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. 2023 Nov 23;103(2):103317. doi: 10.1016/j.psj.2023.103317

Transcriptome-based comparison reveals key genes regulating allometry growth of forelimb and hindlimb bone in duck embryos

Qifan Wu *,†, Hehe Liu *, Qinglan Yang *, Jingjing Qi *, Yang Xi *, Qian Tang *, Rui Wang *, Jiwei Hu *, Liang Li *,1
PMCID: PMC10792745  PMID: 38160613

Abstract

Allometric growth of the forelimb and hindlimb is a widespread phenomenon observed in vertebrates. As a typical precocial bird, ducks exhibit more advanced development of their hindlimbs compared to their forelimbs, enabling them to walk shortly after hatching. This phenomenon is closely associated with the development of long bones in the embryonic stage. However, the molecular mechanism governing the allometric growth of duck forelimb and hindlimb bones is remains elusive.

In this study, we employed phenotypic, histological, and gene expression analyses to investigate developmental differences between the humerus (forelimb bone) and tibia/femur (hindlimb bones) in duck embryos. Our results revealed a gradual increase in weight and length disparity between the tibia and humerus from E12 to E28 (embryo age). At E12, endochondral ossification was observed solely in the tibia but not in the humerus. The number of differentially expressed genes (DEGs) gradually increased at H12 vs. T12, H20 vs. T20, and H28 vs. T28 stages consistent with phenotypic variations. A total of 38 DEGs were found across all 3 stages. Protein-protein interaction network analysis demonstrated strong interactions among members of HOXD gene family (HOXD3/8/9/10/11/12), HOXB gene family (HOXB8/9), TBX gene family (TBX4/5/20), HOXA11, SHOX2, and MEIS2. Gene expression profiling indicated higher expression levels for all HOXD genes in the humerus compared to tibia while opposite trends were observed for HOXA/HOXB genes with low or no expression detected in the humerus. These findings suggest distinct roles played by different clusters within HOX gene family during skeletal development regulation of duck embryo's forelimbs versus hind limbs. Notably, TBX4 exhibited high expression levels specifically in tibia whereas TBX5 showed similar patterns exclusively within humerus as seen previously across other species’ studies. In summary, this study identified key regulatory genes involved in allometric growth of duck forelimb and hindlimb bones during embryonic development. Skeletal development is a complex physiological process, and further research is needed to elucidate the regulatory role of candidate genes in endochondral ossification.

KEY WORDS: duck, forelimb, hindlimb, ossification, embryonic development

INTRODUCTION

The development of limbs in vertebrates has a wide range of allometric characteristics (Cooper, 2019). The diversity of limb skeletal proportion makes possible the diversity of animal behavior, including the dynamic flight of bat forelimbs, the swimming driven by whale fins, and the jumping movement of kangaroo hind limbs (Andrews and Skewes, 2017; Doube et al., 2018; Kelly and Sears, 2011). Many precocial birds, such as duck and swan, experience unique developmental trajectories in which chicks can run after hatching but take a while to fly. At 30 d after hatching, the length of the hind limb and forelimb of ducks are 90% and 60% of the adult value, respectively (Dial and Carrier, 2012).

The longitudinal limb bone growth is driven by endochondral ossification under the control of the growth plate, where chondrocytes undergo a coordinated life cycle of proliferation, matrix production, hypertrophy, and cell death/trans differentiation. (Mark and Zhou, 2016). Many studies have shown that the multiple stages of chondrocyte enlargement are the basis of the difference in bone proportion, and the greatest contribution of bone element elongation comes from the sharp increase of the volume of hypertrophic chondrocytes in the growth plate (Breur et al., 1991; Cooper et al., 2013). In growing mammals, there is a linear relationship between the average hypertrophic chondrocyte volume and bone elongation (Rolian, 2020).

In recent years, the rapid progress of molecular biology research on embryonic bone development has confirmed that signal molecules and transcription factors were the main regulators of basic bone development (Day and Yang, 2008). In developing embryos, limb-specific expression of PITX1, TBX4, and TBX5 has been proven to be an important gene regulating limb morphology (Agarwal et al., 2003; Margulies et al., 2001; Menke et al., 2008). Whole embryo culture, transcriptomics, and RNA interference identified TBX1 and FGF11 as new regulators of mouse limb development (Tejedor et al., 2020). Transcriptome analysis of mouse embryonic homologous tissues showed that 44 differential transcripts such as RDH10, FRZB, TBX18, and HIP showed different expressions between forelimb and hindlimb tissues (Shou et al., 2005).

The embryonic limb skeleton is an excellent model for studying bone development. The growth rates of forelimb and hindlimb bones may differ during the duck embryo stage. This experiment was designed to compare the skeletal development of the forelimb and hind limb in duck embryos from the aspects of bone length, weight, endochondral ossification, and gene expression, to explore the molecular mechanism that causes the difference in bone length.

