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The evolution of HoxD-11 expression in the bird wing: insights from Alligator mississippiensis.

Vargas AO, Kohlsdorf T, Fallon JF, Vandenbrooks J, Wagner GP - PLoS ONE (2008)

Bottom Line: Using degenerate primers we cloned a 606 nucleotide fragment of exon 1 of the alligator HoxD-11 gene and used it for whole-mount in-situ detection in alligator embryos.The ancestral condition for amniotes is that late-phase HoxD-11 expression is absent only in digit 1.HoxD-11 expression in alligator is consistent with the hypothesis that both digit morphology as well as HoxD-11 expression are shifted towards posterior in the bird wing.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, USA. thearchosaur@gmail.com

ABSTRACT

Background: Comparative morphology identifies the digits of the wing of birds as 1,2 and 3, but they develop at embryological positions that become digits 2, 3 and 4 in other amniotes. A hypothesis to explain this is that a homeotic frame shift of digital identity occurred in the evolution of the bird wing, such that digits 1,2 and 3 are developing from embryological positions 2, 3 and 4. Digit 1 of the mouse is the only digit that shows no late expression of HoxD-11. This is also true for the anterior digit of the bird wing, suggesting this digit is actually a digit 1. If this is the case, we can expect closer relatives of birds to show no HoxD-11 expression only in digit 1. To test this prediction we investigate HoxD-11 expression in crocodilians, the closest living relatives of birds.

Methodology/principal findings: Using degenerate primers we cloned a 606 nucleotide fragment of exon 1 of the alligator HoxD-11 gene and used it for whole-mount in-situ detection in alligator embryos. We found that in the pentadactyl forelimbs of alligator, as in the mouse, late expression of HoxD-11 is absent only in digit 1.

Conclusions/significance: The ancestral condition for amniotes is that late-phase HoxD-11 expression is absent only in digit 1. The biphalangeal morphology and lack of HoxD-11 expression of the anterior digit of the wing is like digit 1 of alligator and mouse, but its embryological position as digit 2 is derived. HoxD-11 expression in alligator is consistent with the hypothesis that both digit morphology as well as HoxD-11 expression are shifted towards posterior in the bird wing.

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Identification of the alligator HoxD-11 exon 1 sequence.(EU597806): A) alignment of the deduced alligator amino acid sequence with the HoxD-11 sequence of chicken. In exon 1 the amino acid sequence conservation is 87%. B) Maximum parsimony tree of the aligned exon 1 amino acid sequences from HoxD-11, HoxA-11 and HoxC-11 sequences. C) Neighbor joining tree of the aligned exon 1 amino acid sequences from HoxD-11, HoxA-11 and HoxC-11 sequences. B+C) The numbers at the internal branches represent bootstrap support values. Note that the alligator HoxD-11 sequence, AmiHoxd11, forms a well-supported clade with the human, HsaHoxd11, and the chicken, GgaHoxd11, HoxD-11 sequences, confirming that the alligator sequence is a HoxD-11 ortholog. Furthermore, the alligator sequence is more closely related to the chicken sequence than to the mouse sequence, as expected based on the accepted species phylogeny. D) Expression of the alligator sequence in a stage 12 alligator embryo. The embryo is dissected to show the sharp anterior limit of hindgut expression (1). Expression is also present in genital tubercule (2), distal tail (3) and limb buds (4), all known expression domains of HoxD-11 in chicken and mouse that confirm the alligator sequence is a homolog of HoxD-11.
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pone-0003325-g003: Identification of the alligator HoxD-11 exon 1 sequence.(EU597806): A) alignment of the deduced alligator amino acid sequence with the HoxD-11 sequence of chicken. In exon 1 the amino acid sequence conservation is 87%. B) Maximum parsimony tree of the aligned exon 1 amino acid sequences from HoxD-11, HoxA-11 and HoxC-11 sequences. C) Neighbor joining tree of the aligned exon 1 amino acid sequences from HoxD-11, HoxA-11 and HoxC-11 sequences. B+C) The numbers at the internal branches represent bootstrap support values. Note that the alligator HoxD-11 sequence, AmiHoxd11, forms a well-supported clade with the human, HsaHoxd11, and the chicken, GgaHoxd11, HoxD-11 sequences, confirming that the alligator sequence is a HoxD-11 ortholog. Furthermore, the alligator sequence is more closely related to the chicken sequence than to the mouse sequence, as expected based on the accepted species phylogeny. D) Expression of the alligator sequence in a stage 12 alligator embryo. The embryo is dissected to show the sharp anterior limit of hindgut expression (1). Expression is also present in genital tubercule (2), distal tail (3) and limb buds (4), all known expression domains of HoxD-11 in chicken and mouse that confirm the alligator sequence is a homolog of HoxD-11.

