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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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The expression of HoxD-11 in alligator and chicken limbs.A) The expression of HoxD-11 in the developing forelimb and hind limb of the crocodilian Alligator mississippiensis (staging is according to Ferguson [29]) B) The expression of HoxD-11 in the chicken Gallus gallus (staging according to Hamburger-Hamilton [30]). Alligator forelimb: Early stages 12–14 show extension of HoxD-11 along the limb border, including anterior regions (black arrows). At stage 16 and onwards, there is no detectable expression in the anterior-most digit 1 region. At stage 16 posterior expression is temporarily down-regulated in the region of digits 4 and 5 (black arrow) but is re-expressed by stage 17. At stage 17, only very low expression is detectable in the interdigit between digit 1 and 2 (black arrow). Strong anterior and posterior expression in the wrist and forearm region at stage 14 continues up to stage 17 (white arrows). Alligator hind limb: Stage 12 shows some expression along the anterior margin (white arrow) but this is undetectable in the anterior-most digit 1 region from stage 14 onwards. At stage 16, only very low expression is detectable in the interdigit between digits 1 and 2 (black arrow). Expression in the foreleg is restricted to posterior (stages 12 and 14, black arrow) and absent in the ankle region. Stage 17 and stage 18 show a sharp anterior limit of HoxD-11 expression along the posterior margin of digit 2 (white arrows). Chicken wing: Stage 24 presents anterior expression (black arrow). Expression is undetectable in digit 1 in stage 26 and subsequent stages. No low expression is detectable between interdigits 1 and 2. In stages 26–34, expression of HoxD-11 extends to the anterior border of digit 2 (black arrows). Chicken hind limb: Early stage 24 shows no anterior expression. No expression is ever detected in digit 1 precursor cells or the interdigit between digit 1 and digit 2. In stages 29–34 HoxD-11 shows a sharp anterior limit along the posterior border of digit 2 (black arrows in stage 17–18 of alligator and stages 31–34 of chicken; stage 34 is shown in ventral view). Strong expression is found in the interdigit between digits 2 and 3 (stage 29, black arrow), as in forelimbs. HoxD-11 expression in alligator limbs is consistent with the notion that, as in chicken, normal digit 1 determination occurs at late stages with absent or very low HoxD-11 expression.
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pone-0003325-g004: The expression of HoxD-11 in alligator and chicken limbs.A) The expression of HoxD-11 in the developing forelimb and hind limb of the crocodilian Alligator mississippiensis (staging is according to Ferguson [29]) B) The expression of HoxD-11 in the chicken Gallus gallus (staging according to Hamburger-Hamilton [30]). Alligator forelimb: Early stages 12–14 show extension of HoxD-11 along the limb border, including anterior regions (black arrows). At stage 16 and onwards, there is no detectable expression in the anterior-most digit 1 region. At stage 16 posterior expression is temporarily down-regulated in the region of digits 4 and 5 (black arrow) but is re-expressed by stage 17. At stage 17, only very low expression is detectable in the interdigit between digit 1 and 2 (black arrow). Strong anterior and posterior expression in the wrist and forearm region at stage 14 continues up to stage 17 (white arrows). Alligator hind limb: Stage 12 shows some expression along the anterior margin (white arrow) but this is undetectable in the anterior-most digit 1 region from stage 14 onwards. At stage 16, only very low expression is detectable in the interdigit between digits 1 and 2 (black arrow). Expression in the foreleg is restricted to posterior (stages 12 and 14, black arrow) and absent in the ankle region. Stage 17 and stage 18 show a sharp anterior limit of HoxD-11 expression along the posterior margin of digit 2 (white arrows). Chicken wing: Stage 24 presents anterior expression (black arrow). Expression is undetectable in digit 1 in stage 26 and subsequent stages. No low expression is detectable between interdigits 1 and 2. In stages 26–34, expression of HoxD-11 extends to the anterior border of digit 2 (black arrows). Chicken hind limb: Early stage 24 shows no anterior expression. No expression is ever detected in digit 1 precursor cells or the interdigit between digit 1 and digit 2. In stages 29–34 HoxD-11 shows a sharp anterior limit along the posterior border of digit 2 (black arrows in stage 17–18 of alligator and stages 31–34 of chicken; stage 34 is shown in ventral view). Strong expression is found in the interdigit between digits 2 and 3 (stage 29, black arrow), as in forelimbs. HoxD-11 expression in alligator limbs is consistent with the notion that, as in chicken, normal digit 1 determination occurs at late stages with absent or very low HoxD-11 expression.