MATERIALS AND METHODS

Ethics Statement

The experimental procedures and protocols applied in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan Agricultural University (Permit No. DKY-B20221488) and carried out the approved guidelines.

Experimental Design and Sampling

Fertilized eggs from Nonghua ducks were collected within 6 h after laying and stored at 15°C before being incubated (BLF-440C, Chengdu, China) at 37.8 ± 0.2°C and 60% relative humidity starting from 1 wk after laying. The eggs were provided by the Waterfowl Breeding Farm of Sichuan Agricultural University (Yaan, Sichuan). Embryo weight was recorded daily from E6 onwards while forelimbs’ and hind limbs’ weights were measured every 2 d starting from E10. The weight and length of the humerus, femur, and tibia were recorded every 2 d from 5 ducks between E12 and E28 while bone calcification staining was performed on 2 ducks per time point (E12, E16, E20, E24, and E28). The tissues of the humerus, femur, and tibia were selected for hematoxylin-eosin staining from 5 ducks per time point (E12, E16, E20, E24, and E28). Additionally, transcriptome analysis was performed on humerus’ and tibia's tissues collected from 3 embryos with similar weights at E12, E20, and E28.

Embryonic Bone Calcification Staining

Skeletons of the embryos were stained with Alcian blue 8 GX and alizarin red S (Beijing Solarbio Science & Technology Co. Ltd., Beijing, China) for cartilage and ossified bones, respectively. The staining procedures, adapted from the methods of Dingerkus and Uhler (1977), Yoshifumi and Masaoki (1999), Young et al. (2000), are detailed in Table S1. Skeletal observations were conducted under a dissecting microscope with careful consideration of chondrification and calcification timing. Chondrification was confirmed by Alcian blue 8GX staining resulting in a blue coloration, while calcification was identified by red coloration using alizarin red S.

Paraffin Section

Humerus, femur, and tibia tissues were fixed in 4% paraformaldehyde for over 24 h before decalcifying them in 10% ethylene diamine tetraacetic acid at room temperature for 9 wk followed by embedding them into paraffin blocks. Serial sections measuring 3 μm were prepared for subsequent treatment. Hematoxylin-eosin staining was performed to visualize the changes in the tissues, the nucleus was stained in blue, and the cytoplasm was in red (Rigueur and Lyons, 2014). The sections took panoramic photos with 20× magnification using a Digital Pathology Total Section Scanner (VS120-S6-W, Olympus, Japan).

RNA Extraction and RNA- Sequencing

Total RNA extraction utilized humerus and tibia tissues. Trizol reagent (Invitrogen, Shanghai, China) was employed according to the manufacturer's instructions to isolate total RNA. Poly(A) + messenger RNA (mRNA) was purified using mRNA capture beads, and subsequently fragmented randomly into small fragments through divalent cations present in a fragmentation buffer solution. Then the mRNA was randomly segmented into small fragments by divalent cations in a fragmentation buffer.

These short fragments served as templates for first-strand complementary DNA (cDNA) synthesis utilizing random hexamer primers while second-strand cDNA synthesis involved RNaseH treatment along with DNA polymerase I activity. Short cDNA fragments were purified and then connected with sequencing adapters. After agarose gel electrophoresis, the target fragments of 300 to 500 bp were selected for PCR amplification (Jingjing et al., 2014; Jingjing et al., 2015). The quality and size of the cDNA libraries for sequencing were checked. Then, cDNA libraries were sequenced (Novaseq 6000 Illumina, San Diego, California). Fastqc analyzed raw reads for quality, and high-quality reads with Q > 20 were obtained using the NGSQC Toolkit (version: 2.3.3) (Fumagalli et al., 2014). Finally, functions of the unigenes were annotated based on sequence similarities to sequences in the public UniProt database (Grabherr et al., 2011).

Raw Data Processing

Raw reads underwent quality assessment via Fastqc analysis, high-quality reads having Q-score (integer mapping of the probability of base calling errors) >20 being retained using the NGSQC Toolkit (version:2.3) (Fumagalli et al., 2014). Unigene functions were annotated based on sequence similarities to sequences within the public UniProt database (Shengnan et al., 2019). Hisat2 (v2.1.0) was used to align the clean data to reference the genome of Anas platyrhynchos (RefSeq: GCA_015476345.1). The mapped data was collated and formatted by Samtools. Then the output GTF files were merged into a single unified transcript using the stringTie merge function (Meng et al., 2018). The merged transcripts were compared to the reference annotation using the gffcompare program (v0.10.1, https://ccb.jhu.edu/software/stringtie/gffcompare.shtml) (Feibiao et al., 2019). The quantification of gene expression levels was performed as follows. The gene expression levels were estimated according to fragments per kilobase of transcript per million fragments mapped (CPM). The reads count value was used for DEGs identification and the CPM value was used for all other analyses. DEGs between the experimental groups were identified using the DEseq2 R package. P-value < 0.05 and |log2FC| >1 were set as the screening criteria for significantly differential expression. SIMCA-P software (version 14.1, Umetrics, Umea, Sweden) was used for the principal component analysis of transcriptome data (Thévenot et al., 2015).

