Abstract
The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention1,2, little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the two anatomical structures3-6, yet without providing any underlying developmental mechanism. Here, we have reexamined this question using micro-Computed Tomography (μCT), lineage tracing in three amniote clades and RNA-Seq transcriptional profiling. We show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing center. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, it suggests that a change in the relative position of the cloacal signaling center during evolution has led to an altered developmental route of external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry due to a common evolutionary origin.
To investigate potential interdependencies in the development of limbs and external genitalia, we first determined the location of the two structures during embryogenesis. We focused on mouse4,5 and squamates (lizard and snakes), which show progressive limb-reduction7, yet maintain their external genitalia, the hemipenes8. μCT-reconstructions of mouse, anole lizard (Anolis), python and house snake embryos revealed different anterior-posterior locations of the developing external genitalia, relative to limbs. In mice, the genital tubercle is positioned caudal to the hindlimbs (Fig. 1a), whereas in squamates the paired hemipenes bud from the limbs, or remnants thereof (Fig. 1b-d). The cloaca, a signaling center important for genitalia development6,9,10, is similarly located within the limb-field of squamates (Fig. 1f-h) and expresses Shh (Fig. 1j-l). Thus in squamates all three anatomical structures, limb, hemipenis and cloaca, align at the same anterior-posterior position.
We decided to investigate whether these positional differences would reflect distinct developmental origins of external genitalia. While the cells of the vertebrate limb bud arise through an epithelial-to-mesenchymal transition (EMT) of an epithelial lateral plate mesoderm (LPM) population lining the coelomic cavity11, the developmental origin of external genitalia in vertebrates is still unclear. We developed a lenti-viral lineage tracing approach (see Methods), to systematically follow the two sources previously proposed, the LPM and tail bud10,12,13, in three amniote species, mouse, chicken and anole. Injections into the coelom of embryonic day 9.5 (E9.5) mouse embryos label cells surrounding the coelom as well as the developing hindlimb (Fig. 2a). However, no GFP-positive cells are observed in the genital tubercle, with a sharp boundary of labeled cells extending from the coelomic cavity (Fig. 2a,b). In contrast, injection into the posterior mesenchyme of mouse embryos labels the genital tubercle (Fig. 2c). Tailbud injections label the posterior half, whereas the infra-umbilical mesenchyme gives rise to its anterior part12-14 (Extended Data Fig. 1). In chicken, coelomic injection into stage HH14 embryos labels cells in both limb and genital tubercle (Fig. 2d,e), without any obvious boundary. Tailbud infection also results in GFP-positive genitalia cells, mostly in the posterior tubercle, suggesting that multiple lineages contribute to this species’ genitalia (Fig. 2f). Similar conclusions were reached in a parallel study (Embryonic origin and compartmental organization of the external genitalia, Herrera A. M. & Cohn M. J.). In Anolis, coelomic injections at stage 2-3 result in GFP-positive cells in the limb and the developing genitalia (Fig. 2g,h), whereas no labeled cells are seen in the hemipenes following tailbud injections (Fig. 2i).
As for chicken and mouse limbs11, cells of the Anolis limb, but also the hemipenis, originate via an EMT of the coelomic epithelium (Extended Data Fig. 2a,b). In snakes, we find evidence for similar cellular dynamics. The hemipenes emerge as small buds at a “limb-like” lateral position, juxtaposed to the coelomic cavity (Extended Data Fig. 2c-f). A concomitant basement membrane breakdown, consistent with an EMT of the LPM, is seen in the budding of both mouse limbs and snake hemipenes (Extended Data Fig. 2d,f). Moreover, we find that Tbx4, a gene important for hindlimb development15 is expressed from early on, in both the coelomic epithelium and the mesenchyme of the developing hemipenis (Extended Data Fig. 2g-i). Its forelimb counterpart Tbx5 is expressed later, in the mesenchyme only, in agreement with its genitalia-expression in mammals16 (Extended Data Fig. 2j-l). This suggests that squamate external genitalia initiate with limb-like cellular dynamics, the resulting mesenchymal cell population in modern snakes being converted to a genital fate17. Thus, important differences exist in the developmental origins of external genitalia in amniotes: chicken genitals originate from both LPM and the tailbud, whereas the mouse genital tubercle consists of infra-umbilical mesenchyme and tailbud-descendant cells. In contrast, the hemipenis shares a developmental route with hindlimbs, either through secondary budding as in lizards, or by entirely recruiting the mesenchymal cell population to a genital fate in modern snakes. Given the impact of developmental lineage on an organ’s molecular architecture, we next explored the transcriptomes of emerging genitalia, in the two opposing routes of mammals and squamates.