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)

The expression of HoxD-11 in alligator and chicken limbs.A) The expression of HoxD-11 in the developing forelimb and hind limb of the crocodilian Alligator mississippiensis (staging is according to Ferguson [29]) B) The expression of HoxD-11 in the chicken Gallus gallus (staging according to Hamburger-Hamilton [30]). Alligator forelimb: Early stages 12–14 show extension of HoxD-11 along the limb border, including anterior regions (black arrows). At stage 16 and onwards, there is no detectable expression in the anterior-most digit 1 region. At stage 16 posterior expression is temporarily down-regulated in the region of digits 4 and 5 (black arrow) but is re-expressed by stage 17. At stage 17, only very low expression is detectable in the interdigit between digit 1 and 2 (black arrow). Strong anterior and posterior expression in the wrist and forearm region at stage 14 continues up to stage 17 (white arrows). Alligator hind limb: Stage 12 shows some expression along the anterior margin (white arrow) but this is undetectable in the anterior-most digit 1 region from stage 14 onwards. At stage 16, only very low expression is detectable in the interdigit between digits 1 and 2 (black arrow). Expression in the foreleg is restricted to posterior (stages 12 and 14, black arrow) and absent in the ankle region. Stage 17 and stage 18 show a sharp anterior limit of HoxD-11 expression along the posterior margin of digit 2 (white arrows). Chicken wing: Stage 24 presents anterior expression (black arrow). Expression is undetectable in digit 1 in stage 26 and subsequent stages. No low expression is detectable between interdigits 1 and 2. In stages 26–34, expression of HoxD-11 extends to the anterior border of digit 2 (black arrows). Chicken hind limb: Early stage 24 shows no anterior expression. No expression is ever detected in digit 1 precursor cells or the interdigit between digit 1 and digit 2. In stages 29–34 HoxD-11 shows a sharp anterior limit along the posterior border of digit 2 (black arrows in stage 17–18 of alligator and stages 31–34 of chicken; stage 34 is shown in ventral view). Strong expression is found in the interdigit between digits 2 and 3 (stage 29, black arrow), as in forelimbs. HoxD-11 expression in alligator limbs is consistent with the notion that, as in chicken, normal digit 1 determination occurs at late stages with absent or very low HoxD-11 expression.
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Related In: Results  -  Collection

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pone-0003325-g004: The expression of HoxD-11 in alligator and chicken limbs.A) The expression of HoxD-11 in the developing forelimb and hind limb of the crocodilian Alligator mississippiensis (staging is according to Ferguson [29]) B) The expression of HoxD-11 in the chicken Gallus gallus (staging according to Hamburger-Hamilton [30]). Alligator forelimb: Early stages 12–14 show extension of HoxD-11 along the limb border, including anterior regions (black arrows). At stage 16 and onwards, there is no detectable expression in the anterior-most digit 1 region. At stage 16 posterior expression is temporarily down-regulated in the region of digits 4 and 5 (black arrow) but is re-expressed by stage 17. At stage 17, only very low expression is detectable in the interdigit between digit 1 and 2 (black arrow). Strong anterior and posterior expression in the wrist and forearm region at stage 14 continues up to stage 17 (white arrows). Alligator hind limb: Stage 12 shows some expression along the anterior margin (white arrow) but this is undetectable in the anterior-most digit 1 region from stage 14 onwards. At stage 16, only very low expression is detectable in the interdigit between digits 1 and 2 (black arrow). Expression in the foreleg is restricted to posterior (stages 12 and 14, black arrow) and absent in the ankle region. Stage 17 and stage 18 show a sharp anterior limit of HoxD-11 expression along the posterior margin of digit 2 (white arrows). Chicken wing: Stage 24 presents anterior expression (black arrow). Expression is undetectable in digit 1 in stage 26 and subsequent stages. No low expression is detectable between interdigits 1 and 2. In stages 26–34, expression of HoxD-11 extends to the anterior border of digit 2 (black arrows). Chicken hind limb: Early stage 24 shows no anterior expression. No expression is ever detected in digit 1 precursor cells or the interdigit between digit 1 and digit 2. In stages 29–34 HoxD-11 shows a sharp anterior limit along the posterior border of digit 2 (black arrows in stage 17–18 of alligator and stages 31–34 of chicken; stage 34 is shown in ventral view). Strong expression is found in the interdigit between digits 2 and 3 (stage 29, black arrow), as in forelimbs. HoxD-11 expression in alligator limbs is consistent with the notion that, as in chicken, normal digit 1 determination occurs at late stages with absent or very low HoxD-11 expression.
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