction in the amount of defecation over a 4 h period (Fig. 4F). Intriguingly, we also found an increased incidence of animals where faecal residues blocked the anus (Fig. 4G). We speculated this might result from abnormally wet faeces that adhere more readily to the anus and thus be indicative of increased excretory water loss. Together, these findings suggest altered osmoregulatory performance in the absence of Dachshund, and we decided to assess whether this was a result of perturbed CNC function. To do so, we performed an ex vivo fluid reabsorption assay using isolated guts cultured under paraffin oil, that we established recently (Naseem et al., 2023). We found a significantly reduction in the rate of fluid reabsorption in preparations depleted for dachshund (Fig. 4H). Taken together, these data show that Dachshund is required for normal CNC function and whole-animal water regulation.
Dachshund defines leptophragmata identity
We have previously found that the Dachshund protein is expressed in the distal section of the developing MpTs of Drosophila (Beaven and Denholm, 2022). Furthermore, it has been reported that dachshund mRNA is expressed in the distal sections of the developing MpTs in Tribolium (Prpic et al., 2001). We used in situ HCR to assess dachshund mRNA expression in greater detail. Consistent with the previous report, this revealed dachshund expression in the distal sections of the developing MpTs (Fig. 5A). In late embryos, dachshund expression was restricted to MpTs of the CNC (Fig. 5B). The loops of free MpT, observable by expression of cut, did not show expression of dachshund (Fig. 5B). We also sought to determine whether dachshund is expressed in the developing leptophragmata in late embryos. By co-staining for tiptop mRNA, we found that, dachshund is expressed in both principal (i.e. those expressing cut but not tiptop) and secondary cells (i.e. those expressing cut and tiptop) in the developing CNC (Fig. 5B).
We then defined the pattern of dachshund expression in the mature CNC. We found that dachshund is expressed in the principal cells throughout the perirectal tubules. We also observed expression of dachshund in the tiptop-expressing leptophragmata within the more posterior part of the CNC. Curiously, dachshund did not appear to be expressed in the tiptop-expressing cells within the more anterior part of the CNC (Fig. 5C,D and Fig. S6). This prompted us to consider the distribution of leptophragmata in greater detail. We used the silver staining assay, indicating the ability of a cell to transport and/or accumulate high concentrations of chloride, as a functional marker of the leptophragmata. We used the point where the common trunk of the MpTs joined the CNC as a landmark for the anterior end of the CNC (the common trunk is a segment in which all the free MpTs run together in a single bundle, arrow in Fig. 5E). Using the silver staining assay, we found no stained cells in the anterior region of the CNC (Fig. 5E′), in contrast to the clearly stained leptophragmata in the more posterior part of the CNC (Fig. 5E″). We therefore consider that the leptophragmata are exclusively found in the posterior region of the perirectal tubules, and are distinct from a population of secondary cells in the anterior region of the perirectal tubules. This is consistent with the lack of leptophragmata previously noted in the anterior CNC region of Tenebrio (Grimstone et al., 1968). By assessing nuclear size (using DAPI staining) in tiptop-expressing secondary cells of the perirectal tubules, we also discovered that the nuclei of secondary cells in the posterior CNC are smaller than those of secondary cells in the anterior CNC (Fig. 5F-I), providing an additional marker for leptophragmata and further evidence that leptophragmata are distinct from the secondary cells residing in the anterior perirectal tubules. We observed approximately seven or eight anterior secondary cells in each perirectal MpT, and consider these to be a distinct subpopulation of secondary cell that has not been characterised previously, and that appear to differ from the secondary cells of the free MpT in addition to the leptophragmata, as indicated by their different nuclear size (Fig. 5I). The functions of this anterior subpopulation are currently not known.
We wanted to understand further the role Dachshund plays in the specific differentiation of leptophragmata (in contrast to other secondary cell populations), particularly their physiology and endocrine regulation. Our knowledge of specific proteins that define different secondary cell populations in the Tribolium renal system, such as differentially expressed ion channels, is limited. However, we have shown previously that the hormone receptor Urinate Receptor (Urn8R), which regulates MpT fluid secretion, is specifically expressed in secondary cells within the main segment of the free MpT (Koyama et al., 2021), and could therefore be a potential marker to distinguish secondary cells in different regions.