Gene expression profiling has successfully been applied to questions of developmental and evolutionary origin, of cell types and entire morphological features18-21. We thus dissected early and late stages of developing limbs and genitalia from mouse and anole embryos (Fig. 3a), for comparative RNA-seq analyses. Overall transcriptome similarities were assessed using multidimensional scaling (MDS; Fig. 3b). The transcriptomes dominantly resolve along dimension 1 in a species-dependent manner, as expected for similar tissues in evolutionary distant species22. Dimension 2, however, contains a clear organ identity signal, i.e. limb versus genitalia. This separation is virtually absent in Anolis samples, compared to mouse, reflecting similar transcriptional programs due to a common developmental origin for genitalia and hindlimbs. For hierarchical clustering, we included the early tail bud, as outgroup of the primary body axis, and forelimbs, to account for anterior-posterior differences in the two species’ genitalia (Fig. 3c,d; see Methods). In Anolis, the early hemipenis transcriptome falls within the limb clade, indicative of an almost generic limb molecular architecture, and only later differentiates into a more organ-specific signature (Fig. 3c and Extended Data Fig. 3a). In contrast, mouse genitalia transcriptomes are clearly distinct from limbs, from early on, highlighting the separate developmental origins of the two organs (Fig. 3d and Extended Data Fig. 3b).
To identify genes driving hindlimb- and genitalia-specific transcriptome separation, we used principal component analysis. Principal component 1 (PC1) correlates with species differences, while organ specificity is resolved along PC2 (Fig. 3e), allowing us to identify organ-specific ‘driver’ genes in a species-independent manner. We assessed the contribution of orthologous genes to PC2, according to their absolute loading values (Fig. 3f). Gene Ontology (GO) analysis23 of the top500 genes (Fig. 3f and Supplementary Table 1) identified GO-terms related to transcription factors (TFs) and signaling molecules (Fig. 3g). Gene regulatory networks thus determine limb versus genital organ transcriptomes, but also mirror their developmental origin. Strikingly, TFs/signaling molecule data is sufficient to reproduce the clustering seen with whole-transcriptomes (Fig. 3h,i and Extended Data Fig. 4a,b). Collectively, we find a clear distinction between mouse genitalia and limb transcriptomes, during early and late organogenesis. Such genitalia-specific separation is only seen in late Anolis hemipenes, arguing for a developmental repurposing to a copulatory structure. Importantly, we find transcriptional similarities between early hemipenes and hindlimbs in squamates, illustrating a common developmental origin.
An attractive model for the varied developmental origins of amniote external genitalia would be a repositioning of the cloacal signaling center with respect to different mesenchymal cell populations with progenitor potential. Hence, bringing either hindlimb or tailbud close to the cloaca would allow these lineages to contribute to genital outgrowth. We tested this hypothesis by grafting GFP-transgenic chicken or quail cloacae into the hindlimb bud of wild-type embryos (Fig. 4a and Extended Data Fig. 5a-c). After 1-2 days of incubation, limbs showed ectopic, secondary buds (Fig. 4b,c). GFP-negative cells suggest an inductive effect of the cloaca on the surrounding mesenchyme, rather than simple over-proliferation of the graft itself (Fig. 4c). Similar buds could be induced by grafting beads soaked in known cloacal signaling molecules Shh and Fgf9,10,24,25 (Extended Data Fig. 5d-g). To assess the fate of responding cells, we re-analyzed our RNA-seq data for potential genital versus limb markers. For both species, we performed stage- and organ-specific differential expression analyses. Of the 2003 genes showing an absolute log2 fold-change greater than 1.5 (p-value < 0.05) (Extended Data Fig. 6), we identified 27 that are altered in all four comparisons, 25 of which in the same direction (Fig. 4d). Hierarchical clustering of normalized mouse expression values reveals four stage- and organ-specific signatures, which are largely conserved in Anolis (Fig. 4e). Based on expression patterns and levels (Extended Data Fig. 6 and 7), we chose marker genes to assess transcriptional changes due to ectopic cloacal signals. Indeed, limb cells close to GFP-positive cloacal grafts down-regulate limb markers Lhx9 and Tbx18 (Fig. 4f,g), and ectopically express genital markers Isl126, Gata2 and Runx1 (Fig. 4h and Extended Data Fig. 5j-m). Accordingly, when we graft cloacal tissue into the tailbud mesenchyme, ectopic budding and genital marker expression are equally induced (Fig. 4i-k and Extended Data Fig. 5l-n and 8). Hence, mesenchymal cells from different developmental origins can respond to inductive cloacal signals, generating outgrowths and genitalia-like marker gene expression. Importantly, these results support the idea that changing the relative anterior-posterior position of the cloaca could generate external genitalia with distinct developmental origins during the course of amniote evolution.
