New developmental evidence supports a homeotic frameshift of digit identity in the evolution of the bird wing
- Miguel Salinas-Saavedra1,
- Cristian Gonzalez-Cabrera1,
- Luis Ossa-Fuentes†1,
- Joao F Botelho†1,
- Macarena Ruiz-Flores†1 and
- Alexander O Vargas1Email author
© Salinas-Saavedra et al.; licensee BioMed Central Ltd. 2014
Received: 10 January 2014
Accepted: 7 April 2014
Published: 12 April 2014
The homology of the digits in the bird wing is a high-profile controversy in developmental and evolutionary biology. The embryonic position of the digits cartilages with respect to the primary axis (ulnare and ulna) corresponds to 2, 3, 4, but comparative-evolutionary morphology supports 1, 2, 3. A homeotic frameshift of digit identity in evolution could explain how cells in embryonic positions 2, 3, 4 began developing morphologies 1, 2, 3. Another alternative is that no re-patterning of cell fates occurred, and the primary axis shifted its position by some other mechanism. In the wing, only the anterior digit lacks expression of HoxD10 and HoxD12, resembling digit 1 of other limbs, as predicted by 1, 2, 3. However, upon loss of digit 1 in evolution, the most anterior digit 2 could have lost their expression, deceitfully resembling a digit 1. To test this notion, we observed HoxD10 and HoxD12 in a limb where digit 2 is the most anterior digit: The rabbit foot. We also explored whether early inhibition of Shh signalling in the embryonic wing bud induces an experimental homeotic frameshift, or an experimental axis shift. We tested these hypotheses using DiI injections to study the fate of cells in these experimental wings.
We found strong transcription of HoxD10 and HoxD12 was present in the most anterior digit 2 of the rabbit foot. Thus, we found no evidence to question the use of HoxD expression as support for 1, 2, 3. When Shh signalling in early wing buds is inhibited, our fate maps demonstrate that an experimental homeotic frameshift is induced.
Along with comparative morphology, HoxD expression provides strong support for 1, 2, 3 identity of wing digits. As an explanation for the offset 2, 3, 4 embryological position, the homeotic frameshift hypothesis is consistent with known mechanisms of limb development, and further proven to be experimentally possible. In contrast, the underlying mechanisms and experimental plausibility of an axis shift remain unclear.
The early expression of Shh in the wing bud is also important to the debate on digit identity . A spatio-temporal gradient of posteriorly expressed Shh protein patterns the antero-posterior axis of the limb bud, with greater concentrations and longer exposures determining more posterior digit identities [20–22]. In the mouse, endogenously expressed Shh is absent from the precursors of digits 1, 2, and the anterior half of digit 3  which is also the case for the anterior, middle and posterior digits of the wing, respectively, providing support for 1, 2, 3 [19, 24]. Assuming the evidence for 1, 2, 3 identity is correct, different hypotheses could explain the 2, 3, 4 embryonic position. A decrease in the postero-anterior gradient of Shh signal, either by reduced concentration and/or reduced exposure time, could have induced a homeotic frameshift in evolution, such that cartilages in positions that previously became 2, 3, 4 began developing the adult morphologies of digits 1, 2,3 [5, 19, 25]. Alternatively, a shift in the position of the primary axis occurred, without any re-patterning of cell fates (the “axis shift” hypothesis [24, 26]). Experimental inhibition of early Shh signaling leads to bidactyl wings, in which the posterior digit is missing [27, 28]. In these bidactyl wings, the middle digit develops in line with the primary axis , but this experiment has been interpreted differently, in favour of the homeotic frameshift hypothesis  or the axis shift hypothesis . To clarify this controversy, we have marked cells and fate-mapped them, in both control wings and wings under Shh inhibition.