We assessed the pattern of expression of the Urn8R protein in adult CNCs and free MpTs. We observed expression within secondary cells of the free tubule as reported previously (Koyama et al., 2021; Fig. 6A). We also found that Urn8R is expressed in cells within the anterior part of the perirectal tubules, which correspond to the non-leptophragmata secondary cells identified above (Fig. 6B), but not in leptophragmata (Fig. 6C). On this basis, we hypothesised that Dachshund could function to restrict the expression of Urn8R, repressing (directly or indirectly) its expression specifically within the leptophragmata. We therefore looked at expression of Urn8R upon knockdown of dachshund. We observed clear expression of Urn8R within leptophragmata, when depleting dachshund (Fig. 6D). We have reported previously that the diuretic hormone DH37 signals via Urn8R in secondary cells of the free tubule. Using a fluorescently labelled DH37, we showed the ability of this hormone to bind free tubule secondary cells (Koyama et al., 2021). We found that the labelled DH37 also binds a population of cells in the anterior CNC (Fig. 6E), likely to be the Urn8R-expressing secondary cells, thus indicating that DH37 is competent to bind with the receptor in these cells.
As a further readout of cell identity, we measured the area of leptophragmata nuclei after dachshund depletion, as we had also found this parameter to differ between different secondary cell subpopulations. We found increased area in the dachshund knockdown condition. This provides a further indication that Dachshund functions in specification of leptophragmata identity, and loss of dachshund possibly results in a shift towards anterior CNC secondary cell identity, which we had found to have a larger nuclear size (Fig. 5G-I).
Additionally, we assessed expression of the NHA1 protein, which we previously identified as a proton/potassium antiporter, which is expressed predominantly in the leptophragmata (Naseem et al., 2023). By antibody staining, we observed NHA1 to still be expressed in leptophragmata after knockdown of Dachshund, although there appears to be a significant change in the number of cells expressing NHA1, with apparent loss of expression in the leptophragmata in the anterior region of the complex (Fig. S7).
Overall, our findings suggest Dachshund contributes to specification of leptophragmata identity, and in its absence these cells may adopt an identity closer to the other secondary cell populations in the anterior CNC or free tubule.
The role of Dachshund in distal secondary cell identity is evolutionarily conserved, indicating an ancestral role
The CNC and leptophragmata are derived features of the coleopteran renal system, and it is conceivable that Dachshund was recruited to regulate distal secondary cell identity during the evolution of the system. However, an argument against this comes from our knowledge that Dachshund is expressed in distal MpT cells in Drosophila (an insect without a CNC or leptophragmata) (Beaven and Denholm, 2022). This raises the alternative hypothesis that Dachshund has an ancestral role in patterning identity of the distal secondary cells that predates evolution of the CNC and the unique features of the leptophragmata. With this in mind, we explored the role of Dachshund in Drosophila MpT secondary cell development.
We first co-stained Drosophila embryos with antibodies against Dachshund, along with Teashirt, which is known to mark the secondary cells (Denholm et al., 2013). We found that Dachshund is expressed in the distal, but not the proximal, secondary cells, in addition to the distal principal cells (Fig. 7A). We also looked at the expression of Dachshund in the adult MpTs of Drosophila. At this stage, morphologically distinct bar (distal) and stellate (proximal) secondary cells can be identified in the anterior MpT pair. We marked secondary cells, using a line with Gal4 inserted into a teashirt enhancer region, to drive a GFP-labelled membrane marker (tsh-c724-Gal4>UAS-CD8-GFP). We found that, as in the embryo, Dachshund is expressed in both principal and secondary cells. Interestingly, Dachshund is restricted to the bar cell secondary population (Fig. 7B), suggesting it contributes to distinguishing bar cells compared with stellate cell subtypes. These findings reveal that Dachshund is expressed similarly in the MpTs of Tribolium and Drosophila, and suggest that Dachshund could have functioned to specify an ancestral distal secondary cell type that gave rise to the leptophragmata and bar cells in these two lineages.
We then aimed to test for functional roles of Dachshund in Drosophila secondary cells. Chloride channel-a-Gal4 (Clc-a-Gal4) is expressed in proximal, but not distal, secondary cells (i.e. approximately corresponding to the stellate cells). The Clc-a-Gal4 reporter appears to represent a subset of the endogenous Clc-a expression pattern, as anti-Clc-a stains at least the more proximal bar cells, in addition to stellate cells (Fig. 7C). We used Clc-a-Gal4 to drive expression of Dachshund in the stellate cells, where it would normally be absent. This led to stellate cells losing their normal stellate morphology, becoming smaller and taking on an appearance more reminiscent of bar cells (Fig. 7C,D). This phenotype may be explained by inhibition of stellate cell gene expression by Dachshund, e.g. of RhoGEF64c, which is specifically expressed in stellate cells and the knockdown of which results in a loss of stellate morphology, likely owing to altered F-actin regulation (Xu et al., 2022).
