Desert hedgehogis a mammal-specific gene expressed during testicular and ovarian development in a marsupial
- William A O'Hara†1,
- Walid J Azar†2,
- Richard R Behringer3,
- Marilyn B Renfree2, 4 and
- Andrew J Pask1, 2, 3, 4Email author
© O'Hara et al; licensee BioMed Central Ltd. 2011
Received: 6 June 2011
Accepted: 1 December 2011
Published: 1 December 2011
Desert hedgehog (DHH) belongs to the hedgehog gene family that act as secreted intercellular signal transducers. DHH is an essential morphogen for normal testicular development and function in both mice and humans but is not present in the avian lineage. Like other hedgehog proteins, DHH signals through the patched (PTCH) receptors 1 and 2. Here we examine the expression and protein distribution of DHH, PTCH1 and PTCH2 in the developing testes of a marsupial mammal (the tammar wallaby) to determine whether DHH signalling is a conserved factor in gonadal development in all therian mammals.
DHH, PTCH1 and PTCH2 were present in the marsupial genome and highly conserved with their eutherian orthologues. Phylogenetic analyses indicate that DHH has recently evolved and is a mammal-specific hedgehog orthologue. The marsupial PTCH2 receptor had an additional exon (exon 21a) not annotated in eutherian PTCH2 proteins. Interestingly we found evidence of this exon in humans and show that its translation would result in a truncated protein with functions similar to PTCH1. We also show that DHH expression was not restricted to the testes during gonadal development (as in mice), but was also expressed in the developing ovary. Expression of DHH, PTCH1 and PTCH2 in the adult tammar testis and ovary was consistent with findings in the adult mouse.
These data suggest that there is a highly conserved role for DHH signalling in the differentiation and function of the mammalian testis and that DHH may be necessary for marsupial ovarian development. The receptors PTCH1 and PTCH2 are highly conserved mediators of hedgehog signalling in both the developing and adult marsupial gonads. Together these findings indicate DHH is an essential therian mammal-specific morphogen in gonadal development and gametogenesis.
Desert hedgehog (DHH) is a member of the hedgehog gene family which act as secreted intercellular signal transducers . Hedgehog was first identified as a segment polarity gene in Drosophila and has since been identified as a key regulator of pattern formation in embryonic and adult development in many vertebrate and invertebrate species. In addition, the hedgehog gene has undergone duplications in both invertebrates and vertebrates to produce a number of orthologues [2–6]. Mammals have three hedgehog orthologues, Sonic (Shh), Indian (Ihh), and desert (Dhh) hedgehog [3, 4, 7–9]. All three share a striking homology with the Drosophila orthologue . Each mammalian orthologue has a unique, largely non-overlapping expression pattern, except in the gut where Ihh and Shh are co-expressed, and in the adult ovaries where Ihh and Dhh are co-expressed [11, 12]. Shh has an essential role in early fetal development, and is required for correct formation of the limbs, phallus, somites and neural tube [8–10]. Ihh has a more restricted developmental role, and is essential for chondrocyte development . Dhh is essential for the maintenance of the male germ line and spermatogenesis . Dhh is also expressed in Schwann cells, and appears to play a role in nerve sheath formation [15, 16].
Hedgehog actions are mediated at the cell surface by a multi-component receptor complex comprising the patched (PTCH) receptors and smoothened (SMO) protein . Both proteins have orthologues in Drosophila that are also involved in hedgehog signal transduction and pattern formation. Initially, all three hedgehog protein functions were thought to be mediated through the PTCH1 receptor in mammals. However, a second orthologue was identified, PTCH2, which shares significant sequence homology with PTCH1. PTCH1 and PTCH2 both bind all hedgehog family members with similar affinities, and each forms a complex with SMO. However, the expression patterns of PTCH1 and PTCH2 do not entirely overlap, suggesting some degree of functional specialization . While PTCH1 is widely expressed throughout the mouse embryo, PTCH2 is most predominant in the skin and in testis .