HoxD expression in a limb that has lost digit I does not resemble the wing
Inhibition of Shh signalling produces an experimental homeotic frameshift in the wing
If wing digit identity were 2, 3, 4, coinciding directly with embryological position, both the homeotic frameshift and axis shift hypotheses would be unnecessary. This could be the case if support for 1, 2, 3, were equivocal. Our data from the rabbit foot produced no evidence to question the use of HoxD expression as support for 1, 2, 3. The MAD (Most Anterior Digit) hypothesis claimed that upon loss of digit 1, a most anterior digit 2 would take over the HoxD signature expression of digit 1, as a result of intrinsic properties of Shh signalling and HoxD regulation [17, 18]. If this were indeed such an inescapable outcome of limb development, HoxD expression in the wing would be trivially as expected for 2, 3, 4. However, the rabbit foot suffices to demonstrate that the MAD hypothesis does not always apply. At best, the MAD hypothesis is still possible, but unsupported by any actual empirical case. Our result is further relevant considering that HoxD regulation is largely understood from studies made in the mouse, a fairly close relative of the rabbit. It could be argued that the rabbit is too distantly related to birds and thus irrelevant to discuss the plausibility of events in the evolution of that lineage. However, developmental biologists constantly integrate information from mouse and chicken, even though they are distant relatives. This practice is informative because molecular mechanisms of limb development (Shh expression, HoxD cluster structure) are highly conserved. The closest living relatives of birds are the Crocodylia, but they cannot test the MAD hypothesis since none presents reduction of digit 1. Digit 1 reduction is found among squamates, but these have accumulated unusually large numbers of transposable elements in their Hox clusters . In fact, the mammalian HoxD cluster is more comparable to that of birds. For now, the rabbit foot provides the only available data for HoxD expression in limbs where loss of digit 1 is non-controversial. Even if a small field with no HoxD10-12 is still present in the rabbit foot, it is clearly larger in the bird wing, engulfing the PIDM of the anterior digital ray. Thus, comparison to the rabbit foot provides no support for the occurrence of digit 1 reduction in the wing.
Any trait that is proposed to mark the identity of a given digit is strengthened when the association is maintained across different limbs: The greater the sample, the better, demonstrating great evolutionary conservation. Lack of HoxD10, HoxD11 and HoxD12 in digit 1 has been confirmed in the chicken foot and both the hand and foot of the mouse . Lack of HoxD11 in digit 1 has also been confirmed in the hand and foot of the alligator and of the three-toed skink [15, 37]. The new data from the rabbit foot further supports the use of HoxD expression to identify digit 1. As with the HoxD genes, an extended sample of limbs, including limbs that lost digit 1, will test the validity of other genes suggested as markers of digit 1 identity, such as Zic2 and Lhx9. These are expressed in the anterior digit of the wing and digit 1 of the chicken foot . Further limb sampling could also strengthen the case that digits 1,2, and most of digit 3 are derived from cells that do not express Shh, as in both the hand and foot of mouse and chicken [19, 23, 24]. For now, HoxD expression remains the best-documented line of developmental evidence to support 1, 2, 3.
Developmental evidence for 1, 2, 3 should not be simply dismissed. A recent quantitative study presented parsimony analysis, allegedly supporting 2, 3, 4 identity in the lineage leading to birds ever since early tetanuran dinosaurs like Allosaurus. However, developmental evidence was only used to assume 2, 3, 4, “a priori” in Archaeopteryx, (the only Avialae included in that analysis) and thus code all morphological traits as if present on digits 2, 3, 4 of this taxon. This assumption managed to reverse the result of parsimony analysis of morphological data, which otherwise supports the traditional 1, 2, 3 identification of tetanuran digits . Current molecular-developmental evidence for 1, 2, 3 questions this and any other analysis constructed on the assumption that development univocally supports 2, 3, 4, which only reflects information regarding embryological position.