We also carried out the converse experiment in which we depleted Dachshund in the bar cells. To do this, we knocked down dachshund within all tubule secondary cells by expression of a shRNA targeting dachshund, and assessed bar cell morphology in adult MpTs. Although bar cells with relatively normal morphology and distribution were still observed upon dachshund depletion in some tubules, abnormal bar cells were also frequently observed, including abnormally large cells, abnormally small cells and cells that appeared to be extruding from the tubule. We consider it likely that these are dying cells, as staining for DNA was very diffuse, indicative of disrupted nuclei that characterise apoptotic cells. Stellate cells were unaffected, in line with a role for Dachshund specifically in bar cells (Fig. 7E,F).
We also found a significant reduction in bar cell number in dachshund shRNA expressing MpTs at all time points tested. We wondered whether bar cells were progressively lost in dachshund-depleted MpTs, i.e. whether Dachshund functions in bar cell maintenance. Although we found a slight tendency towards fewer bar cells as adult MpTs matured, the difference between timepoints is not significant (Fig. 7G). Overall, our findings indicate that Dachshund confers a distal/bar cell identity to the secondary cells in Drosophila MpTs, akin to its role in conferring leptophragmata identity in Tribolium. Furthermore, it provides evidence to homologise leptophragmata and bar cells, and supports the idea that Dachshund confers distal secondary cell identity more widely in insect MpTs.
DISCUSSION
Tiptop and Dachshund establish leptophragmata identity
Leptophragmata are a derived and highly specialised cell type of the CNC, whose activities as the main site for ion exchange drive CNC function (Naseem et al., 2023). Here, we show that leptophragmata constitute a subpopulation of MpT secondary cells, a cell type found widely in insect MpTs, including those species without a CNC. We find that leptophragmata identity is established by the transcription factor Tiptop, akin to the requirement for Teashirt/Tiptop transcription factors for secondary cell identity in Drosophila (Denholm et al., 2013). Leptophragmata are distinct from other secondary cells in the free MpT region of Tribolium and in the anterior perirectal MpT, as well as from secondary cells of other species, both in their morphology and function. This posed the question of how different secondary cell subtypes, including the leptophragmata, the secondary cells in the anterior CNC and free MpT region, are established in Tribolium, and how they have evolved between species. A screen for further transcription factors that control leptophragmata differentiation identified a role for Dachshund. We find that Dachshund controls features of leptophragmata morphology, such as their cytoskeletal arrangement and nuclear size, and acts to regulate gene expression, which distinguishes leptophragmata from other secondary cell subtypes. Loss of Dachshund leads to loss of leptophragmata characteristics and their adoption of characteristics resembling the non-leptophragmata secondary cell subtypes. This includes de-repression of the diuretic hormone receptor Urn8R, which is expressed in secondary cells of the free tubule (Koyama et al., 2021) and anterior CNC, but not in leptophragmata (Fig. 8A).
The upstream developmental mechanisms by which these transcription factors become expressed await further investigation. In Drosophila, the unique molecular identity of secondary cells, including their expression of Teashirt/Tiptop, is a consequence of their distinct developmental origin compared with that of principal cells. The secondary cells in Drosophila originate from a subpopulation of caudal visceral mesoderm; they integrate into the developing MpTs and begin to express Teashirt (Campbell et al., 2010; Denholm et al., 2003). It seems likely that a common developmental process is conserved in Tribolium. There is less evidence of how the expression of Dachshund comes to be patterned in the secondary cells. In Drosophila, specification of distal MpT identity (including expression of Dachshund) occurs very early in principal cell precursors under the control of Wingless/Wnt signalling, which is expressed asymmetrically in the principal cell precursors (Beaven and Denholm, 2022). It is conceivable that secondary cell identity (in both Drosophila and Tribolium) could be determined according to whether secondary cells integrate into a Dachshund positive or negative population of principal cells, possibly through interactions with their new principal cell neighbours. Such an interaction has precedent; apical-basal polarity of the principal cells is required for correct polarisation of the secondary cells in Drosophila (Campbell et al., 2010).
