Reconstruction and in vivo analysis of the extinct tbx5 gene from ancient wingless moa (Aves: Dinornithiformes)
© Huynen et al.; licensee BioMed Central Ltd. 2014
Received: 15 February 2014
Accepted: 25 March 2014
Published: 14 May 2014
The forelimb-specific gene tbx5 is highly conserved and essential for the development of forelimbs in zebrafish, mice, and humans. Amongst birds, a single order, Dinornithiformes, comprising the extinct wingless moa of New Zealand, are unique in having no skeletal evidence of forelimb-like structures.
To determine the sequence of tbx5 in moa, we used a range of PCR-based techniques on ancient DNA to retrieve all nine tbx5 exons and splice sites from the giant moa, Dinornis. Moa Tbx5 is identical to chicken Tbx5 in being able to activate the downstream promotors of fgf10 and ANF. In addition we show that missexpression of moa tbx5 in the hindlimb of chicken embryos results in the formation of forelimb features, suggesting that Tbx5 was fully functional in wingless moa. An alternatively spliced exon 1 for tbx5 that is expressed specifically in the forelimb region was shown to be almost identical between moa and ostrich, suggesting that, as well as being fully functional, tbx5 is likely to have been expressed normally in moa since divergence from their flighted ancestors, approximately 60 mya.
The results suggests that, as in mice, moa tbx5 is necessary for the induction of forelimbs, but is not sufficient for their outgrowth. Moa Tbx5 may have played an important role in the development of moa’s remnant forelimb girdle, and may be required for the formation of this structure. Our results further show that genetic changes affecting genes other than tbx5 must be responsible for the complete loss of forelimbs in moa.
To determine the molecular basis of winglessness in moa, we present the first reconstruction and characterization of an ancient multi-exon gene, tbx5, a highly conserved Tbox motif-containing transcription factor known to be essential for forelimb initiation in nearly all animals. Tbx5 is an essential member of a tightly regulated gene network that includes the Hox genes (reviewed in ), fgf8, fgf10[4–6], ANF , and sall4. Of these, tbx5 is perhaps the best characterized [5, 6, 8–10]. Knockouts of tbx5 in mice, or morpholino-induced tbx5 knockdowns in zebrafish, result in the complete loss of forelimbs [11, 12]. Similarly, natural occurrences of limblessness in humans and zebrafish have been attributed to mutations in tbx5. In humans, most tbx5 mutations are found in the highly conserved DNA-binding Tbox region and result in Holt-Oram syndrome (HOS), characterized by reduction of the forelimbs and associated heart anomalies . A similar phenotype is shown by the zebrafish tbx5 mutant heartstrings.
The spectrum of mutations in Tbx5 that contribute to Holt-Oram syndrome continues to grow [9, 14, 15]. Tbx5 mutations have now been found in more than 70% of patients with a strict clinical diagnosis of HOS . Interestingly, a number of Tbx5 coding sequence mutations result in relatively mild heart defects but severe forelimb abnormalities [14, 17, 18].
As the forelimb phenotypes of mouse tbx5 knockouts very closely resemble the phenotype seen in moa, we reconstruct, build, and characterize moa tbx5 to determine the molecular basis of winglessness in this extinct ratite.
Moa tbx5construction and characterization
Moa Tbx5 activates fgf10 and ANFpromoters
Moa Tbx5 induces forelimb features in chicken hindlimbs
Phenotypes of chick embryo RCAS moa tbx5 at stage E16
Percentage (n = 9)
Feather induction in leg
Digit 4 converted to wing-type digit 3
Determination of tbx5promoter activity in moa by analysis of forelimb-specific exon 1 sequences
Complementary DNAs from ostrich and kiwi heart and forelimb, respectively, were A-tailed and 5’ nested RACE was carried out to isolate tbx5 exon 1 sequences (Additional file 1: Figure S9). Primers designed to exon 1 sequences obtained from forelimb tbx5 only amplified from ostrich or kiwi forelimb RNA (Figure 2). Primer walking using the chicken genome allowed the isolation of ~350 bp of forelimb-specific exon 1. Isolation of this sequence from emu, cassowary, kiwi, ostrich, rhea, tinamou, and moa shows very little difference between the ratites, with only 4.7% difference shown between moa and rhea, thought to have diverged approximately 80 mya (Additional file 1: Figures S10-12; ).
Loss of flight is common in birds . Studies on Pacific Island rails show that flightlessness can occur rapidly with some birds becoming large and flightless within three million years . For ratites however, it is unknown how long it took for each species to lose flight capabilities. It has been proposed that all lost their flight independently during the Cretaceous Tertiary (KT) boundary approximately 65 mya , a theory that challenges the vicariance biogeography model where flightless ratites are presumed to have ‘rafted’ apart with the breakup of Gondwana .
Unlike the other ratites, moa have lost all elements of the forelimb skeleton, save for a finger-like bone, the scapulocoracoid, that once was part of the forelimb girdle. The scapulocoracoid consists of a short coracoid and a long tapering scapula. It is more easily identified in Dinornis, Anomalopteryx, and Pachyornis, but was very small due to the loss of the coracoidal fossa in Megalapteryx, Euryapteryx, and Emeus. Moa do not have a glenoid fossa for articulation to the humerus .