In the mouse, Dhh is expressed in the presumptive testis from E11.5 through to adult stages. Dhh was initially thought to be specifically expressed in the testis in the pre-Sertoli cells and so it was suggested it may be directly modulated by Sry [1, 10, 19] or its downstream partner Sox9 [20–22]. Dhh-null male mice are sex reversed [21, 23], their gonads are small and devoid of sperm and the mice develop as phenotypic females due to a lack of male steroid hormone production [20, 21]. Leydig cell numbers are dramatically decreased in the Dhh null gonad, but are not entirely absent . Dhh appears to be an important regulator of Leydig cell development since its over-expression in the somatic cells can induce Leydig cell development . In contrast to the Dhh-null males, female mice lacking the Dhh gene develop normally and are fertile . While Dhh does not appear to be important for ovarian development, Dhh mRNA has been detected in the granulosa cells of preantral and antral follicles suggesting it can be activated in an SRY-independent manner [1, 12, 24–26]. In humans, mutations in DHH cause a range of phenotypes in 46, XY male patients from mild  to complete gonadal dysgenesis, including bilateral streak gonads, normally developed Müllerian ducts, and female external genitalia .
The majority of genes involved in testicular differentiation are highly conserved among the vertebrates . However, no DHH orthologue has been identified in birds, although a paralogue (annotated as DHH) is present in the zebrafish (Danio), anole lizard (Anolis) and African clawed frog (Xenopus) genomes http://www.ensembl.org. Here we describe high conservation of DHH, PTCH1 and 2 in the genome of a marsupial, the tammar wallaby (Macropus eugenii). Marsupials, have been evolving independently of eutherian mammals for 130- 148 million years . To determine when DHH evolved its role in mammalian testicular development, we examined the expression of DHH and its receptors PTCH1 and 2 during marsupial gonad development.
Tammar wallabies of Kangaroo Island, South Australia origin were maintained in our breeding colony in open grassy enclosures. Husbandry, handling and experiments were in accordance with the National Health and Medical Research Council of Australia/Commonwealth Scientific and Industrial Research Organization/Australian Research Council (1990) guidelines and approved by the University of Melbourne Animal Experimentation Ethics Committees.
Pregnancies were initiated by removal of the pouch young (RPY) from animals carrying a blastocyst in embryonic diapause [31, 32]. The day of RPY is designated Day 0 of pregnancy and births occur 26-27 days later . Fetuses were removed from the uterus, dissected and tissues snap frozen in liquid nitrogen for mRNA expression analyses or fixed in 4% paraformaldehyde overnight at 4°C for protein localization. The sex of the fetus or pouch young was determined by the presence of scrotal bulges in males or mammary primordia in females  or by PCR for SRY . The developing gonadal ridge first becomes apparent around day 22 of gestation in the fetal tammar wallaby . We examined gonads from day 23 of gestation through to adulthood in both males and females.
Cloning of a tammar wallaby Dhh, PTCH1 and PTCH2
A full length DHH cDNA clone was isolated from a lambda-Zap II cDNA library (constructed from total RNA combined from male and female day 5 postpartum pouch young). The cDNA library was constructed by Clontech Laboratories. Libraries were plated to a density of 120, 000 plaque-forming units per 22 × 22 cm Nunc plate. The library was screened with a full-length mouse Dhh cDNA probe at 60°C in Church's buffer .
PTCH1 and 2 sequences were partially isolated from the tammar wallaby genome trace-archives http://www.ensembl.org. PCR and RACE was used to fully characterise the open reading frame (primers listed in Additional file 1) of each receptor. PCR fragments were cloned into p-GEM-T-Easy plasmid (Promega) and sequenced to verify. RACE was performed according to manufacturers instructions (Roche).