Do Tiptop and Dachshund function as part of a single transcriptional complex in defining leptophragmata identity? From Drosophila, it is known that Teashirt/Tiptop transcription factors regulate different sets of genes in a context-dependent manner, depending on the presence of other transcriptional co-regulators (Bessa et al., 2002; Datta et al., 2011; de Zulueta et al., 1994; Gallet et al., 1998; Taghli-Lamallem et al., 2007). Interestingly, Dachshund has been shown to block the transcription of a target gene of the Teashirt-Yorkie-Homothorax complex, in the Drosophila eye disc (Brás-Pereira et al., 2015; Peng et al., 2009). It is conceivable that similar regulation occurs in MpT secondary cells and leptophragmata: Tiptop may activate urn8r transcription (and possibly other targets too) in secondary cells of the free tubule and anterior CNC, with Dachshund acting to repress this specifically in leptophragmata (Fig. 8A). This type of cross-regulation might also extend to the control of genes underpinning the distinct morphology of leptophragmata.
Dachshund regulates the physiological functions of the CNC
Our data suggest that Dachshund regulates the transcriptional control of urn8r, and Dachshund may regulate other genes expressed in leptophragmata that underpin their unique morphology and functions. A central role for the regulation by Dachshund of physiologically important targets in the CNC is supported by the dachshund knockdown experiments, which lead to defects in CNC osmoregulation and a failure of the animal to tolerate desiccation stress (Fig. 4E-H). However, it is likely other transcription factors act in parallel to Dachshund, as some features of leptophragmata identity remain upon Dachshund depletion, such as their ability to accumulate/transport high levels of chloride ions (Fig. 4D). Alternatively this could result from incomplete knockdown of Dachshund after dsRNA injection. We are now working to resolve mRNA expression data of the CNC at single cell resolution, to identify further regulators of leptophragmata identity. Furthermore, although a role for Dachshund in leptophragmata is clear, Dachshund is also expressed in perirectal principal cells, which is indicative of a requirement for Dachshund in this subpopulation of principal cells. It remains to be resolved whether defects in osmoregulation and tolerance to desiccation stress seen in the absence of Dachshund reflect a requirement in leptophragmata and/or principal cells.
Why expression of the Urn8R hormone receptor would be actively repressed within the leptophragmata remains another interesting question for future investigation. Distinct physiological functions of insect tubules, including calcium homeostasis, fluid secretion and reabsorption are segregated to discreet regions along their length (Dow et al., 1994; O'Donnell and Maddrell, 1995; Sozen et al., 1997; Wessing and Eichelberg, 1978). Decoupling of endocrine control of secondary cell activity within different regions might provide a finer level of control. This could be even more important in species with CNCs, such as Tribolium, where the perirectal MpTs serve a highly specialised function. Our previous results show that activating the Urn8R pathway in free tubule secondary cells has a diuretic effect by stimulating fluid secretion into the MpT lumen, which is then expelled into the gut (Koyama et al., 2021; Fig. 8A). It would make sense to prevent simultaneous upregulation of secretion by the perirectal MpTs, as this would recycle fluid from the rectum back to the haemolymph, thereby counteracting the diuretic effect. This may thus explain why it is necessary to repress Urn8R expression in the leptophragmata. Our data do not address whether distinct endocrine signals regulate leptophragmata activity but, given the high metabolic costs of the system, it is likely that activity is regulated in alignment with the physiological needs of the animal. Single cell expression data would also help identify candidate endocrinological control mechanisms.
Evidence that leptophragmata originated from an ancestral distal secondary cell
There is growing evidence that the physiological functions of beetle MpTs are radically different compared with other insects, of which the emergence of the specialised leptophragmata cell is one aspect. These evolutionary changes are likely to underly the ability of some tenebrionid beetle species to survive in very dry environments, thus contributing to their remarkable evolutionary success. It is known that major changes to the endocrinological control of MpT cell types have occurred in beetles, including the loss (at genome level) of the kinin hormone pathway that regulates secondary cells in other species, and the restriction of another hormonal pathway active in all principal cells in other species, to only a small subset of principal cells in beetles (Halberg et al., 2015). In the case of the Urn8-receptor pathway, we recently found that the diuretic hormones DH37 and DH47 signal to the free tubule secondary cells to stimulate secretion in beetle species of Polyphaga, including Tribolium (Koyama et al., 2021). However, in the more basal beetle group Adephaga, and in other insects, including Drosophila, there are indications this pathway instead controls principal cell activity (Cannell et al., 2016; Koyama et al., 2021).
