Open Access Research article

Desert hedgehog is a mammal-specific gene expressed during testicular and ovarian development in a marsupial

William A O'Hara1, Walid J Azar2, Richard R Behringer3, Marilyn B Renfree24 and Andrew J Pask1234*

Author Affiliations

1 Department of Molecular and Cellular Biology, The University of Connecticut, Storrs CT 06269, USA

2 Department of Zoology, The University of Melbourne, Victoria 3010, Australia

3 Department of Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA

4 The Australian Research Council Centre of Excellence in Kangaroo Genomics, Australia

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BMC Developmental Biology 2011, 11:72  doi:10.1186/1471-213X-11-72

Published: 1 December 2011

Additional files

Additional file 1:

Primes used to PCR clone and check splice variants of the genes described.

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Additional file 2:

Table of full-length hedgehog and PTCH sequences used for phylogenetic analyses.

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Additional file 3:

a. Schematic diagram of the alternative splice variants detected for tammar wallaby PTCH2 relative to the human PTCH2 structure. Primers spanned exons 16-22 (red bar) and 7 splice variants (including the full length transcript) were isolated. Tammar PTCH2 has two additional introns in exon 17 and 22 (blue arrow heads) and one additional exon (21-Me; red arrow). b. Table showing the relative homologies of the epitope to which the DHH antibody was raised (recombinant mouse (Rm) Dhh amino acids 199-396) to tammar wallaby DHH, SHH and IHH. Homology is significantly lower with SHH and IHH. c. Western Blot of DHH antibody a band at 43 kDa, which is the predicted size of the tammar wallaby DHH protein in its uncleaved form. Antibody cross-reactivity with SHH or IHH would create bands at 48 and 45 kDa respectively.

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Additional file 4:

Alignment of tammar Dhh protein sequence with four eutherian mammals. Dark shading indicates agreement in at least 60% of the sequences, light shading indicates amino acid similarity to consensus. Double dashed area represents conserved sequence necessary for secreted DHH. Asterisked region represents conserved catalytic site.

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Additional file 5:

Alignment of tammar Ptch1 protein sequence with four eutherian mammals. Dark shading indicates agreement in at least 60% of the sequences, light shading indicates amino acid similarity to consensus. Double dashed areas represent putative trans-membrane binding domains, with species (Ptch1 unless indicated) showing highest sequence identity indicated in parentheses. Any conserved domains are mentioned above the relative sequence. Glycosylation sites are denoted with a cross.

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Additional file 6:

a. Alignment of tammar Ptch2 protein sequence with four eutherian mammals. Dark shading indicates agreement in at least 60% of the sequences, light shading indicates amino acid similarity to consensus. 70 amino acid stretch maintained in Tammar is italicized. Double dashed areas represent putative trans-membrane binding domains, with species showing highest sequence identity indicated in parentheses. Any conserved domains are mentioned above the relative sequence. b. Alternative splice variants of PTCH2. Primers were designed to span the region corresponding to exons 18-22 of the human PTCH2 gene. RT-PCR was carried out in day 3, 7 and 14 post partum testes. Day 3 PCR produced four bands of ~1.2 Kb, 1.05 Kb, 950 bp and 860 bp. We sequence verified that the 1.2 Kb fragment was the full-length transcript and that the 950 bp transcript was a Δ-21a PTCH2 isoform. The identity of the missing exons in the 1.05 Kb and 860 bp fragments is shown in Additional File 9. These slice variants were developmentally regulated, with the smaller two isoforms not seen in the day 7 or 14 testis and the larger two isoforms appear to change in their relative abundance between stages.

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Additional file 7:

a-c. Phylogenetic trees showing divergence of DHH(A), PTCH1(B), and PTCH2(C) in model organisms in which the genes have been completely sequenced. Mm = mouse, La = elephant, Me = tammar wallaby, Cf = dog, Tt = dolphin, Ss = pig, Gg = gorilla, Hs = human, Pp = Chimpanzee, Bt = cow, Dr = zebrafish, Tn = Tetraodon, Ol = Oryzias. Zebrafish is included in b and c as a known outlier.

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Additional file 8:

Phylogenetic tree showing clustering of all complete sequenced HH proteins (Indian (IHH), Sonic (SHH), desert (DHH), Echidna (EHH), TwiggyWinkle (TWHH)). EHH and TWHH each contain only 1 member, and have both been shown to cluster within IHH and SHH groups respectively. The fish DHH orthologues (FHH) form a separate cluster from the mammalian DHH genes. EHH, TWHH and reported fish DHH orthologues (FHH) are highlighted in red. Node labels are in the format: PROTEIN_Genus_species.

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Additional file 9:

Immunohistochemistry of DHH, PTCH1 and PTCH2 in the tammar wallaby testis at key developmental time points. Red/brown staining indicates protein distribution while the heamatoxalin counterstain appears blue. It is important to note that DHH is a highly secreted molecule and staining does not imply cell of origin. DHH was initially present at high levels throughout the indifferent gonad (d24 fetus), by D1pp Dhh is confined to the aggregating seminiferous cords (AC). Scale bars = 36 μm.

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Additional file 10:

Immunohistochemistry of DHH, PTCH1 and PTCH2 in the tammar wallaby ovary at key developmental time points. At day 9pp when the ovary is forming a cortex and medulla, there was widespread staining for DHH, PTCH1 and PTCH2 throughout the ovary. By D72pp DHH and PTCH1 were concentrated in the germ cell nests (CGN) and PTCH2 was largely in the interstitium. In the adult ovary, DHH was found in the granulosa cells (GC) of follicles at all stages of development, and in the oocyte cytoplasm. Staining was also observed in the corpus luteum (CL). Scale bars = 40 μm at D9, D72 and 160 μm in the corpus luteum.

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