Gene Function and Pathway Enrichment Analysis

Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs was achieved using KOBAS 3.0.11 (http://kobas.cbi.pku.edu.cn/) (Xie et al., 2011). Human was used as a reference species. Q-value < 0.05 was considered significant. R package and related packages are used to realize data visualization.

Gene Expression Pattern Analysis

The gene expression pattern was analyzed by Short Time-series Expression Miner software (v1.3.13, Redwood shore, California)  (Ernst and Bar-Joseph, 2006). The “Log normalize data” method was adopted in the strategy of expression quantity transformation. Other options remained at default settings since they have yielded optimal results when applied to both biological and simulated data. The expression level used in this study was the previously calculated CPM value. The P-value of the clustered profile was less than 0.05, which was considered significant (Zhongxian et al., 2018).

RESULTS

Development of Limb Bones

We documented the weight fluctuations of ducks embryos during their embryonic stage. The embryo weight exhibited a gradual increase in the early embryonic stage, followed by a rapid surge from E14 (Figure S1A). The growth pattern of hindlimb weight mirrored that of body weight, while the growth rate of forelimb weight gradually diminished in the late embryonic stage (Figure S1B).

Similar developmental discrepancies were observed in both the forelimb and hindlimb bones of duck embryos (Figure 1A). The tibia and femur experienced substantial weight gain from E12 to E28, whereas the humerus displayed a slower weight increment after E24 (Figure 1B). Tibia and humerus length demonstrated rapid augmentation from E12 to E28, with humerus length exhibiting slower growth after E24 (Figure 1C). The weight ratios of the tibia and humerus at E12, E20, and E28 were recorded as 1.48, 2.05, and 5.25 respectively (Figure S2), displaying significant differences among different embryonic stages (P < 0.01). The ratios of tibia to humerus length at E12, E20, and E28 were 1.48, 1.68, and 2.46 (Figure S2), respectively, displaying significant differences among different embryonic stages (P < 0.01). As embryonic age increased, disparities in both weight and length between tibia and humerus became more pronounced.

Figure 1.

Figure 1

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Development of limb bones in duck embryo. (A) Morphological changes of forelimbs and hindlimbs in embryonic ducks. Abbreviations: E, embryo age; F, forelimb; H, hindlimb. (B) Changes in bone weight of limbs during the embryonic period. The error bars on the graphs represent std. dev. (C) Changes in bone length of limbs during the embryonic period. The error bars on the graphs represent std. dev. (D) Bone calcification staining at 5-time points during the embryonic period. Abbreviations: F, femur; H, humerus; T, tibia.

Calcification and Histomorphological Analysis of Limb Bones

Compared to other bone regions, limb bones initiated cartilage-to-bone transformation earlier during development. Ossification centers had already emerged in both the humerus and tibia at E12, calcification was nearly complete towards the end of the embryo formation process (Figure 1D). Calcification rates appeared similar between forelimbs and hind limbs bones.

We delineated the 5 histological developmental stages for a duck's humerus, femur, and tibia; sectional photographs depicted histological structures within the middle portions of these 3 bones (Figure 2). Generally, ossification (calcification) occurs more rapidly in the tibia compared to the humerus and femur. The interior of the humerus and femur were all hypertrophic chondrocytes at E12, indicating that calcification had not yet commenced. However, the bone marrow cavity has appeared in the tibia, suggesting that calcification had been initiated. At E20, hypertrophic chondrocytes completely disappeared and bone matrix formed in both the humerus and tibia with embedded osteocytes. At E28, cortical bone was formed around the humerus and femur while trabecular bone appeared in the middle of the tibia (Figure 2).

Figure 2.

Figure 2

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Histological changes of limb bones in embryonic ducks. These pictures show the middle of each bone. Abbreviations: BC, blood cell; BM, bone matrix; CB, cortical bone; CC, calcified cartilage; F, femur; H, humerus; HC, hypertrophic chondrocyte; MC, marrow cavity; MT, muscle tissue; O, osteocyte; T, tibia; TB, trabecular bone.