The timing of forelimb loss in moa is difficult to determine as a result of limited fossil material. However, by applying mutation rates to phylogenies constructed from Holocene moa, it has been suggested that the last common ancestor of Holocene moa lived 5.8-18.5 mya [25, 26]. Furthermore, the discovery of moa leg bones 16–19 mya that are morphologically very similar to those from the Holocene, suggests that these moa were very similar to their later relatives and were also likely to be wingless at that time . As the tinamou/moa split is thought to have occurred 60 mya , this places the time taken for moa to develop winglessness at approximately 40 mya. Although by no means conclusive, it is worth noting that a fossil toe bone of Late Cretaceous age (80–65 mya) from New Zealand’s North Island may possibly be from a very large bird .
Flightlessness and forelimb loss is thought to occur by gradual deminishment as a result of changes in expression of a number of select genes. However, high conservation between the tinamou and moa forelimb-specific exon 1 suggests that in moa tbx5 continues to be expressed in the forelimb region despite possibly 16–19 mys of winglessness. Due to the complex nature of limb development, it has been proposed that limbs can be lost but not regained [29–31]. Collin and Cipriani (2003)  go on to suggest that unused genes remain in a functional state in genome for ~6 my but are almost certain to lose their function at about 10 my. This proposal has been challenged by studies on squamates where it has been shown that lost digits at least are recoverable through evolutionary time [33–35]. One reason for this may be that the pleiotropic nature of some genes may act to keep them functional . For example, avian teeth were lost 70–80 million years ago, but the avian genome still harbours the genes required for tooth formation and birds are still able to do so, as has been shown by the recovery of crocodilian-like teeth in mutant talpid 2 chickens . Similarly, despite having been eyeless for over 1 million years, crosses between different eyeless cavefish populations have been shown to restore vision suggesting that even for complex structures such as eyes, very few loci are required for their redevelopment .
The reasons for forelimb-specific expression of tbx5 in moa are unclear, but may suggest a role for Tbx5 in maintaining the scapulocoracoid. In flighted birds the scapulocoracoid is always separated into a coracoid and scapula that collectively function to operate flight muscles important for the wing’s downward stroke. The retention of a remnant of this structure in moa for so long may well suggest that in moa the scapulocoracoid has evolved a new function. Our results support observations made in mice showing that Tbx5 is necessary for the induction of forelimbs but not sufficient for continued forelimb development [39, 40].
Ratite bloods, embryos, and tissues
A ~7 day post-fertilization kiwi embryo and kiwi embryonic heart were kindly made available to us by Dr. Suzanne Bassett, Otago University, New Zealand. Fertilized ostrich eggs were obtained from Kadesh Ltd, Tajo Ostrich Centre, Kumeu, Auckland, New Zealand, and incubated at 37°C with alternate clockwise/anticlockwise 180° rotations every 12 hours. Ostrich eggs were backlit to measure air-cell size reduction during embryonic growth and embryos were harvested after two weeks. (corresponding to approximate Hamburger Hamilton stage 38, (HH38; [43, 44]) when most organ and limb building events are taking place). Emu, rhea, ostrich, and cassowary DNAs were a kind gift from Dr Joy Halverson, Zoogen Services, Sacramento, California, US. Kiwi blood was provided by Dr Murray Potter, Massey University, Palmerston North, New Zealand.
Bone samples, kindly provided by the Otago Museum, the Auckland Institute and Museum, Canterbury Museum, and Massey University, were obtained for D. novaezealandiae, D. robustus, E. curtus, E. crassus, and M. didinus (see Additional file 1: Table S1).
All animal work was conducted according to relevant national and international guidelines. In particular, chick embryo work followed guidelines for animal work enforced by Nagoya University. Chicken embryos were sacrificed at HH stage 40. For the ostrich embryo work, given the early stage of sacrifice, no ethics approval was required. All waste material, including chicks, chick eggshell, and yolks were autoclaved and disposed of as industrial waste.
Nucleic acid extraction and manipulation
Total RNA was isolated from approximately 100 mg of proximal forelimb or heart tissue using TRIzol® (Invitrogen) according to the manufacturers instructions. Genomic DNA was recovered from extant ratite blood by standard phenol/chloroform extraction and ethanol precipitation . Ancient DNA (aDNA) was extracted from about 50 mg of moa bone shavings by incubation with rotation overnight at 56°C in 0.5 M EDTA/0.01% Triton X100 with ~2 mg of proteinase K. The mix was then extracted with phenol:chloroform and chloroform and the acqueous layer was purified using a Qiagen Dneasy® Blood and Tissue Kit. The silica bound aDNA was eluted with ~40 ul of 0.01% Triton X100 and stored at -20°C.
Reverse transcription of RNA
Approximately 5 ug of total RNA was reverse transcribed at 41°C for 60 min in 20 ul volumes with random 7-mer or oligodT primers and purified by phenol:chloroform extraction and ethanol precipitation.