Complete cDNA sequences for annotated, full-length hedgehog and patched family members were obtained from the Ensembl database (Ensembl identification numbers listed in Additional file 2) and aligned using MUSCLE http://www.ebi.ac.uk. Trees with fewer than 20 nodes were constructed by aligning sequences using MUSCLE and assembling tree with Mr.Bayes 3.1.2 at 100, 000 generations, sampled every 100 with burnin set to the first 250. Larger trees were aligned with TCoffee http://www.ebi.ac.uk and assembled with Mr.Bayes 3.1.2 at 1, 000, 000 generations, sampled every 1000 with burnin set to the first 250. All trees were viewed and ordered in FigTree 1.3.1.
Total RNA was extracted from frozen tissues using the GenElute Total RNA mini kit (Sigma). RNA was DNase treated using the DNAfree reagent (Ambion). DNA free RNA was reverse transcribed using a dT primer and the superscript III kit according to manufacturers instructions (Invitrogen). All RT-PCR derived products were sequence verified as described above.
Tissues were fixed in 4% PFA overnight and embedded in paraffin wax according to standard methods. Deparaffinised tissue sections (8 μm) were treated with 3% hydrogen peroxide in methanol for 5 min to quench endogenous peroxidase activity. Antigen retrieval was achieved by placing slides in boiling 0.05 M Tris HCL pH 9.0 for 20 min. Primary antibodies were applied to sections as follows; DHH (anti-mouse DHH, goat polyclonal IgG, R&D Systems, cat.#AF196), 1:400, PTCH1 (anti-human PTCH1, rabbit polyclonal IgG, Abcam, cat.#AB39266) 1:400 and PTCH2 (anti-human PTCH2, rabbit polyclonal IgG, Lifespan BioSciences, cat#LS-B301) 1:400. The DHH antibody was verified by Western Blot to detect a single protein of 43 kDa (the predicted size of the tammar wallaby DHH protein; result not shown Additional file 3). Furthermore, DHH is the primary hedgehog ligand expressed in the marsupial gonad . While the PTCH1 and 2 antibodies were not validated by western blot in the tammar wallaby, the PTCH1 and PTCH2 antibodies were raised against epitopes that share 99 and 100% homology respectively with the analogous tammar target proteins, but that show no cross reactivity. In addition, no other homologous sequences exist within the tammar genome with which the antibody could non-specifically cross react . Primary antibodies were incubated at 4°C overnight. Depending on the primary antibody, the secondary antibody was either an anti-goat IgG raised in rabbit (Millipore) or an anti-rabbit IgG raised in goat (Santa Cruz Biotechnology), both conjugated to biotin and used at a 1:250 dilution. Signal was amplified using the ABC kit (Vector Labs) and visualised using 3-amino-9-ethylcarbazole (AEC) (Vector Labs) and sections were counterstained with haematoxylin. Immunohistochemistry was performed on at least 3 independent samples at each stage. Negative controls were performed as above with the primary antibody omitted.
Fluorescence In SituHybridisation Mapping
Chromosomes were prepared from peripheral blood according to standard methods . Chromosome in situ suppression (CISS) hybridisation of the genomic lambda clones was performed, with minor modifications . The largest DHH cDNA clone was labelled with digoxigenin using nick translation. The labelled probe was incubated with 50 ng of tammar wallaby Cot1 DNA before hybridisation to tammar-fixed metaphase chromosome spreads for 48 h at 37°C. Slides were washed in 0.2 × SSC at 60°C and hybridisation detected with anti-DIG-mouse antibody (Serva), followed by tetramethyl rhodamine iso-thiocyanate (TRITC) conjugated goat-anti-mouse antibody (Serva). Following hybridisation, the slides were counterstained with DAPI (4, 6-diamidino-2-phenylindole) to visualise the chromosomes.
RT-PCR was carried out according to standard methods using oligo dT primed cDNA (superscript III, Invitrogen) and 30 PCR cycles. No-template negative controls were included in each round, and in each case showed no amplification (data not shown). A single sample was used for each amplification.