Overview of Transcriptome Dataset

A total of 18 cDNA libraries were synthesized for transcriptome RNA-sequencing (RNA-seq). The RNA-seq yielded a total of 381.50 million (M) clean reads corresponding to 113.97 gigabases (Gb) of clean data with over 91.70% bases scoring Q30 (the percentage of bases with a mass value of 30 or greater) (Table S2). Additionally, 92.61% of clean reads from ducks were properly mapped to the Anas platyrhynchos reference genome (Table S3). Principal component analysis revealed that PC1 accounted for 28.5% variation among transcripts (Figure 3), demonstrating good repeatability within sample groups and reliable sample quality.

Figure 3.

Figure 3

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Principal component analysis of transcriptome data of limb bones. Abbreviations: H, humerus; T, tibia.

Differential Expressed Gene Analysis

We compared gene expression between different embryonic ages within each bone type resulting in H12 vs. H20 and H20 vs. H28 having 2473 and 1637 DEGs, respectively (Figure S3). Similarly, T12 vs. T20 and T20 vs. T28 showed 2579 and 1039 DEGs, respectively (Figure S3). Compared with E12 vs. E20, the DEGs of the humerus and tibia were reduced at E20 vs. E28, which may be attributed to peak calcification occurring between E12 and E20 (Figure 1D).

The gene expression of the humerus and tibia at the same embryonic age was compared. H12 vs. T12, H20 vs. T20, and H28 vs. T28 exhibited 207, 613, and 706 DEGs, respectively (Figure 4A). The number of DEGs in the tibia and humerus gradually increased at all 3-time points, consistent with the progressive weight and length differences observed between these skeletal elements. This suggests that gene expression may play a pivotal role in driving developmental disparities between the humerus and tibia. We identified a set of 38 common DEGs across these 3 time points through comparative analysis (Figure 4B). Gene ontology enrichment analysis revealed their involvement in key biological processes such as embryonic skeletal system morphogenesis, limb morphogenesis, cartilage development, and transcriptional regulation (Figure 4C). Furthermore, mapping these shared DEGs to various KEGG pathways highlighted their relevance to organic systems, disease mechanisms, and metabolism (Figure S4).

Figure 4.

Figure 4

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Analysis of differentially expressed genes in embryonic tibia and humerus. (A) Volcano plots of genes in groups H12 vs. T12, H20 vs. T20, and H28 vs. T28. (B) The common DEGs between H12 vs. T12, H20 vs. T20, and H28 vs. T28. (C) GO enrichment analysis of the shared DEGs. Abbreviations: H, humerus; T, tibia.

Protein-protein Interaction Network Analysis

To gain further insights into potential interactions among the identified 38 DEGs, we performed a protein-protein interaction network analysis of the 38 DEGs using the STRING Database (version 11.0) with a filter threshold of 0.4. Notably strong interactions were observed among members of HOXD gene family (HOXD3, HOXD8, HOXD9, HOXD10, HOXD11, and HOXD12), HOXB gene family (HOXB8 and HOXB9), TBX gene family (TBX4, TBX5, and TBX20), as well as HOXA11, SHOX2 and MEIS2 (Figure 5). These 14 genes are mainly involved in embryonic skeletal system morphogenesis, limb morphogenesis, and DNA-binding transcription factor activity (Figure 4C), indicating their crucial role in the regulation of duck limb bone development.

Figure 5.

Figure 5

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Protein-protein interaction (PPI) network analysis of the 38 DEGs. The thicker the lines, the stronger the interaction between the 2 proteins. The more the number of protein nodes, the larger the sphere.

Gene Expression Pattern Analysis

Cluster analysis was conducted on candidate genes expression at E12, E20, and E28 (Figure 6). Candidate genes were mainly clustered into 2 groups: one exhibiting higher expression levels in the humerus compared to those in the tibia at each time point; while another group showed opposite patterns. It suggests that the sustained differential expression of candidate genes is a significant factor contributing to the weight and length disparities between the humerus and tibia during embryonic development. We further examined the expression profiles of candidate genes at 3 different embryonic time points (Figure S5, Figure 6) and observed similar expression trends in most genes between the humerus and tibia. However, TBX4 exhibited an opposite expression trend in these 2 bones, while HOXB9 was expressed exclusively in the tibia but not in the humerus.

Figure 6.

Figure 6

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Gene Expression Pattern Analysis of candidate genes. The expression level used in the heat map was the log scale of the CPM value. The number in the expression profile represents the profile number. The horizontal axis represents 3 embryonic time points, and the vertical axis represents the gene expression level. Colored boxes indicate that the P-value < 0.05. Abbreviations: H, humerus; T, tibia.

DISCUSSION

During the embryonic period, there was a gradual increase in the difference multiples of humerus and tibia lengths (Figure 1C, Figure S2). The elongation of long bones is primarily driven by the intrachondral ossification process (Geiger et al., 2014; Rolian, 2020). Histological examination revealed that endochondral ossification in the tibia commenced at E12, preceding that of the humerus. Furthermore, there was a progressive increase in the number of differentially expressed genes between these 2 bones at 3 time points. These findings suggest potential candidate genes involved in regulating the process of endochondral ossification, thereby influencing differential growth rates between forelimb and hindlimb bones.