The evidence for 1, 2, 3 strengthens the case that the position of the primary axis is not related to digit identity by any direct mechanism of causation [38, 39]. The argument has been made that the primary axis is non-significant to the extent that digits are simply 1, 2, 3, and no homeotic frameshift hypothesis is necessary . We think this view is extreme: While the primary axis is not directly related to digit identity, it remains a reliable indicator of relative position among the cartilaginous elements of the embryonic limb. Additionally, our fate maps confirm that in control and cyclopamine-treated wings, the position of a digit with regard to the primary axis directly reflects the earlier antero-posterior position of its precursors at autopod patterning stages. In non-controversial limbs, the digit cartilage at the primary axis consistently gives rise to digit 4, rather than digit 3. Thus, an explanation is still required on how morphological identity and gene expression in the wing have shifted their position towards posterior. Accumulated knowledge on the molecular-developmental mechanisms of digit patterning through a spatio-temporal posterior gradient of Shh signaling has provided a framework in which a homeotic frameshift is readily conceivable [5, 25]. It is further significant that reduced Shh signaling using cyclopamine in fact produces such an experimental frameshift. This discards characterizations of the frameshift as an “awkward” or “ad hoc” auxiliary hypothesis, with no reason of being beyond explaining an apparent incongruence of data . Previously, reduced Shh signaling had been argued to favour the “axis shift” hypothesis. In this context, it was suggested that the presence of a posterior necrotic zone in the early wing could explain the loss of posterior digits in evolution . However, our experimental frameshifts actually suggest the alternative that more posterior morphologies failed to develop, as the result of posterior cells becoming re-specified to more anterior identities. In fact, a large anterior necrotic zone is present in both fore and hind limb buds of mouse and chicken that is unrelated to any evolutionary loss of anterior digits . Mechanistic plausibility is an important pre-requisite for acceptance of any hypothesis, which is now confirmed for the homeotic frameshift. In contrast, the possible mechanisms underlying an “axis shift” remain unclear, and it is yet to be proven experimentally possible. Experimental re-creation of evolutionary events (“synthetic experimental evolution” ) is an important new component of evolutionary biology. Because evolution and developmental experiments emerge as twin outputs of the same underlying mechanisms, they often illuminate each other in concrete ways . Our experiments support continued inquiry into regulatory mechanisms that relate Shh signalling and HoxD expression in the limb [44, 45]. It is conceivable that specific mutations responsible for inducing the homeotic frameshift may be identified in birds.
Gene cloning and whole-mount in situ hybridization
To make in situ probes, specific gene segments were cloned using exact primers (designed from http://www.ensembl.org/index.html) for Chicken, Mouse and Rabbit. The chosen segments of HoxD11 and HoxD12 for rabbit and mouse were identical in sequence (100% homology). The following probes were used:
Chicken (Gallus gallus)
Rabbit (Oryctolagus cuniculus) and mouse (Mus musculus)
Embryos were collected at day 7 of incubation and fixed during 2 hr to O/N with 4% paraformaldehyde (PFA). Rabbit embryos were collected at 14 dpf, a stage that corresponds with mouse at 13 dpf. Embryos were dehydrated in a methanol series and stored at -20°C. Rehydrated chicken embryos were treated with 6% peroxide solution into PBT by 30 minutes. Mouse and rabbit were rehydrated and treated with acetylation solution (triethanolamine, acetic anhydride and chloridric acid) for 10 and 40 minutes, respectively. Whole mount in situ hybridization was carried out .