Quantitative PCR was similarly performed on oligo dT primed, reverse transcribed mRNA was prepared as outlined above. Quantitative PCR was carried out using the IQ Sybr green master mix (BioRad) in 20 ul reactions using primers for tammar DHH, PTCH1 and PTCH2 (Table 1) and normalised to β-actin as previously described . Each PCR was performed on a single sample in triplicate (except for the adult ovary which was verified on three independent samples) and only those samples with a standard deviation of less than 1 cycle considered for analyses. Relative changes in gene expression were analysed using the methods described by Pfaffl [39, 42]. The variance in cycle threshold (ΔCt) was normalised across samples (with beta actin levels set to 100%) and inversely plotted as a percentage of the earliest ΔCt.
Isolation of the tammar wallaby DHHcDNA
Eight independent cDNA clones of DHH were isolated from the tammar wallaby pouch-young cDNA library. The two cDNA clones containing the longest 5' and 3' UTRs and overlapping with each other in the coding region were sequenced. The combined length of the two DHH clones was 2.38 kb with a predicted open reading frame of 1, 197 bp. This encoded a protein of 399 amino acids with a predicted molecular weight of 44 kD. There were several common features between the predicted tammar DHH protein and that of mouse . A short stretch of N-terminal residues (25 for the tammar and 22 for the mouse) were highly hydrophobic and presumed to function as a signalling peptide. There was a conserved 6 amino acid stretch, CGPGRG, after the signal peptide used to generate the secreted form of DHH. Drosophila hedgehog protein and vertebrate Shh, and Dhh proteins are processed into two smaller secreted peptides by an auto-proteolytic process, both in vitro and in vivo . The catalytic site, GCF, was conserved in the tammar DHH protein, suggesting a similar auto-proteolytic mechanism may also occur to produce a 19.9 kD N-terminal peptide (amino acids 1-200) and a 21.6 kD C-terminal peptide (amino acids 201-403). Furthermore, the tammar and mouse DHH proteins share 96% identity in the N-terminal peptide and 94.5% in the C-terminal peptide, implying that both regions have been highly conserved during evolution (Additional file 4).
PTCH1 and 2 were partially isolated from the tammar wallaby genome http://www.ensembl.org. PCR and RACE was used to fill in missing portions to complete the open reading frame for each gene. PTCH1 was seven amino acids longer than its human orthologue and shares 83% sequence homology and 96% amino acid similarity. Conservation was particularly high (85%-100%) in the transmembrane domains and there was complete conservation of the putative glycosylation sites (Additional file 5).
Phylogenetic relationships of DHH, PTCH1 and PTCH2 to eutherian and non-mammalian vertebrates
A phylogenetic analysis of DHH sequences grouped them into four main clusters, one for primates, another elephant, mouse, and hyrax, a third for pig, dog, and dolphin; tammar was an outlier (Additional file 7a). These groupings are largely consistent with the accepted mammalian phylogenetic tree and all eutherian clusters show equal divergence.
The PTCH analyses were less inclusive as only a few species had complete open reading frame sequences available. However, the PTCH1 phylogenetic tree mimicked standard mammalian groupings, with the tammar clustering with other mammals, and zebrafish as the outlier (Additional file 7b). Phylogenetic analysis of PTCH2 grouped tammar PTCH2 with that of other mammals, but as the most divergent lineage. PTCH2 was more divergent between species than DHH or PTCH1 (Additional file 7c).
All full-length proteins used for phylogenetic analyses were obtained from Ensembl and are listed in Additional file 2.