In the comparison of the humeral and tibial transcriptome, differential expression of multiple homeobox genes belonging to the HOXA, HOXB, and HOXD gene clusters was observed (Figure 6). These gene clusters are located on distinct chromosomes in the Anas platyrhynchos genome without any interconnection (Li et al., 2021). Among the candidate genes identified in this study, different HOX gene clusters exhibited different expression patterns during embryonic development of the tibia and humerus. Specifically, HOXA11 and HOXB8/9 were found to be expressed in the embryonic tibia but showed low or no expression in the humerus (Figure 6). In mice, mutations in HOXA11 and HOXD11 have been reported to severely affect radius and ulna formation in forelimbs (Boulet and Capecchi, 2004, Kherdjemil et al., 2016, Wellik and Capecchi, 2003). Deletion of the HOXB gene cluster significantly influences animal bone development (Carlson et al., 2001, Medina-Martinez et al., 2000, Zakany and Duboule, 2007). The regulation of continuous IGF2BP1 expression by the HOXB gene cluster has been shown to increase body size by approximately 15% in Peking ducks compared with mallard ducks after hatching (Zhou et al., 2018). Furthermore, our previous Genome-wide association study revealed a significant association between duck tibia length and the HOXB cluster.

During the embryonic stage, the expression of all candidate HOXD genes (HOXD3/8/9/10/11/12) are higher in the humerus compared to the tibia (Figure 6). HOXD genes play a crucial role in limb growth and patterning, and their activation is governed by intricate transcriptional regulation, resulting in collinear expression domains both spatially and temporally (Dlugaszewska et al., 2006; Tarchini and Duboule, 2006). The expression quantities of HOXD genes exhibit significant variations between chicken and mouse as well as during fore- and hindlimb bud development, with particularly pronounced differences observed in chicken (Yakushiji-Kaminatsui et al., 2018).

The T-domain transcription factors, TBX4 and TBX20, exhibit high expression levels in the embryonic tibia, while their expression is significantly lower in the humerus. Conversely, TBX5 demonstrates an inverse pattern of expression (Figure 6). TBX5 plays a crucial role in forelimb bud formation and continued outgrowth in chickens and mice (Rallis et al., 2003; Minguillon et al., 2005). The TBX4 transcription factor is crucial for normal hindlimb and vascular development in vertebrates (Naiche, 2003; Krause et al., 2004; Menke et al., 2008). It suggests that TBX4 and TBX5 have extensive regulatory effects on limb development in different species. No studies have been found on the relationship between Tbx20 and limb development.

Among other candidate genes, the expression levels of SHOX2 and MEIS2 in the humerus were found to be higher compared to those in the tibia (Figure 6). SHOX2 plays a crucial role in promoting chondrocyte proliferation and maturation specifically in the proximal limb skeleton (Yu et al., 2007). Genetic interactions between SHOX2 and HOX genes have been demonstrated during the regional growth and development of mouse limbs (Neufeld et al., 2014). In humans and mice, inadequate expression or inactivation of the SHOX2 gene can result in limb dwarfism and deformities (Gahunia et al., 2009; Xu et al., 2019). Differential expression patterns of MEIS2 during limb development have been associated with variations in vertebrate limb morphology diversification (Capdevila et al., 1999; Dai et al., 2014).

CONCLUSION

This study once again confirms the crucial roles of TBX5 and TBX4 in the growth of forelimb and hindlimb bones, respectively, in vertebrates. Furthermore, we have identified a significant association between the HOXD gene family (HOXD3/8/9/10/11/12), as well as the HOXB gene family (HOXB8/9), with allometric patterns observed in embryonic duck tibia and humerus.

ACKNOWLEDGMENTS

This study was funded by the China Agricultural Research System of MOF and MARA (CARS-42-4), and the Key Technology Support Program of Sichuan Province (2021YFYZ0014, 2021JDJQ0008).

DISCLOSURES

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2023.103317.

Appendix. Supplementary materials

mmc1.docx (434.2KB, docx)