In summary, we show substantial variation in external genitalia development in extant amniote species. We propose that repositioning the cloaca can recruit different mesenchymal cell populations, either through spatial or heterochronic changes in the dynamics of their emergence. In squamates, the hindlimb is the dominant source, with modern snakes entirely repurposing a mesenchymal bud to a genital fate. In mice, limbs and genitals have discrete developmental origins, the LPM and the ventral and tailbud mesenchyme, respectively, with chicken showing an intermediate state. Moreover, we find that similarities in limb and genitalia transcriptomes are dependent on the cellular source of the primordia from which they emerge. Specifically, there is a higher degree of early transcriptome congruence in species deriving their intromittent organs from limb anlagen. Strikingly, the ability of different mesenchymal cell populations to respond to cloacal, genitalia-inducing signals seems conserved in extant species. It is therefore tempting to speculate that a limb-derived state could represent the ancestral condition in the evolution of external genitalia, as suggested by their position relative to limbs during turtle development17,27 and the bifid genitalia of basal mammals28,29. As such, a developmental continuity between limbs and genitalia could have turned into an ‘evolutionary continuity’ in mammals, as the two organs spatially separated due to a relative repositioning of the cloaca30. Once shared developmental trajectories could thus help to explain molecular similarities still noticeable in species that develop the two organs from distinct cellular sources3,4,19.
METHODS SUMMARY
μCT-scan reconstructions were used to visualize overall embryonic morphology. For lineage tracing, GFP-expressing lentiviruses were injected into the coelomic cavity or tailbud of mouse, chicken and Anolis embryos. RNA-seq was performed on mouse and Anolis embryonic tissue, using an Illumina HiSeq 2000. Normalized gene read-counts were used for downstream analyses. For grafting experiments, GFP-transgenic chicken or quail cloacal tissue was grafted ectopically into hindlimbs or tailbuds of wild-type recipient chicken. Marker gene expression and grafted tissue were assessed with in situ hybridization and immunohistochemistry. See full Methods for details and any associated references.
METHODS
Tissue sample collection
All embryos were collected in accordance with the appropriate Institutional Animal Care and Use Committee guidelines. Timed-pregnant CD1 and C57BL/6 females were purchased from Charles River. Gravid Anolis females were purchased from Candy Quality Reptiles, La Place, LA (IACUC #26-11 and #28-14). Anole housing and egg incubation were done as previously described31. To collect early stage pre-oviposition embryos, females were euthanized by intraperitoneal Euthasol injection and eggs dissected from the oviduct. Snake husbandry and egg collection have been described before32. Fertilized white leghorns chicken eggs were obtained from Charles River Laboratories and incubated at 38°C. For staging of mouse embryos, noon on the day of the vaginal plug was considered as embryonic day 0.5 (E0.5). For chicken and anole embryos, staging was performed according to Hamburger and Hamilton33 or Sanger and colleagues31, respectively. Embryonic tissue was dissected in cold PBS and either fixed in 4% PFA and processed for cryo-embedding or CT scanning, or else directly processed for RNA extraction or stored in RNAlater (Qiagen).
CT scanning and image processing
Embryos were fixed in 4% PFA and stored in 100% ethanol. Staining was done for 48h in 30% PTA (1% (w/v) phosphotungstic acid in water) and 70% ethanol34. Specimens were rinsed and stored in 70% ethanol until image acquisition. CT scans were carried out on a Bruker Skyscan 1173 or a Nikon (Metris) X-tek HMXST225, at 50-57kV, 115-145uA and 810-1000ms exposure time. Voxel sizes ranged from 0.0023 to 0.0056, with 1500 to 2400 total projections. Post-processing of scan data was done in VGStudio MAX 2.2 (Volume Graphics). For 3D re-construction of the cloaca, serial TIFF stacks produced by VGStudio were read into the Imaris software package (Bitplane) and the endodermal epithelium was used as a guide to manually outline the extent of the cloacal volume.