Mentions: A genomic fragment was amplified by PCR with a primer pair targeting the conserved 5′ sequence of the HoxD-11 coding sequence and a part of the homeobox (see Material and Methods). These primers target a sequence that corresponds to nucleotides 22 to 690 of the chicken HoxD-11 coding sequence, but include the intron between exon 1 and 2. We obtained a PCR product of approximately 900 nucleotides and sequenced 819 nucleotides from the 5′ end of this sequence. This sequence contains the complete exon 1 of 606 nucleotides and the adjacent intron sequence with a putative 5′ splice site AG/GTAGGT (the G/G is the putative exon-intron boundary). The translated exon 1 sequence has 87% sequence conservation with the corresponding part of the chicken HoxD-11 gene (Figure 3A). A phylogenetic analysis of this and published paralog group 11 amino acid sequences reveals strong support for the hypothesis that the alligator sequence is a HoxD-11 ortholog. Our sequence forms a well supported clade with the chicken HoxD-11 sequence and together with the human HoxD-11 sequence is separated by a well supported node from HoxA-11 and HoxC-11 sequences (Figure 3 B, C). Furthermore, in situ hybridization revealed expression in all structures where HoxD-11 is known to be expressed in other amniotes, as can be observed in Figure 3D. The specimen is dissected to show the sharp anterior limit of hindgut expression (Figure 3D, 1) expression in the genital tubercle (Figure 3D, 2), distal tail (Figure 3D, 3) and limbs (Figure 3D 4, Figure 4). We thus conclude that we have isolated the exon 1 and 5′ part of the intron of alligator HoxD-11 gene (Genbank accession # EU597806).


The evolution of HoxD-11 expression in the bird wing: insights from Alligator mississippiensis.

Vargas AO, Kohlsdorf T, Fallon JF, Vandenbrooks J, Wagner GP - PLoS ONE (2008)

Identification of the alligator HoxD-11 exon 1 sequence.(EU597806): A) alignment of the deduced alligator amino acid sequence with the HoxD-11 sequence of chicken. In exon 1 the amino acid sequence conservation is 87%. B) Maximum parsimony tree of the aligned exon 1 amino acid sequences from HoxD-11, HoxA-11 and HoxC-11 sequences. C) Neighbor joining tree of the aligned exon 1 amino acid sequences from HoxD-11, HoxA-11 and HoxC-11 sequences. B+C) The numbers at the internal branches represent bootstrap support values. Note that the alligator HoxD-11 sequence, AmiHoxd11, forms a well-supported clade with the human, HsaHoxd11, and the chicken, GgaHoxd11, HoxD-11 sequences, confirming that the alligator sequence is a HoxD-11 ortholog. Furthermore, the alligator sequence is more closely related to the chicken sequence than to the mouse sequence, as expected based on the accepted species phylogeny. D) Expression of the alligator sequence in a stage 12 alligator embryo. The embryo is dissected to show the sharp anterior limit of hindgut expression (1). Expression is also present in genital tubercule (2), distal tail (3) and limb buds (4), all known expression domains of HoxD-11 in chicken and mouse that confirm the alligator sequence is a homolog of HoxD-11.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553264&req=5