Fate-mapping of wing buds
Broiler chicken eggs were incubated at 38°C for 3–3, 5 days and stages were selected . Limb buds cells were labeled with DiI (1, 1-dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchlorate; Sigma-Aldrich) and DiO (3, 3′-dioctadecyloxacarbocyanine, perchlorate; Sigma-Aldrich), fluorescent lipophilic dyes that label the cell membrane and do not leak into neighbouring cells. Both dyes were prepared  (DiI 1% in 100% Ethanol, DiO 1% in dimethylformamide) and administered [34, 49] by pressure-injection using a Picospritzer®III (Parker Hannifin Corporation; General Valve) and a pulled micropipette with an open tip made with a 0.78 mm (inner diameter) borosilicate capillary. For labelling wing digits, the embryos were selected and injected in ovo at stage HH18-19 into the sub-apical region . Immediately after injection the limb bud was photographed in light microscope (Olympus SZX10), cyclopamine was applied, and the embryo put back to incubate at 38°C until HH31, in which the injected wing was photographed under a fluorescence microscope (Olympus BX61) and again under light microscope. This procedure was delivered to both control and cyclopamine-treated embryos. In all these experiments, the cells were labelled in the right limb bud and measurements were taken of the dye dot size and position at the time of administration and after the incubation period. The size range of the injected dye dots was 60–100 μm. Position of injected dots within the limb bud was determined in relation to the position of neighbouring somites [24, 34].
Cyclopamine was applied as in previous studies [25, 27, 28]. Eggs were incubated at 38°C and windowed at day 3–3.5 to obtain embryos spanning stages 18–19 according to Hamburger and Hamilton . We delivered 5 μl of 1 mg/ml solution of Cyclopamine (LC Laboratories) in 45% 2-hydropropyl-b-cyclodextrin (HBC; Sigma) into the amniotic cavity, in direct contact with the embryo [25, 27, 28]. Presumably, previous procedure of injecting DiO or DiI could have somewhat delayed cyclopamine application, with most applications being delivered towards stage 19.
Embryos were fixed in Dent’s fixation solution (4:1 methanol:DMSO) for two hours at room temperature, dehydrated in methanol and left overnight at −80°C to postfix. Large embryos were skinned before immersion in the fixative. Embryos were bleached for 24 h at room temperature in Dent’s bleaching (4:1:1 methanol:DMSO:H2O2). Primary antibody was diluted in PBST, 5% normal goat serum (NGS) and 5% DMSO. The Antibody used was anti collagen type 9 (1:20, DSHB). Immunolabeling was carried out for 48 h at 4°C in agitation. Primary antibodies were washed six times (1 hour each) in PBST and incubated overnight at 4°C with Alexa Fluor 488 goat anti-mouse IgG (H + L) (A-11001, Molecular Probes) or Alexa Fluor 596 goat anti-mouse IgG (H + L) (A-11031, Molecular Probes) as secondary antibodies. The secondary antibodies were washed another six times (1 hour each) in PBST and cleared in Scale  for at least five days. Embryos were photographed in stereoscopic fluorescent microscope Olympus MVX10 with a Qimaging camera.
This work was funded by a Fondecyt grant 1120424 (Government of Chile) to AOV. We thank Sergio Soto-Acuña for Figure 2.00.
- Holmgren N: Studies on the phylogeny of birds. Acta Zool. 1955, 36: 243-328. 10.1111/j.1463-6395.1955.tb00381.x.View ArticleGoogle Scholar