Polymerase Chain Reaction (PCR)
DNA was amplified from approximately 1–20 ng of DNA as outlined in . A number of PCR-based methods were used to obtain the kiwi tbx5 intron/exon boundaries required to allow primer design for moa amplification. These included: Single primer PCR - A single primer was used in a standard PCR reaction at low annealing temperatures for one cycle to allow random priming and then amplified for ~35 cycles with the annealing temperature set at the primer’s Tm. Hairpin primer ligation PCR - hairpin primers containg a PstI compatible overhang were ligated to PstI-digested DNA before standard PCRs were carried out using the hairpin primer and a tbx5-specific primer. Medium range PCR - amplification across the smaller tbx5 introns was carried out usng Elongase® (Invitrogen) or Expand Long Template PCR System (Roche) as outlined by the manufacturer. Inverse PCR - Inverse PCR was carried out to obtain intron sequences directly from moa. Biefly, moa aDNA was denatured, then dephosphorylated with shrimp alkaline phosphatase (SAP) before fresh phosphates were added with T4 Polynucleotide Kinase. The aDNA was then circularised with CircligaseTM ssDNA Ligase (Epicentre®) and subjected to rolling circle amplification using TemplifyTM (Amersham). The amplified aDNA was then subjected to standard PCR using inverse primers.
Construction strategy for moa tbx5
To isolate moa tbx5 sequences we initially obtained full-length tbx5 cDNA sequences for kiwi and ostrich. Comparison with the chicken genome identified the intron/exon boundaries and primers were then designed to recover moa tbx5 exon sequences. To obtain moa tbx5 intron/exon boundaries, these boundaries were first isolated from kiwi. Primers were then designed to kiwi tbx5 intron sequences and conserved ostrich/kiwi/chicken tbx5 exon sequences to obtain moa tbx5 intron/exon boundary sequences. Most primers designed to kiwi tbx5 intron sequences successfully amplified from moa aDNA. For a few introns however, primers successful for amplification from moa required additional intron sequences from rhea, and/or ostrich.
Nested 5’ RACE
Total RNA isolated from embryonic ostrich and kiwi forelimb and kiwi heart were A-tailed with terminal transferase (Invitrogen) and amplified with H5FdT (5’- AATCGGACAAACTGGTCCTTGCAACdT20) and ex2R2 (5’- GGTGAGCGACTTGCTGGTG), followed by H5F (5’- AATCGGACAAACTGGTCCTTGCAAC) and ex2R3 (5’- CAAAGCCTTCCTCCGTAT). Amplified products were TA cloned into pGEM®T-Easy (Promega) and sequenced with m13F (5’-TGTAAAACGACGGCCAGT) or m13R (5’-CAGGAAACAGCTATGACC). Full-length forelimb-specific exon 1 transcripts were then obtained by cDNA walking using primers designed to conserved upstream regions of the chicken genome.
PCR products were purified by centrifugation through dry Sephacryl S200HR, sequenced using ABI BigDye® Terminator v3.1 chemistry, then analysed and aligned in SequencherTM 5.0 (Gene Codes Corporation).
Ancient DNA procedures
In accordance with criteria suggested for the verification of aDNA sequences , a number of samples were extracted and sequenced at a separate ancient DNA facility at Massey University, Auckland, New Zealand.
Activation of fgf10 and ANFpromoters by moa Tbx5
The ability of moa Tbx5 to bind to and activate downstream fgf10 and ANF promoters was determined using a luciferase assay. HEK293T cells were seeded into 24-well plates at a density of 5 × 104 cells/well twenty four hours before transfection. The following vectors were transfected into HEK293T cells using XtremeGENE HP (Roche); 6 kb of mouse sequence immediately upstream of fgf10, or 0.7 kb of mouse sequence immediately upstream of the ANF promoter , both fused to luciferase, pCAGGS containing chick or moa tbx5, CMX-β-galactosidase, and an empty control vector (pcDNA3.1). Forty-eight hours after transfection, the cells were lysed, and luciferase activity was measured using a Luminescencer-JNR (ATTO). β-galactosidase activities were measured to standardize the efficiency of transfection. Results are expressed as the average of three samples including standard deviation.
Miss-expression of moa tbx5in chicken
Electroporation into the chick hindlimb field was carried out as described previously . Briefly, 2 ug/ml of RCAS-moa tbx5 plasmid was injected into the prospective hindlimb field at Hamburger Hamilton (HH) stage 14 by glass capillary. Electric pulses (8 V, 60 ms pulse-on, 50 ms pulse-off, three repetitions) were applied using an CUY21-EDIT electroporator (NAPA GENE) with platinum electrodes. Electroporated embryos were harvested at HH stage 40 and stained with Victoria blue. Victoria blue staining was carried out as described in .
Availability of supporting data
Supporting tables and figure are available as additional files. Tbx5 sequences are deposited in NCBI’s GenBank [GenBank Acc Nos. KJ584152-KJ584161].
Grateful thanks to Paul Scofield (Canterbury Museum) and Brian Gill (Auckland Museum) for assistance. Thanks to Tim Heupink for helpful comments. This work was carried out with support from the Australian Research Council (Grant number DP110101364; The molecular evolution of wings in ratites).
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