These evolutionary changes also appear to relate to the movement of water. It is known for many insects that secondary cells are a major site of water flow, facilitated by expression of aquaporin water channels (Cabrero et al., 2020; Kaufmann et al., 2005). By contrast, leptophragmata are considered impermeable to water; an important feature to prevent water being drawn from the haemolymph into the CNC, which would counter the ability of the CNC to establish a high ionic concentration (Grimstone et al., 1968; Koefoed, 1971).The ability to transport water may have been specifically lost in leptophragmata, possibly by Dachshund acquiring the ability to inhibit aquaporin gene transcription. There is some evidence that water transport mechanisms may have been altered or lost altogether from the secondary cells of beetles (Cabrero et al., 2020; Nagae et al., 2013), and future analysis of aquaporin expression in Tribolium should help resolve this.
Although cellular function has clearly changed dramatically in beetles, we find evidence that the same fundamental cell types are present within the MpTs of Tribolium and Drosophila. This set of cell types, including a distal subtype of secondary cell, therefore seems to have existed in the last common ancestor of Coleoptera and Diptera. Furthermore, the differentiation of MpT cell types, and the maintenance of their identity, appear to be controlled by the same core transcriptional regulators in these different species. For example, Cut, Teashirt/Tiptop and Dachshund are expressed in the principal cells, secondary cells and distal MpT, respectively, with these factors even undergoing similar changes in their expression patterns during development (Beaven and Denholm, 2022; Denholm et al., 2013; King and Denholm, 2014). In both species, Teashirt/Tiptop is expressed in all secondary cells, and Dachshund is expressed in a distal subtype of these cells (leptophragmata in Tribolium and bar cells in the tubules of Drosophila; Fig. 8A,B). This suggests that the leptophragmata originated from a secondary cell subtype already present in the common ancestor of Diptera and Coleoptera, and that the core transcription factors specifying the identity of this cell type have been conserved. Changes to the molecular networks downstream to these transcription factors, however, are likely to have been significantly remodelled, underpinning the evolution of the unique characteristics of leptophragmata. Although Drosophila bar cells are genetically distinct from the more proximal (stellate) cells (Sozen et al., 1997), we have little understanding of the ancestral or extant functions of the distal secondary cells for any insect. It is known that the initial segment of the MpT, in which the bar cells reside, functions in calcium homeostasis (Chintapalli et al., 2012; Wessing and Eichelberg, 1978), and the specialised role of the bar cells could relate to this.
Our findings provide insights into the evolutionary processes that have given rise to an unusual renal cell in a major beetle lineage. More broadly, this can inform ongoing debates into how new cell types arise, and supports the paradigm that cell types can be defined by the core set of transcription factors they express, which tend to be conserved over evolutionary time (Almeida et al., 2021; Arendt et al., 2016; Posnien et al., 2023; Rebeiz et al., 2015). In contrast, the downstream pathways that ultimately confer phenotypic differentiation of the cell appear to be highly labile to evolutionary change. This study uncovers the developmental mechanisms underlying the establishment of cell identity for an unusual renal cell in the beetle CNC: leptophragmata. Leptophragmata are derived cell types that have evolved novel characteristics, underpinning CNC function. Our finding that leptophragmata are derived from ancestral MpT secondary cells whose identity, but not downstream characteristics, is established by the same patterning mechanisms found in insects without leptophragmata, sheds light on how novel cell types evolve.
MATERIALS AND METHODS
Insect culturing and sample preparation
Drosophila melanogaster were cultured on standard media at 25°C. w1118 Drosophila embryos were collected on grape juice agar plates with yeast paste. Embryos were dechorionated in 50% bleach for ∼4 min, rinsed in water and fixed for ∼25 min in 4% formaldehyde in PBS/heptane on a rotor. Formaldehyde was replaced by methanol and vitelline membranes removed by shaking, before rinsing embryos in methanol. Embryos were then processed or stored in methanol at −20°C. Adult MpTs were dissected in PBS and fixed for ∼20 min in 4% formaldehyde in PBS. The following stocks were used: tsh-c724-Gal4 (Sozen et al., 1997), UAS-CD8-GFP (Lee and Luo, 1999), UAS-dachshund (3rd) (Shen and Mardon, 1997), UAS-dac-shRNA (dacHMS01435, Bloomington 35022) and w*; UAS-GFP(Cytosolic); Clc-A-GAL4 (from Julian Dow and Anthony Dornan, University of Glasgow, UK; Clc-A-GAL4 is VDRC_ID 202625).