- Hinchliffe JR: “One, two, three” or “two, three, four”: an embryologist’s view of the homologies of the digits and carpus of modern birds. Beginnings of birds. Edited by: Hecht M, Ostrom JH, Viohl G, Wellnhofer P. 1985, Eichstätt: Freunde des Jura-Museum, 141-147.Google Scholar
- Burke C, Feduccia A: Developmental patterns and the identification of homologies in the Avian Hand. Science. 1997, 278: 666-668. 10.1126/science.278.5338.666.View ArticleGoogle Scholar
- Gauthier JA: Saurischian monophyly and the origin of birds. Mem Calif Acad Sci. 1986, 8: 1-55.Google Scholar
- Wagner GP, Gauthier JA: 1, 2, 3 = 2, 3, 4: a solution to the problem of the homology of the digits in the avian hand. Proc Natl Acad Sci U S A. 1999, 96: 5111-5116. 10.1073/pnas.96.9.5111.PubMedPubMed CentralView ArticleGoogle Scholar
- Rauhut OWM: The interrelationships and evolution of basal theropod dinosaurs. Spec Pap Palaeontol. 2003, 69: 1-213.Google Scholar
- Carrano MT, Benson RBJ, Sampson SD: The phylogeny of Tetanurae (Dinosauria: Theropoda). J Syst Palaeontol. 2012, 10: 211-300.View ArticleGoogle Scholar
- Galis F, Kundrát M, Sinervo B: An old controversy solved: bird embryos have five fingers. Trends Ecol Evol. 2003, 18: 7-9. 10.1016/S0169-5347(02)00018-6.View ArticleGoogle Scholar
- Galis F, Kundrát M, Metz JAJ: Hox genes, digit identities and the theropod/ bird transition. J Exp Zool B Mol Dev Evol. 2005, 304: 198-205.PubMedView ArticleGoogle Scholar
- Xu X, Clark JM, Mo J, Choiniere J, Forster CA, Erickson GM, Hone DWE, Sullivan C, Eberth DA, Nesbitt S, Zhao Q, Hernandez R, Jia C, Han F, Guo Y: A Jurassic ceratosaur from China helps clarify avian digit homologies. Nature. 2009, 459: 940-944. 10.1038/nature08124.PubMedView ArticleGoogle Scholar
- Seki R, Kamiyama N, Tadokoro A, Nomura N, Tsuihiji T, Manabe M, Tamura K: Evolutionary and Developmental Aspects of Avian-Specific Traits in Limb Skeletal Pattern. Zool Sci. 2012, 29: 631-644. 10.2108/zsj.29.631.PubMedView ArticleGoogle Scholar
- Vargas A, Wagner G, Gauthier J: Limusaurus and bird digit identity. Nat Prec. 2009,http://precedings.nature.com/documents/3828/version/1,Google Scholar
- Xu X, Mackem S: Tracing the evolution of Avian Wing Digits. Curr Biol. 2013, 23: 538-544. 10.1016/j.cub.2013.04.071.View ArticleGoogle Scholar
- Vargas AO, Fallon JF: Birds have dinosaur wings: The molecular evidence. J Exp Zool B Mol Dev Evol. 2005, 304: 86-90.PubMedView ArticleGoogle Scholar
- Vargas AO, Kohlsdorf T, Fallon JF, Van den Brooks J, Wagner GP: The evolution of HoxD11 expression in the bird wing: insights from alligator mississippiensis. PLoS One. 2008, 3: e3325-10.1371/journal.pone.0003325.PubMedPubMed CentralView ArticleGoogle Scholar
- Wang Z, Young RL, Xue H, Wagner GP: Transcriptomic analysis of avian digits reveals conserved and derived digit identities in birds. Nature. 2011, 477: 583-586. 10.1038/nature10391.PubMedView ArticleGoogle Scholar
- Woltering JM, Duboule D: The origin of digits: expression patterns versus regulatory mechanisms. Dev Cell. 2010, 18: 526-532. 10.1016/j.devcel.2010.04.002.PubMedView ArticleGoogle Scholar
- Capek D, Metscher BD, Müller GB: Thumbs down: a molecular-morphogenetic approach to avian digit homology. J Exp Zool B Mol Dev Evol. 2014, 322: 1-12. 10.1002/jez.b.22545.PubMedView ArticleGoogle Scholar