pone-0003325-g003: Identification of the alligator HoxD-11 exon 1 sequence.(EU597806): A) alignment of the deduced alligator amino acid sequence with the HoxD-11 sequence of chicken. In exon 1 the amino acid sequence conservation is 87%. B) Maximum parsimony tree of the aligned exon 1 amino acid sequences from HoxD-11, HoxA-11 and HoxC-11 sequences. C) Neighbor joining tree of the aligned exon 1 amino acid sequences from HoxD-11, HoxA-11 and HoxC-11 sequences. B+C) The numbers at the internal branches represent bootstrap support values. Note that the alligator HoxD-11 sequence, AmiHoxd11, forms a well-supported clade with the human, HsaHoxd11, and the chicken, GgaHoxd11, HoxD-11 sequences, confirming that the alligator sequence is a HoxD-11 ortholog. Furthermore, the alligator sequence is more closely related to the chicken sequence than to the mouse sequence, as expected based on the accepted species phylogeny. D) Expression of the alligator sequence in a stage 12 alligator embryo. The embryo is dissected to show the sharp anterior limit of hindgut expression (1). Expression is also present in genital tubercule (2), distal tail (3) and limb buds (4), all known expression domains of HoxD-11 in chicken and mouse that confirm the alligator sequence is a homolog of HoxD-11.
Mentions: A genomic fragment was amplified by PCR with a primer pair targeting the conserved 5′ sequence of the HoxD-11 coding sequence and a part of the homeobox (see Material and Methods). These primers target a sequence that corresponds to nucleotides 22 to 690 of the chicken HoxD-11 coding sequence, but include the intron between exon 1 and 2. We obtained a PCR product of approximately 900 nucleotides and sequenced 819 nucleotides from the 5′ end of this sequence. This sequence contains the complete exon 1 of 606 nucleotides and the adjacent intron sequence with a putative 5′ splice site AG/GTAGGT (the G/G is the putative exon-intron boundary). The translated exon 1 sequence has 87% sequence conservation with the corresponding part of the chicken HoxD-11 gene (Figure 3A). A phylogenetic analysis of this and published paralog group 11 amino acid sequences reveals strong support for the hypothesis that the alligator sequence is a HoxD-11 ortholog. Our sequence forms a well supported clade with the chicken HoxD-11 sequence and together with the human HoxD-11 sequence is separated by a well supported node from HoxA-11 and HoxC-11 sequences (Figure 3 B, C). Furthermore, in situ hybridization revealed expression in all structures where HoxD-11 is known to be expressed in other amniotes, as can be observed in Figure 3D. The specimen is dissected to show the sharp anterior limit of hindgut expression (Figure 3D, 1) expression in the genital tubercle (Figure 3D, 2), distal tail (Figure 3D, 3) and limbs (Figure 3D 4, Figure 4). We thus conclude that we have isolated the exon 1 and 5′ part of the intron of alligator HoxD-11 gene (Genbank accession # EU597806).

Bottom Line: Using degenerate primers we cloned a 606 nucleotide fragment of exon 1 of the alligator HoxD-11 gene and used it for whole-mount in-situ detection in alligator embryos.The ancestral condition for amniotes is that late-phase HoxD-11 expression is absent only in digit 1.HoxD-11 expression in alligator is consistent with the hypothesis that both digit morphology as well as HoxD-11 expression are shifted towards posterior in the bird wing.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, USA. thearchosaur@gmail.com

ABSTRACT

Background: Comparative morphology identifies the digits of the wing of birds as 1,2 and 3, but they develop at embryological positions that become digits 2, 3 and 4 in other amniotes. A hypothesis to explain this is that a homeotic frame shift of digital identity occurred in the evolution of the bird wing, such that digits 1,2 and 3 are developing from embryological positions 2, 3 and 4. Digit 1 of the mouse is the only digit that shows no late expression of HoxD-11. This is also true for the anterior digit of the bird wing, suggesting this digit is actually a digit 1. If this is the case, we can expect closer relatives of birds to show no HoxD-11 expression only in digit 1. To test this prediction we investigate HoxD-11 expression in crocodilians, the closest living relatives of birds.

Methodology/principal findings: Using degenerate primers we cloned a 606 nucleotide fragment of exon 1 of the alligator HoxD-11 gene and used it for whole-mount in-situ detection in alligator embryos. We found that in the pentadactyl forelimbs of alligator, as in the mouse, late expression of HoxD-11 is absent only in digit 1.

Conclusions/significance: The ancestral condition for amniotes is that late-phase HoxD-11 expression is absent only in digit 1. The biphalangeal morphology and lack of HoxD-11 expression of the anterior digit of the wing is like digit 1 of alligator and mouse, but its embryological position as digit 2 is derived. HoxD-11 expression in alligator is consistent with the hypothesis that both digit morphology as well as HoxD-11 expression are shifted towards posterior in the bird wing.

Show MeSH