The Tribolium castaneum San Bernardino (SB) strain was used, with cultures maintained on wholemeal flour with yeast. Embryos were collected by allowing adults to lay on plain wheat flour for 3 days at 30°C, before removing embryos with a 300 μm gauge sieve. Embryos were fixed as detailed for Drosophila embryos (above) with the following modification for removal of vitelline membranes: embryos were briefly rinsed in heptane and transferred onto double-sided tape with a paintbrush. They were then bathed in PBS and removed from their vitelline membranes by hand using a pulled glass needle.
For larval/adult preparations of the CNC, guts with associated MpTs were dissected in PBS by pulling the posterior tip of the larval or adult abdomen away from the body, which exposes the gut, CNC and MpTs, before fixing for ∼20 min in 4% formaldehyde in PBS. In some cases, in order to avoid damaging attachments between the MpTs and the gut, dissection was performed more carefully by first opening up the abdomen of the adult by cutting the cuticle with spring scissors (Fine Science Tools, 15000-08).
dsRNA synthesis and injection
For knockdown, dsRNA targeting specific genes was generated as follows. In most cases templates were amplified from genomic DNA. This was extracted from five adult beetles by freezing in 200 μl lysis buffer [100 mM Tris-HCl (pH 9), 100 mM EGTA and 1% SDS] for 30 min before thawing and homogenising. A further 600 μl of lysis buffer was added and the sample was incubated at 70°C for 30 min. Cooled 8 M potassium acetate (150 μl) was added and incubated on ice for 20 min. The sample was then centrifuged for 20 min at 13,000 rpm (17,000 g) at 4°C). The equivalent of 0.9 of the supernatant volume of cold isopropanol, was added to the supernatant to precipitate the DNA and centrifuged for 10 min at 13,000 rpm at 4°C. The pellet was washed with 70% ethanol and air dried. The pellet was resuspended in 100 μl TE buffer (QIAGEN) and 100 μl of phenol-chloroform was added, vortexed and centrifuged for 15 min (at 13,000 rpm, 4°C). The upper phase was removed and, to this, 5 μl of 3 M sodium acetate and 250 μl of ice-cold 100% ethanol were added, and left at −20°C overnight. The sample was centrifuged for 15 min (at 13,000 rpm, 4°C), the pellet washed in 70% ethanol and air dried. The pellet was finally resuspended in TE buffer. For GFP and Amp (Ampicillin resistance gene - beta-lactamase) dsRNA, plasmids containing the relevant sequence were used to generate the template. Templates were generated by PCR, using primers (Table S3) to which T7 promoter sequences had been added at the 5′ ends. The following primers were used.
dsRNA was synthesised from these templates using the MEGAscript T7 transcription kit (Invitrogen) and precipitated using LiCl. dsRNA pellets were finally resuspended in injection buffer (1.4 mM NaCl, 0.07 mM Na2HPO4, 0.03 mM KH2PO4 and 4 mM KCl), and the approximate concentration of dsRNA was determined using a nano-drop, measuring optical density using a factor of 45 ng-cm/μl. The following concentrations of dsRNA were injected: tiptop (1), 200 ng/μl; tiptop (2), 850-3700 ng/μl; PvuII-PstI homology 13, 1100 ng/μl; tailup, 1200-1800 ng/μl; even skipped, 1975 ng/μl; twist, 6151 ng/μl; elbow B, 970 ng/μl; net, 445 ng/μl; engrailed-like, 1480 ng/μl; SoxD, 4195 ng/μl; disconnected, 3750 ng/μl; dachshund, 440-4440 ng/μl; GFP, 3500 ng/μl; Amp, 1350 ng/μl. For tiptop dsRNA experiments, either tiptop (2) or a mixture of tiptop (1) and tiptop (2) were used, which are previously used target sequences (Shippy et al., 2008).
For maternal injection, female beetles were glued on their backs with Marabu Fixogum rubber cement. Pulled glass capillaries (Narishige GD-1, 1×90 mm) with the tips broken were mounted in a needle manipulator and dsRNA was front-loaded using a syringe. Beetles were anaesthetised using CO2 and injected close to the posterior end in the softer cuticle beneath the elytra. Injected females, together with uninjected males, were placed on plain wheat flour with yeast. After a few days they were moved to fresh flour, and eggs laid were collected and allowed to hatch into larvae. For larval injections, final instar larvae were selected (based on size) and injected as for adult females, except they were glued down ventrally and injected dorsally into the softer cuticle between body segments. Injected larvae were placed on sieved wholemeal flour with yeast and allowed to mature into adults.