- Tamura K, Nomura N, Seki R, Yonei-Tamura S, Yokoyama H: Embryological evidence identifies wing digits in birds as digits 1, 2, and 3. Science. 2011, 331: 753-757. 10.1126/science.1198229.PubMedView ArticleGoogle Scholar
- Riddle RD, Johnson RL, Laufer E, Tabin CJ: Sonic hedgehog mediates the polarizing activity of the ZPA. Cell. 1993, 75: 1401-1416. 10.1016/0092-8674(93)90626-2.PubMedView ArticleGoogle Scholar
- Drossopoulou G, Lewis KE, Sanz-Ezquerro JJ, Nikbakht N, McMahon AP, Hofmann C, Tickle C: A model for anteroposterior patterning of the vertebrate limb based on sequential long- and short-range Shh signalling and Bmp signalling. Development. 2000, 127: 1337-1348.PubMedGoogle Scholar
- Yang Y, Drossopoulou G, Chuang PT, Duprez D, Marti E, Bumcrot D, Vargesson N, Clarke J, Niswander L, McMahon A, Tickle C: Relationship between dose, distance and time in Sonic-Hedgehog-mediated regulation of anteroposterior polarity in the chick limb bud. Development. 1997, 124: 4393-4404. 23PubMedGoogle Scholar
- Harfe BD, Scherz PJ, Nissim S, Tian H, McMahon AP, Tabin CJ: Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell. 2004, 118: 517-528. 10.1016/j.cell.2004.07.024.PubMedView ArticleGoogle Scholar
- Towers M, Signolet J, Sherman A, Sang H, Tickle C: Insights into bird wing evolution and digit specification from polarizing region fate maps. Nat Commun. 2011, 2: 426-PubMedView ArticleGoogle Scholar
- Vargas AO, Wagner GP: Frame-shifts of digit identity in bird evolution and cyclopamine-treated wings. Evol Dev. 2009, 11: 163-169. 10.1111/j.1525-142X.2009.00317.x.PubMedView ArticleGoogle Scholar
- Chatterjee S: Counting the fingers of birds and dinosaurs. Science. 1998, 280: 355a-10.1126/science.280.5362.355a.View ArticleGoogle Scholar
- Chen JK, Taipale J, Cooper MK, Beachy PA: Inhibition of Hedgehog signalling by direct binding of cyclopamine to Smoothened. Genes Dev. 2002, 16: 2734-2748.Google Scholar
- Scherz PJ, McGlinn E, Nissim S, Tabin CJ: Extended exposure to sonic hedgehog is required for patterning the posterior digits of the vertebrate limb. Dev Biol. 2007, 308: 343-354. 10.1016/j.ydbio.2007.05.030.PubMedPubMed CentralView ArticleGoogle Scholar
- Dahn RD, Fallon JF: Interdigital regulation of digit identity and homeotic transformation by modulated BMP signaling. Science. 2000, 289: 438-441. 10.1126/science.289.5478.438.PubMedView ArticleGoogle Scholar
- Suzuki T, Hasso SM, Fallon JF: Unique SMAD1/5/8 activity at the phalanx –forming region determines digit identity. Proc Natl Acad Sci U S A. 2008, 105: 4185-4190. 10.1073/pnas.0707899105.PubMedPubMed CentralView ArticleGoogle Scholar
- Sears KA, Bormet AK, Rockwell A, Powers LE, Cooper LN, Wheeler MB: Developmental basis of mammalian digit reduction: a case study in pigs. Evol Dev. 2011, 13: 533-541. 10.1111/j.1525-142X.2011.00509.x.PubMedView ArticleGoogle Scholar
- Holmgren N: An embryological analysis of the mammalian carpus and its bearing upon the question of the origin of the tetrapod limb. Acta Zool. 1952, 33: 1-115. 10.1111/j.1463-6395.1952.tb00360.x.View ArticleGoogle Scholar
- Hamrick MW: Developmental mechanisms of digit reduction. Evol Dev. 2002, 4: 247-248. 10.1046/j.1525-142X.2002.02012.x.PubMedView ArticleGoogle Scholar
- Vargesson N, Clarke JD, Vincent K, Coles C, Wolpert L, Tickle C: Cell fate in the chick limb bud and relationship to gene expression. Development. 1997, 124: 1909-1918.PubMedGoogle Scholar