Immunohistochemistry and In Situ Hybridization
Fixed embryos were embedded in 7.5% gelatin / 15% sucrose or dehydrated in sucrose gradients and embedded in OCT. Sectioning was performed on a Leica CM3000 cryostat. For immunohistochemistry, sections were incubated with primary antibodies in PBST (PBS/BSA 0.2%, Triton 0,1% / SDS 0,02%) overnight, washed 2×10min in PBST and incubated for 1h with secondary antibodies. To detect genital tubercle and ectopic limb Isl1 expression, as well as Lmx1b, signal was amplified using the TSA Plus Cy3 kit (Perkin Elmer). Primary antibodies used were anti β (Santa Cruz), Laminin (Sigma), GFP (Abcam), QCPN, Lmx1b and Isl1 (all Developmental Studies Hybridoma Bank). In Situ Hybridization was performed using standard protocols32,35. Fluorescent images were acquired on a Zeiss LSM10 inverted confocal miscroscope. Bright-field images were acquired on a Nikon Eclipse E1000. Whole-mount images were acquired on a Leica MZ FL III. Images were globally processed for color balance and brightness using Adobe Photoshop.
Lineage tracing analyses
For lentiviral lineage tracing, viral particles harboring ubiquitously expressing GFP cassettes (UbiC-GFP or hPGK-GFP) were produced by transient transfection in 293T cells as described elsewhere36. Viral particles were then injected into either the coelomic cavitiy, or the tailbud mesenchyme of mouse E9.5 embryos, chicken HH14 or Anolis st. 2-3. For mouse experiments, timed-pregnant CD1 females were anaesthetized using isoflurane and surgery, in utero visualization of embryos and virus injection was done as previously outlined37. For chicken embryos, eggs were lowered and windowed and virus injected using a pressure injector. For Anolis lineage tracing, we developed a novel whole-embryo ex ovo culturing system, using media conditions previously described for squamate organ cultures38. Briefly, we prepared culture dishes with an indentation, by pouring 1% Agar Noble (BD Difco) dissolved in culture medium into cell culture dishes containing a modeling clay, egg-shaped casting mold. Once solidified, the mold was removed and st. 2-3 Anolis embryos, dissected in 2× PBS, were placed with their yolk intact in the resulting cavity and covered with culture medium. 10% ink in 1× PBS/PenStrep was mouth-pipetted underneath the embryo, for better visualization, and lentiviral particles were injected using a pressure injector. To increase viral infection rate, embryo plates were kept for 12-16h at 37°C in a humidified chamber, before switching them to 28°C. Additional tailbud injections were performed using DiI. Embryos survived for up to 12 days. Only live specimens showing overall normal morphology were considered for further analysis.
To assess GFP+ cell contribution, embryos were dissected, fixed in 4% PFA, gelatin-embedded and cryo-sectioned. Sections were stained for GFP and imaged on a Zeiss LSM10 inverted confocal miscroscope. For quantifications, 4-5 embryos per condition and species were imaged on multiple sections spanning the respective organs. GFP+ cell counting was performed in ImageJ, using the “ITCN” plugin written by Thomas Kuo (UCSB). A total of 59331 GFP+ cells were counted (mouse: 33853; chicken: 23710, anole: 1768). Counts were averaged over multiple sections and normalized on tissue area measured. The resulting ratios of the two tissues are given as a percentage of total GFP+ cells per area.
RNA sample preparation and sequencing
Total RNA was extracted from freshly dissected or RNAlater-preserved tissue using the Arcturus PicoPure RNA Isolation Kit (Life Technologies) and enriched for mRNA fraction using MPG mRNA Purification Kit (PureBiotech). Multiplexed RNA-seq libraries were produced using the SPIA cDNA synthesis kit and the Ovation Ultralow DR Multiplex system (NuGen). Sequencing was done on an Illumina HiSeq 2000, with eight samples multiplexed per lane. Base calling was performed using the Illumina software. Over 561M 50bp reads were generated, with an average of 23.4 reads per sample (median = 22.0M reads). For all tissues, biological duplicates were sequenced.