REFERENCES

  1. Agarwal P., Wylie J.N., Galceran J., Arkhitko O., Li C., Deng C., Grosschedl R., Bruneau B.G. Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo. Development. 2003;130:623. doi: 10.1242/dev.00191. [DOI] [PubMed] [Google Scholar]
  2. Andrews R.M., Skewes S.A. Developmental origin of limb size variation in lizards. Evol. Dev. 2017;19:136–146. doi: 10.1111/ede.12221. [DOI] [PubMed] [Google Scholar]
  3. Boulet A.M., Capecchi M.R. Multiple roles of Hoxa11 and Hoxd11 in the formation of the mammalian forelimb zeugopod[J] Development. 2004;131(2):299–309. doi: 10.1242/dev.00936. [DOI] [PubMed] [Google Scholar]
  4. Breur G.J., Vanenkevort B.A., Farnum C.E., Wilsman N.J. Linear relationship between the volume of hypertrophic chondrocytes and the rate of longitudinal bone growth in growth plates. J. Orthop. Res. 1991;9:348–359. doi: 10.1002/jor.1100090306. [DOI] [PubMed] [Google Scholar]
  5. Capdevila J., Tsukui T., Esteban C.R., Zappavigna V., Belmonte J.C.I. Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin. Mol. Cell. 1999;4:839–849. doi: 10.1016/s1097-2765(00)80393-7. [DOI] [PubMed] [Google Scholar]
  6. Carlson M.R.J., Komine Y., Bryant S.V., et al. Expression of Hoxb13 and Hoxc10 in developing and regenerating Axolotl limbs and tails[J] Dev. Biol. 2001;229(2):396–406. doi: 10.1006/dbio.2000.0104. [DOI] [PubMed] [Google Scholar]
  7. Cooper K.L. Developmental and evolutionary allometry of the mammalian limb skeleton. Integrat. Comparat. Biol. 2019;59:1356–1368. doi: 10.1093/icb/icz082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper K.L., Oh S., Sung Y., Dasari R.R., Sung Y. Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions. Nature. 2013;495:375–378. doi: 10.1038/nature11940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dai M., Wang Y., Fang L., Irwin D.M., Zhu T., Zhang J., Zhang S., Wang Z. Differential expression of Meis2, Mab21l2 and Tbx3 during limb development associated with diversification of limb morphology in mammals. PLoS One. 2014;9 doi: 10.1371/journal.pone.0106100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Day T.F., Yang Y. Wnt and hedgehog signaling pathways in bone development. J. Bone Joint Surg. Am. 2008;90:19–24. doi: 10.2106/JBJS.G.01174. [DOI] [PubMed] [Google Scholar]
  11. Dial T.R., Carrier D.R. Precocial hindlimbs and altricial forelimbs: partitioning ontogenetic strategies in mallards (Anas platyrhynchos) J. Exp. Biol. 2012;215:3703–3710. doi: 10.1242/jeb.057380. [DOI] [PubMed] [Google Scholar]
  12. Dingerkus G., Uhler L.D. Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage. Stain Technol. 1977;52:229–232. doi: 10.3109/10520297709116780. [DOI] [PubMed] [Google Scholar]
  13. Dlugaszewska B., Silahtaroglu A., Menzel C., Kübart S., Cohen M., Mundlos S., Tümer Z., Kjaer K., Friedrich U., Ropers H.-H. Breakpoints around the HOXD cluster result in various limb malformations. J. Med. Genet. 2006;43:111–118. doi: 10.1136/jmg.2005.033555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Doube M., Felder A.A., Chua M.Y., Lodhia K., Kłosowski M.M., Hutchinson J.R., Shefelbine S.J. Limb bone scaling in hopping macropods and quadrupedal artiodactyls. R. Soc. Open Sci. 2018;5 doi: 10.1098/rsos.180152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ernst J., Bar-Joseph Z. STEM: a tool for the analysis of short time series gene expression data. BMC Bioinformat. 2006;7:191. doi: 10.1186/1471-2105-7-191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Feibiao S., Lanmei W., Wenbin Z., Zaijie D. Long noncoding RNA and mRNA expression profiles following igf3 knockdown in common carp, Cyprinus carpio. Sci Data. 2019;6 doi: 10.1038/sdata.2019.24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fumagalli M., Vieira F.G., Linderoth T., Nielsen R. ngsTools: methods for population genetics analyses from next-generation sequencing data. Bioinformatics. 2014;30:1486–1487. doi: 10.1093/bioinformatics/btu041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gahunia H.K., Babyn P.S., Kirsch S., Mendoza-Londono R. Imaging of SHOX-associated anomalies. Semin. Musculoskelet. Radiol. 2009;13:236–254. doi: 10.1055/s-0029-1237691. [DOI] [PubMed] [Google Scholar]
  19. Geiger M., Forasiepi A.M., Koyabu D., Sánchez-Villagra M.R. Heterochrony and post-natal growth in mammals: an examination of growth plates in limbs. J. Evol. Biol. 2014;27:98–115. doi: 10.1111/jeb.12279. [DOI] [PubMed] [Google Scholar]