- Towers M, Mahood R, Yin Y, Tickle C: Integration of growth and specification in chick wing digit-patterning. Nature. 2008, 452: 882-886. 10.1038/nature06718.PubMedView ArticleGoogle Scholar
- Di-Poï N, Montoya-Burgos JI, Miller H, Pourquié O, Milinkovitch MC, Duboule D: Changes in Hox genes structure and function during the evolution of the squamate body plan. Nature. 2010, 464: 99-103. 10.1038/nature08789.PubMedView ArticleGoogle Scholar
- Young RL, Caputo V, Giovannotti M, Kohlsdorf T, Vargas AO, May GE, Wagner GP: Evolution of digit identity in the three-toed Italian skink Chalcides chalcides: a new case of digit identity frame shift. Evol Dev. 2009, 11: 647-658. 10.1111/j.1525-142X.2009.00372.x.PubMedView ArticleGoogle Scholar
- Cohn MJ, Lovejoy CO, Wolpert L, Coates MI: Branching, segmentation and the metapterygial axis: pattern versus process in the vertebrate limb. Bioessays. 2002, 24: 460-465. 10.1002/bies.10088.PubMedView ArticleGoogle Scholar
- Sheth R, Marcon L, Bastida MF, Junco M, Quintana L, Dahn R, Kmita M, Sharpe J, Ros MA: Hox genes regulate digit patterning by controlling the wavelength of a turing-type mechanism. Science. 2012, 338: 1478-1480.View ArticleGoogle Scholar
- Thulborn RA: Theropod dinosaurs, progenesis and birds: homology of digits in the manus. Neues Jb Geol Paläontol Abh. 2006, 242: 205-241.Google Scholar
- Fernández-Terán MA, Hinchliffe JR, Ros MA: Birth and death of cells in limb development: a mapping study. Dev Dyn. 2006, 235: 2521-2537. 10.1002/dvdy.20916.PubMedView ArticleGoogle Scholar
- Wake D, Wake MH, Specht C: Homoplasy: From Detecting Pattern to Determining Process and Mechanism of Evolution. Science. 2011, 331: 1032-1035. 10.1126/science.1188545.PubMedView ArticleGoogle Scholar
- Davidson EH: Emerging properties of animal gene regulatory networks. Nature. 2010, 468: 911-920. 10.1038/nature09645.PubMedPubMed CentralView ArticleGoogle Scholar
- Montavon T, Garrec JFL, Kerzberg M, Duboule D: Modelling HOX genes regulation in digits: reverse collinearity and the molecular origin of thumbness. Genes Dev. 2008, 22: 236-259.View ArticleGoogle Scholar
- Tarchini B, Duboule D: Control of HoxD collinearity during early limb development. Dev Cell. 2006, 10: 93-103. 10.1016/j.devcel.2005.11.014.PubMedView ArticleGoogle Scholar
- Acloque H, Wilkinson DG, Nieto MA: In situ hybridization analysis of chick embryos in whole‒Mount and tissue sections. Methods Cell Biol. 2008, 87: 169-185.PubMedView ArticleGoogle Scholar
- Hamburger V, Hamilton HL: A series of normal stages in the development of the chick embryo. Dev Dyn. 1992, 195: 231-272. 10.1002/aja.1001950404.PubMedView ArticleGoogle Scholar
- Li S, Muneoka K: Cell migration and chick limb development: chemotactic action of FGF-4 and the AER. Dev Biol. 1999, 211: 335-347. 10.1006/dbio.1999.9317.PubMedView ArticleGoogle Scholar
- Sato K, Koizumi Y, Takahashi M, Kuroiwa A, Tamura K: Specification of cell fate along the proximal-distal axis in the developing chick limb bud. Development. 2007, 134: 1397-1406. 10.1242/dev.02822.PubMedView ArticleGoogle Scholar
- Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A: Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci. 2011, 14: 1481-1488. 10.1038/nn.2928.PubMedView ArticleGoogle Scholar
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