Genomic localisation of the tammar wallaby DHHcDNA
Expression of DHHin the tammar wallaby
A limited quantitative real-time PCR profile confirmed the presence of DHH at all stages of gonad development in both testes and ovaries (Figure 4b-d). DHH mRNA levels were similar in the developing male and female gonads at all stages examined, but in adult gonads ovarian expression was low (Figure 4b). PTCH1 expression was consistent throughout gonadal development in males and females, with lowest levels seen in the adult ovary (Figure 4c). In contrast, PTCH2 levels remained constant in the gonad of both males and females at all stages examined including in the adult (Figure 4d). All three genes were also expressed in the prostate and adrenal albeit at lower levels than in the developing gonads.
DHH, PTCH1 and PTCH2 protein distribution during gonadogenesis
We have shown that DHH is a highly conserved mammal specific hedgehog paralogue with conserved expression during mammalian gonadogenesis. DHH and its receptors PTCH1 and 2 are highly conserved at the protein level and are expressed in an analogous pattern to that seen in the mouse gonad. However, DHH was expressed in the developing marsupial ovary in contrast to the mouse, in which it is testis-specific during development.
Phylogenetic analysis of the hedgehog gene family across vertebrates shows that non-mammalian DHH genes in fish form a distinct subgroup, distantly related to mammalian DHH genes, indicating they have had an independent evolutionary origin. We have re-named this sub-group fishy hedgehog (FHH) to emphasise their distinction from the DHH genes. This suggests that the evolution of mammalian DHH is a recent event (Figure 2) making it quite unique among the gonadal differentiation genes, all of which have orthologues in the non-mammalian vertebrates with the notable exception of the sex determination switch gene SRY, which is also mammal specific . Despite its recent origin, DHH was extremely highly conserved between marsupials and eutherians, suggesting it quickly adopted an essential function in mammalian reproduction.
The hedgehog receptors PTCH1 and 2 were highly conserved between marsupials, eutherians and non-mammalian vertebrates. Marsupial PTCH2 was the most divergent (especially in the C-terminal region, consistent with findings in other vertebrate species ) but still shared 89% amino acid similarity with eutherian orthologues. The tammar PTCH2 C-terminus contained a 70 amino acid additional exon not found in eutherian PTCH2 proteins. Interestingly, significant homology to the additional tammar exon was identified in the human PTCH2 genomic sequence, in intron 21, and shared 70% identity at the nucleotide level and 79% amino acid similarity with the tammar additional exon (hereafter referred to as exon 21a). The level of conservation of this exon between marsupials and humans was much higher than that of non-functional intronic DNA, suggesting functional conservation of the sequence. Translation of the human sequence revealed a premature stop codon at amino acid 35, so its inclusion in the transcript would lead to a PTCH2 receptor with a severely truncated intracellular signalling domain (Figure 1b). Such an isoform, lacking exon 22, identical to the one predicted from the inclusion of the human putative exon 21a, has been previously identified (the Δ-22 isoform). The human Δ-22 PTCH2 isoform is the only one capable of acting as a strong inhibitor of SHH induction, similar in function to PTCH1 . It appears that the ability to produce such an isoform was derived from a stochastic nonsense mutation in the original exon 21a leading to a truncated protein. The tammar does not have a premature stop (exon 21a is an intact ORF) and so this tammar PTCH2 isoform does not share redundancy with PTCH1 function. The degree of conservation of this region in humans suggests that it has only recently become non-functional in primate evolution. It is intriguing then, that this sequence could not be identified in any other mammalian PTCH2 loci, but only in the tammar, opossum, and human. These findings suggest that the exon was present in the ancestral PTCH2 gene and has been independently lost in different eutherian lineages (Figure 1c). We also identified several PTCH2 isoforms that appear to be dynamically regulated at specific developmental time points. This is also consistent with findings in humans that identified PTCH2 isoforms lacking exons 9 and 10 (PTCH2-Δ9-10). Taken together, these data suggest that PTCH2 has divergent species-specific roles in development, while PTCH1 is likely to maintain a highly conserved function in hedgehog signal transduction. Furthermore, it suggests that the human PTCH2 Δ-22 isoform may have evolved to compensate for a loss of PTCH1 in tissues in which they are co-expressed.