  20. Grabherr M.G., Haas B.J., Yassour M., Levin J.Z., Thompson D.A., Amit I., Adiconis X., Fan L., Raychowdhury R., Zeng Q., Chen Z., Mauceli E., Hacohen N., Gnirke A., Rhind N., Di Palma F., Birren B.W., Nusbaum C., Lindblad-Toh K., Friedman N., Regev A. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 2011;29:644–652. doi: 10.1038/nbt.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jingjing J., Deepa P., Qian W., Jung K.M., Jun L., Jun-Lin Y., Limsoon W., In-Cheol J., Nam-Hai C., Rajani S. Next generation sequencing unravels the biosynthetic ability of spearmint (Mentha spicata) peltate glandular trichomes through comparative transcriptomics. BMC Plant Biol. 2014;14:1–15. doi: 10.1186/s12870-014-0292-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jingjing J., Jung K.M., Savitha D., Gambino T.J., Jun-Lin Y., Limsoon W., Rajani S., Nam-Hai C., In-Cheol J. The floral transcriptome of ylang ylang (Cananga odorata var. fruticosa) uncovers biosynthetic pathways for volatile organic compounds and a multifunctional and novel sesquiterpene synthase. J. Exp. Bot. 2015;66:3959–3975. doi: 10.1093/jxb/erv196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kelly E.M., Sears K.E. Limb specialization in living marsupial and eutherian mammals: constraints on mammalian limb evolution. J. Mammal. 2011;92:1038–1049. [Google Scholar]
  24. Kherdjemil Y., Lalonde R.L., Sheth R., et al. Evolution of Hoxa11 regulation in vertebrates is linked to the pentadactyl state[J] Nature. 2016;539(7627):89–92. doi: 10.1038/nature19813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krause A., Zacharias W., Camarata T., Linkhart B., Law E., Lischke A., Miljan E., Simon H.G. Tbx5 and Tbx4 transcription factors interact with a new chicken PDZ-LIM protein in limb and heart development. Dev. Biol. 2004;273:106–120. doi: 10.1016/j.ydbio.2004.05.024. [DOI] [PubMed] [Google Scholar]
  26. Li J., Zhang J., Liu J., et al. A new duck genome reveals conserved and convergently evolved chromosome architectures of birds and mammals[J] Gigascience. 2021;10(1):142. doi: 10.1093/gigascience/giaa142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Margulies E.H., Kardia S.L.R., Innis J.W. A comparative molecular analysis of developing mouse forelimbs and hindlimbs using serial analysis of gene expression (SAGE)[J] Genome Res. 2001;11(10):1686–1698. doi: 10.1101/gr.192601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Medina-Martinez O., Bradley A., Ramírez-Solis R. A large targeted deletion of Hoxb1-Hoxb9 produces a series single-segment anterior homeotic transformations[J] Dev. Biol. 2000;222(1):71–83. doi: 10.1006/dbio.2000.9683. [DOI] [PubMed] [Google Scholar]
  29. Meng L., Jinjie Z., Nianwu T., Hang S., Jinling H. Horizontal gene transfer from bacteria and plants to the arbuscular mycorrhizal fungus Rhizophagus irregularis. Front. Plant Sci. 2018;9:701. doi: 10.3389/fpls.2018.00701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Menke D.B., Guenther C., Kingsley D.M. Dual hindlimb control elements in the Tbx4 gene and region-specific control of bone size in vertebrate limbs. Development. 2008;135(15):2543–2553. doi: 10.1242/dev.017384. [DOI] [PubMed] [Google Scholar]
  31. Minguillon C., Buono J.D., Logan M.P. Tbx5 and Tbx4 are not sufficient to determine limb-specific morphologies but have common roles in initiating limb outgrowth. Dev. Cell. 2005;8:75–84. doi: 10.1016/j.devcel.2004.11.013. [DOI] [PubMed] [Google Scholar]
  32. Naiche L.A. Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois. Development. 2003;130(12):2681–2693. doi: 10.1242/dev.00504. [DOI] [PubMed] [Google Scholar]
  33. Neufeld S.J., Wang F., Cobb J. Genetic interactions between Shox2 and Hox genes during the regional growth and development of the mouse limb. Genetics. 2014;198:1117–1126. doi: 10.1534/genetics.114.167460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rallis, C., B. G. Bruneau, J. Del Buono, C. E. Seidman, J. G. Seidman, S. Nissim, C. J. Tabin, and M. P. O. Logan. 2003. Tbx5 is required for forelimb bud formation and continued outgrowth, 1477-9129. [DOI] [PubMed]
  35. Rigueur D., Lyons K.M. Whole-mount skeletal staining[M] Methods Mol. Biol. 2014;1130:113–121. doi: 10.1007/978-1-62703-989-5_9. Chicago, Illinois. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rolian C. Endochondral ossification and the evolution of limb proportions. Wiley Interdiscip. Rev. Dev. Biol. 2020;9:e373. doi: 10.1002/wdev.373. [DOI] [PubMed] [Google Scholar]