Immunohistochemistry and in situ hybridisation
For immunohistochemistry, PBST+BSA (PBS with 0.3% Triton X-100 and 0.5% bovine serum albumin) was used to dilute antibodies and perform wash steps. The following antibodies were used: anti-Urn8R (rabbit, 1:200) (Koyama et al., 2021), anti-Tiptop (rat, 1:100) (Laugier et al., 2005), anti-Teashirt (rabbit, 1:3000, from S. Cohen, IMCB, Singapore), anti-Dachshund (mouse, 1:100, mAbdac1-1-c from DSHB), anti-alpha-Tubulin (mouse, 1:20, AA4.3, DSHB) to stain microtubules, anti-GFP (goat, 1:500, ab6673, Abcam), anti-Clc-a (rabbit, 1:2000) (Cabrero et al., 2014) and anti-NHA1 (polyclonal rabbit, 1:500) (Naseem et al., 2023). Secondary antibodies from Jackson ImmunoResearch of appropriate species tagged with Alexa Fluor 488, Cy3 or Cy5 fluorophores were used at 1:200 (z11-165-152, 111-175-144, 712-545-153, 15-545-150, 715-175-150, 715-165-150). DAPI (Molecular Probes) was used at 1:1000 to stain DNA. Alexa Fluor 488- 568- or 647-conjugated phalloidin (Life Technologies, A12379, A12380, A22287) were used at 1:200 to stain F-actin. Samples were mounted in 85% glycerol and 2.5% propyl gallate.
Fluorescent in situ hybridisation was carried out using the hybridisation chain reaction v3.0 (HCR v3.0) technique (Choi et al., 2018), with a probe set size of 20 for cut, tiptop and dachshund (Molecular Instruments). A protocol from Bruce and colleagues was used (Bruce et al., 2021). Samples were mounted in 85% glycerol and 2.5% propyl gallate.
Sliver staining assay
For the silver staining assay, guts were dissected in water and transferred to 1% silver nitrate in water, where they were incubated in the light for ∼4 min. They were then rinsed in water before fixing for ∼20 min in 4% formaldehyde in PBS. Samples were rinsed in PBS and mounted in DPX new (Merck).
Microscopy and image analysis
Fluorescence images were taken using either a Nikon A1R or Zeiss LSM800 confocal microscope. Maximum intensity projection images were generated using Fiji. Samples from the silver staining were imaged with a Zeiss Axioplan light microscope with a Leica DFC 425C camera. To obtain an indication of the size of leptophragmata nuclei, the area was measured from a single z-section at approximately the midpoint of the nucleus. The perimeter of the nucleus was defined manually, and the area calculated using the freehand selections tool in Fiji. To calculate the staining intensity of dachshund mRNA from HCR samples, images were captured using equivalent settings. The region of the nucleus was defined in a single z-section, using the tiptop stained channel, and the mean staining intensity in the dachshund channel was calculated using Fiji.
Ligand-receptor binding assay
A synthetic analogue of Diuretic Hormone 37 (DH37) with an N-terminal cysteine was synthesised by Cambridge Peptides (Birmingham, UK) at a purity of >90%, in order to conjugate a TMR-C5-maleimide Bodipy dye (Bio-Rad), to make fluorescent TMR-C5-maleimide-SPTISITAPIDVLRKTWAKENMRKQMQINREYLKNLQamide (DH37-F). The ex vivo receptor-binding assay was performed as described previously (Halberg et al., 2015; Koyama et al., 2021). Perirectal tubules attached to the perinephric membrane were carefully dissected from the rectum under Schneider's and Tribolium saline (1:1) and then mounted on poly-L-lysine-covered 35 mm glass bottomed dishes. Next, the tissues were set up in a matched-pair protocol, in which one batch was incubated in the appropriate insect saline plus the labelled neuropeptide analogue (10−6 M) and DAPI (1 µg/ml), while the other was incubated in DAPI alone; the latter batch was used to adjust baseline filter and exposure settings to minimise auto-fluorescence during image acquisition. Images were subsequently recorded on a Zeiss LSM 900 confocal microscope using baseline filter and exposure settings.
Physiological assays
For the physiological assays, either 1 µg/µl dachshund dsRNA or Amp dsRNA (control) were injected into age-matched final larval instar larvae using a Nanoject II injector (Drummond Scientific). Animals 10-14 days after eclosion were used for experiments. To assess tolerance to desiccation, animals were transferred to a 96-well plate in a container filled with silica gel beads (Sigma-Aldrich) to produce a low-humidity environment (approximate relative humidity 5%, measured by a custom-build hygrometer). Mortality rates of the treated adults were assessed over a 16-day period by viewing under a stereo-microscope (Koyama et al., 2021).