Read mapping and transcriptome analyses
Initial read mapping was performed with the RNA-Seq unified mapper (RUM)39, using mouse NCBI37/mm9 and Anolis AnoCar2.0 genome assemblies, with UCSC mm9 refseq and ASU_Acar v2.140 annotation files, respectively. This resulted in 10265 orthologuos genes between mouse and Anolis. An in-house improved annotation for Anolis, generated prior to the publication of Eckalbar et al., yielded concordant results in all downstream analyses. To account for species-specific differences in non-uniquely mapping (NU) reads, we redistributed NU reads based on the number of uniquely mapping (U) reads mapping to the respective loci, following a logic outlined before41. Multidimensional scaling analysis was carried out on normalized read counts using the ‘edgeR’ bioconductor package42. Genes differentially expressed between early fore- and hindlimb samples were determined in ‘edgeR’, and genes showing consistent changes in mouse and Anolis were excluded from further analyses, to dampen potential anterior-posterior differences between the organs. For hierarchical clustering, correlation coefficient and principal component analyses, we calculated ‘transcripts per million’ (TPM)43 values, which were then log2-transformed. Hierarchical clustering was done using the ‘pvclust’ R package44, with 1000 iterations of multi-scale bootstrap re-sampling, and approximately unbiased (AU) p-values are provided in the graph. Heat maps of correlation coefficients were plotted with the ‘lattice’ R package. Principal component analysis was done with the ‘prcomp’ function in the ‘stats’ R package and ‘GOseq’45 was used for GO-term enrichment analysis. Pair-wise differential expression analysis between early and late budding stages, in mouse and Anolis limbs and genitalia, was done in ‘edgeR’, and Venn diagram of genes with an absolute log(fold-change) > 1.5 and p-value < 0.05 was visualized using ‘VennDiagram’46.
Grafting experiments
For heteroptopic, homochronic cloacal grafts, donor and recipient embryos were incubated to reach stage HH17-20. Donor embryos were either GFP-transgenic chicken47, purchased from Clemson University, or quail, purchased from Strickland GameBird Farm. Cloacas were dissected in ice-cold PBS and grafted using tungsten needles to a proximal-ventral position, to mimic the squamate configuration, and removed from the apical ectodermal ridge to avoid Shh-induced digit duplications10, or the tailbud in wild-type recipient chicken. Successful grafts were incubated for 1-3 additional days, dissected and screened for the appearance of ectopic outgrowths. Donor versus recipient tissue was discriminated using either GFP or QCPN antibody staining on cryo-sections, or GFP fluorescence for whole-mount embryos. Sham surgery or grafting of GFP-positive limb mesenchyme did not cause any comparable outgrowths. Cloaca-induced outgrowths never stained positive for Alcian blue at later stages, indicating that they were not digit duplications (data not shown). For bead experiments, Affi-Gel Blue Gel beads (150-300μm; Bio-Rad) were washed in PBS and incubated for 1-2h at room temperature, in PBS with recombinant proteins (SHH, FGF2, FGF8; all R&D Systems) at concentrations of 0.1-1μg/μl. Soaked beads were briefly washed in PBS and grafted to limb and tail buds, as outlined for the cloacal grafts. Control grafts using beads soaked in PBS with bovine serum albumin (BSA) did not yield any observable outgrowths.
Extended Data
Supplementary Material
Acknowledgements
The authors thank D. Duboule, H. Kaessmann, A. Necsulea, B. Okaty, G. Rey and G.P. Wagner for discussions, M.A. de Bakker for the snake Tbx5 probe and A.M. Herrera and M.J. Cohn for discussing and sharing unpublished results. μCT scans were performed at the Center for Nanoscale Systems, Harvard University (supported by NSF award ECS-0335765) and at the Museum of Comparative Zoology. Next-Generation Sequencing was performed at the HMS Biopolymers Facility and computational analyses were run on the Orchestra Cluster, HMS Research Computing. P.T. was supported by post-doctoral fellowships from the Swiss National Science Foundation, EMBO and the Human Frontiers Science Program. A.C.G. was supported by a post-doctoral fellowship from the Swiss National Science Foundation. This work was supported by NIH grant R37-HD032443 to C.J.T.
Footnotes
Supplementary Information is linked to the online version of the paper at www.nature.com/nature.
Author Contributions
P.T., J.G. and C.J.T conceived the project and designed the experiments. P.T. performed most experiments and computational analyses. E.S. prepared CT scans and helped with statistical analyses. T.S. helped with CT scans, Anolis husbandry and embryo collection. A.C.G. produced lenti-viruses and A.C.A. helped with grafting experiments. J.K.H., O.P. and J.G. initiated snake analyses. O.P. contributed snake embryos. J.G. contributed to chick lineage tracing experiments. P.T., J.G. and C.J.T wrote the paper, with comments from co-authors.
Author Information
Sequencing data has been deposited in the Gene Expression Omnibus under accession number GSE60373. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper.
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