  37. Shengnan L., Xinyan H., Ruixin T., Yanting G., Junzhen C., Zhen L., Xinyan Z., Ling K., Duoliang R., Hongqiong Z., Xiaohong Z., Jinquan W., Lining X., Jianbo Y., Gang Y., Qiang F., Huijun S. RNA-Seq-based transcriptomic profiling of primary interstitial cells of Cajal in response to bovine viral diarrhea virus infection. Vet. Res. Commun. 2019;43:143–153. doi: 10.1007/s11259-019-09754-y. [DOI] [PubMed] [Google Scholar]
  38. Shou S., Scott V., Reed C., Hitzemann R., Stadler H.S. Transcriptome analysis of the murine forelimb and hindlimb autopod. Dev. Dyn. 2005;234:74–89. doi: 10.1002/dvdy.20514. [DOI] [PubMed] [Google Scholar]
  39. Tarchini B., Duboule D. Control of Hoxd genes’ collinearity during early limb development. Dev. Cell. 2006;10:93–103. doi: 10.1016/j.devcel.2005.11.014. [DOI] [PubMed] [Google Scholar]
  40. Tejedor G., Laplace-Builhé B., Luz-Crawford P., Assou S., Barthelaix A., Mathieu M., Kissa K., Jorgensen C., Collignon J., Chuchana P., Djouad F. Whole embryo culture, transcriptomics and RNA interference identify TBX1 and FGF11 as novel regulators of limb development in the mouse. Sci. Rep. 2020;10:3597. doi: 10.1038/s41598-020-60217-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thévenot E.A., Roux A., Xu Y., Ezan E., Junot C. Analysis of the human adult urinary metabolome variations with age, body mass index, and gender by implementing a comprehensive workflow for univariate and OPLS statistical analyses. J. Proteome Res. 2015;14:3322–3335. doi: 10.1021/acs.jproteome.5b00354. [DOI] [PubMed] [Google Scholar]
  42. von der Mark K., Zhou X. Cartilage, Volume 1: Physiology and Development. Thousand Oaks; Los Angeles: 2016. Cell fate of growth plate chondrocytes in endochondral ossification: cell death or reprogramming into osteogenic lineage? pp. 115–142. [Google Scholar]
  43. Wellik D.M., Capecchi M.R. Hox10 and Hox11 genes are required to globally pattern the mammalian skeleton[J] Science. 2003;301(5631):363–367. doi: 10.1126/science.1085672. [DOI] [PubMed] [Google Scholar]
  44. Xie C., Mao X., Huang J., Ding Y., Wu J., Dong S., Kong L., Gao G., Li C.-Y., Wei L. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 2011;39:W316–W322. doi: 10.1093/nar/gkr483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Xu J., Wang L., Li H., Yang T., Zhang Y., Hu T., Huang Z., Chen Y. Shox2 regulates osteogenic differentiation and pattern formation during hard palate development in mice. J. Biol. Chem. 2019;294:18294–18305. doi: 10.1074/jbc.RA119.008801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yakushiji-Kaminatsui N., Lopez-Delisle L., Bolt C.C., Andrey G., Beccari L., Duboule D. Similarities and differences in the regulation of HoxD genes during chick and mouse limb development. PLoS Biol. 2018;16 doi: 10.1371/journal.pbio.3000004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yoshifumi N., Masaoki T. Development of the skeleton in Japanese quail embryos. Dev. Growth Differ. 1999;41:523–534. doi: 10.1046/j.1440-169x.1999.00454.x. [DOI] [PubMed] [Google Scholar]
  48. Young A.D., Phipps D.E., Astroff A.B. Large-scale double-staining of rat fetal skeletons using Alizarin Red S and alcian blue. Teratology. 2000;61:273–276. doi: 10.1002/(SICI)1096-9926(200004)61:4<273::AID-TERA5>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  49. Yu L., Liu H., Yan M., Yang J., Long F., Muneoka K., Chen Y. Shox2 is required for chondrocyte proliferation and maturation in proximal limb skeleton. Dev. Biol. 2007;306:549–559. doi: 10.1016/j.ydbio.2007.03.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zakany J., Duboule D. The role of Hox genes during vertebrate limb development[J] Curr. Opin. Genet. Dev. 2007;17(4):359–366. doi: 10.1016/j.gde.2007.05.011. [DOI] [PubMed] [Google Scholar]
  51. Zhongxian X., Tiandong C., Feng L., Kai T., Qing Z., Kumar M.S., Yifei D., Mingzhou L., Diyan L. The temporal expression patterns of brain transcriptome during chicken development and ageing. BMC Genom. 2018;19:1–12. doi: 10.1186/s12864-018-5301-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zhou Z., Li M., Cheng H., Fan W. An intercross population study reveals genes associated with body size and plumage color in ducks. Nat. Commun. 2018;9:2648. doi: 10.1038/s41467-018-04868-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

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