DHH, PTCH1 and PTCH2 mRNA and protein were present throughout gonadal development in both males and females, from early development through to adult stages. The presence of ligand and both receptor proteins throughout gonadal development is consistent with findings in mouse testis, but not ovary  and suggests a conserved role for hedgehog signalling in mammalian gonad formation. These findings are also consistent with the observed disruption to normal gonadal patterning and significant reduction in the expression of the downstream target gene GLI1, in the tammar when hedgehog signalling is ablated in vitro .
In the testis, DHH could be seen within the pre-Sertoli cells of the aggregating cords. Once the testes differentiated, DHH staining was concentrated in the Sertoli cells, especially at the basal lamina of the cord. This protein distribution is similar to that reported in mouse [49, 24, 49] and suggests it is critical for testicular patterning. However, there were some differences in PTCH distribution from predicted mouse patterns. PTCH1 staining was similar to that of DHH, and was distributed mainly within the seminiferous cords (containing germ cells and Sertoli cells). This was in contrast to the interstitial expression seen for Ptch1 in the developing mouse testis  but similar to the expression of Ptch2 . Conversely, PTCH2 staining in the tammar was more reminiscent of Ptch1 distribution in the mouse testis  and was located throughout the gonad but concentrated in the interstitium and Leydig cells. This suggests there may have been a reversal in the roles of these receptors in marsupial testicular development relative to the mouse. Since detailed localisation of the PTCH receptors during gonad development in other mammals and vertebrates is not available we cannot determine which profile is more typical during development.
There was discrete staining of DHH, PTCH1 and PTCH2 proteins in the adult testis. DHH was concentrated in the differentiating germ cells, but restricted to the post pachytene primary spermatocyte stage through to the mature sperm. There was faint PTCH1 and PTCH2 staining throughout the testis but the proteins were concentrated in the Leydig cells and Sertoli cells respectively. This is consistent with in situ results in the adult mouse testis , suggesting a conserved role for these genes in maintaining testicular function and spermatogenesis in all therian mammals.
Unlike in the mouse, in which Dhh is testis-specific in early development , DHH was expressed in the developing tammar ovary throughout development. Activation of hedgehog signalling in the developing mouse ovary leads to Leydig cell development . However, early ovarian development was not affected by the presence of DHH in the tammar, despite the presence of similar mRNA and protein levels of both ligand and receptors as in the developing testis. These findings show that SRY is not needed for DHH activation in the developing gonad. In the juvenile ovary, DHH was abundant in the oocytes consistent with the suggested role for DHH in maintaining the germ line . In the adult ovary, DHH was broadly co-localized with PTCH1 and 2, in follicles and the corpus luteum suggesting it may be important for normal folliculogenesis and steroidogenesis, consistent with recent findings in the mouse . As in the testis, PTCH2 appeared to be the predominant receptor throughout ovarian development and in the adult.
These data support a conserved role for hedgehog signalling in gonadal development but show that in marsupials this pathway may be significant for early patterning of the ovary as well as for the testis. These results, in conjunction with our phylogenetic analysis of hedgehog family members in all vertebrates, suggests that DHH is unique to mammals and is a conserved member of the gonadal development pathway.
We thank Drs Chai-an Mao, Jenny L. Harry and Deanne J. Whitworth for help with the initial cloning of the DHH gene in the tammar wallaby. We also thank Kerry Martin and Scott Brownlees for assistance with the animals. This study was supported by The University of Connecticut Faculty Large Grant, a National Health and Medical Research Council project grant to MBR and AJP, an R D Wright Fellowship to AJP, the Australian Research Council Centre of Excellence in Kangaroo Genomics, a Federation Fellowship to MBR and an NIH research grant (HD30284) to RRB. There is no financial or other potential conflict of interest.
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