To assess the effects of dachshund knockdown on excretory behaviour in intact living animals, dsRNA-injected animals were starved for 2 days followed by re-feeding a standard Tribolium medium supplemented with 0.05% (w/w) Bromophenol Blue (BPB) sodium salt (Sigma-Aldrich) overnight as described previously (Koyama et al., 2021; Naseem et al., 2023). This was created by mixing the standard Tribolium medium with BPB and a small amount of water to create a uniform paste, which was left to dry at room temperature overnight. The dried BPB-labelled Tribolium medium was then ground to a fine powder, creating a consistency similar to that of the standard medium. Beetles were then placed into individual wells of a 96-well plate fitted with a small piece of filter paper and the number of BPB-labelled deposits produced by each animal over a 4 h period was quantified. Next, the treated adults were recovered and raised in the same BPB medium for an additional 7 days, after which the number animals exhibiting defective defecation (i.e. their anus was blocked by excretory product likely due to excess water content) was quantified under a stereo microscope.
For the ex vivo fluid reabsorption assay, water reabsorption rates from the rectal complex were measured using a previously described protocol (Naseem et al., 2023). The control and dachshund knockdown animals were dissected carefully, keeping the head, gut, tubules and CNC intact under Schneider's medium. MpTs were then broken from the entry point (common trunk) to the CNC. The dissected animals were carefully transferred to another dish containing paraffin oil (molecular grade) with a wax layer at the bottom, and gently stretched (to keep intact the fore-, mid-, hind-gut, MpTs and CNC) and pinned at the head and anal cuticle. Two drops (5 μl and 0.3 μl) of premixed 3× Tribolium saline+Schneider's solution (1:1) supplemented with 100 µmol/l of amaranth (Sigma-Aldrich) were dropped under paraffin oil and their diameter measured. The 5 μl drop was coaxed into place around the midgut and MpTs, and the 0.3 μl drop around the rectal complex with the help of a capillary pulled glass rod, ensuring contact with the head and anal cuticle was avoided. MpTs were gently drawn out from the saline drop and then the open ends of MpTs were wrapped around the pin with the help of fine forceps. The preparation was left for 2 h – the maximum time the system remained stable. The change in volume of the initial and final drop were measured as volume (v)=(π x diameter3)/6, and the rate of water absorbance by the CNC was calculated by the following formula: Jfluid=Δv/Δt, where Jfluid is the fluid reabsorption rate (nl/min), Δv is the change in volume (nl) and Δt is the duration of the experiment (min).
Acknowledgements
We thank Matt Benton for the Tribolium stock; the Bloomington Drosophila Stock Center and Resource Center (National Institutes of Health grants P40OD018537 and 2P40OD010949-10A1) and the Vienna Drosophila Resource Center (VDRC) for Drosophila stocks; Julian Dow and Anthony Dornan for Clc-a-Gal4 and anti-Clc-a antibody; and Anisha Kubasik-Thayil (IMPACT imaging facility, University of Edinburgh) for assistance with imaging.
Footnotes
Author contributions
Conceptualization: R.B., K.V.H., B.D.; Methodology: R.B., B.D.; Validation: R.B., B.D.; Formal analysis: R.B., K.V.H., B.D.; Investigation: R.B., T.K., M.T.N., K.V.H., B.D.; Resources: K.V.H., B.D.; Writing - original draft: R.B., B.D.; Visualization: R.B., K.V.H., B.D.; Project administration: B.D.; Funding acquisition: R.B., K.V.H., B.D.
Funding
This work was funded by the Leverhulme Trust (RPG-2019-167), the Carnegie Trust for the Universities of Scotland (70425), the Biotechnology and Biological Sciences Research Council (BB/N001281/1 and BB/X014703/1), the Villum Fonden (15365), the Danmarks Frie Forskningsfond (9064-00009B), the Ragna Rask-Nielsens Foundation (KH0622) and by a research infrastructure grant from Carlsbergfondet (CF19-0353). Open Access funding provided by the University of Edinburgh. Deposited in PMC for immediate release.
Data availability
All relevant data can be found within the article and its supplementary information
Peer review history
The peer review history is available online at https://journals.biologists.com/dev/lookup/doi/10.1242/dev.202994.reviewer-comments.pdf
Special Issue
This article is part of the Special Issue ‘Uncovering developmental diversity’, edited by Cassandra Extavour, Liam Dolan and Karen Sears. See related articles at https://journals.biologists.com/dev/issue/151/20
References
Competing interests
The authors declare no competing or financial interests.