Identification of Transcription Factor Networks during Mouse Hindlimb Development

: Mammalian hindlimb development involves a variety of cells and the regulation of spatiotemporal molecular events, but regulatory networks of transcription factors contributing to hindlimb morphogenesis are not well understood. Here, we identified transcription factor networks during mouse hindlimb morphology establishment through transcriptome analysis. We used four stages of embryonic hindlimb transcription profiles acquired from the Gene Expression Omnibus database (GSE30138), including E10.5, E11.5, E12.5 and E13.5, to construct a gene network using Weighted Gene Co-expression Network Analysis (WGCNA), and defined seven stage-associated modules. After filtering 7625 hub genes, we further prioritized 555 transcription factors with An-imalTFDB3.0. Gene ontology enrichment showed that transcription factors of different modules were enriched in muscle tissue development, connective tissue development, embryonic organ development, skeletal system morphogenesis, pattern specification process and urogenital system development separately. Six regulatory networks were constructed with key transcription factors, which contribute to the development of different tissues. Knockdown of four transcription factors from regulatory networks, including Sox9, Twist1, Snai2 and Klf4, showed that the expression of limb-development-related genes was also inhibited, which indicated the crucial role of transcription factor networks in hindlimb development.

[1]  B. Smeets,et al.  Mechanical Regulation of Limb Bud Formation , 2022, Cells.

[2]  Anna B. Osipovich,et al.  A developmental lineage-based gene co-expression network for mouse pancreatic β-cells reveals a role for Zfp800 in pancreas development , 2021, Development.

[3]  P. Tschopp,et al.  Assessing evolutionary and developmental transcriptome dynamics in homologous cell types , 2021, bioRxiv.

[4]  Ines Desanlis,et al.  Transcriptional Trajectories in Mouse Limb Buds Reveal the Transition from Anterior-Posterior to Proximal-Distal Patterning at Early Limb Bud Stage , 2020, Journal of developmental biology.

[5]  M. Kadota,et al.  Developmental hourglass and heterochronic shifts in fin and limb development , 2020, bioRxiv.

[6]  O. Andreassen,et al.  A global overview of pleiotropy and genetic architecture in complex traits , 2019, Nature Genetics.

[7]  T. Kaplan,et al.  Unraveling the transcriptional regulation of TWIST1 in limb development , 2018, PLoS genetics.

[8]  Hui Hu,et al.  AnimalTFDB 3.0: a comprehensive resource for annotation and prediction of animal transcription factors , 2018, Nucleic Acids Res..

[9]  M. Torres,et al.  Coordination of limb development by crosstalk among axial patterning pathways. , 2017, Developmental biology.

[10]  M. Bonaguidi,et al.  Genome-wide analysis of facial skeletal regionalization in zebrafish , 2017, Development.

[11]  N. Ahituv,et al.  Limb development: a paradigm of gene regulation , 2017, Nature Reviews Genetics.

[12]  B. Song,et al.  Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate , 2016, PLoS genetics.

[13]  A. Visel,et al.  A unique stylopod patterning mechanism by Shox2-controlled osteogenesis , 2016, Development.

[14]  D. Agrawal,et al.  Key transcription factors in the differentiation of mesenchymal stem cells. , 2016, Differentiation; research in biological diversity.

[15]  Han Liu,et al.  Whole transcriptome expression profiling of mouse limb tendon development by using RNA‐seq , 2015, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[16]  M. Guerquin,et al.  Transcriptomic analysis of mouse limb tendon cells during development , 2014, Development.

[17]  Vincent J. Henry,et al.  OMICtools: an informative directory for multi-omic data analysis , 2014, Database J. Biol. Databases Curation.

[18]  T. He,et al.  BMP signaling in mesenchymal stem cell differentiation and bone formation. , 2013, Journal of biomedical science and engineering.

[19]  M. Kmita,et al.  Decoupling the function of Hox and Shh in developing limb reveals multiple inputs of Hox genes on limb growth , 2013, Development.

[20]  J. Cobb,et al.  Shox2 regulates progression through chondrogenesis in the mouse proximal limb , 2012, Journal of Cell Science.

[21]  S. Vokes,et al.  Integration of the transcriptional networks regulating limb morphogenesis. , 2012, Developmental biology.

[22]  Véronique Duboc,et al.  Pitx1 is necessary for normal initiation of hindlimb outgrowth through regulation of Tbx4 expression and shapes hindlimb morphologies via targeted growth control , 2011, Development.

[23]  I. Ovcharenko,et al.  Global Gene Expression Analysis of Murine Limb Development , 2011, PloS one.

[24]  Véronique Duboc,et al.  Regulation of limb bud initiation and limb‐type morphology , 2011, Developmental dynamics : an official publication of the American Association of Anatomists.

[25]  P. Kraus,et al.  Generation of mice with a novel conditional null allele of the Sox9 gene , 2011, Biotechnology Letters.

[26]  R. Kirby,et al.  Updated National Birth Prevalence estimates for selected birth defects in the United States, 2004-2006. , 2010, Birth defects research. Part A, Clinical and molecular teratology.

[27]  Steve Horvath,et al.  WGCNA: an R package for weighted correlation network analysis , 2008, BMC Bioinformatics.

[28]  P. Geetha-Loganathan,et al.  Wnt signaling in limb organogenesis , 2008, Organogenesis.

[29]  M. Buckingham,et al.  The role of Pax genes in the development of tissues and organs: Pax3 and Pax7 regulate muscle progenitor cell functions. , 2007, Annual review of cell and developmental biology.

[30]  Denis Duboule,et al.  The role of Hox genes during vertebrate limb development. , 2007, Current opinion in genetics & development.

[31]  P. Hurlin,et al.  Activities of N-Myc in the developing limb link control of skeletal size with digit separation , 2007, Development.

[32]  Y. Kawakami,et al.  The role of TGFβs and Sox9 during limb chondrogenesis , 2006 .

[33]  R. Schweitzer,et al.  Pitx1 determines the morphology of muscle, tendon, and bones of the hindlimb. , 2006, Developmental biology.

[34]  G. Neri,et al.  Limb anomalies: Developmental and evolutionary aspects. , 2002, American journal of medical genetics.

[35]  P. Beachy,et al.  Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function. , 2001, Developmental biology.

[36]  C. Sheeba,et al.  The Roles of T-Box Genes in Vertebrate Limb Development. , 2017, Current topics in developmental biology.

[37]  Toshihisa Komori,et al.  Regulation of bone development and extracellular matrix protein genes by RUNX2 , 2009, Cell and Tissue Research.

[38]  W. Woodward,et al.  Embryonic limb mesenchyme micromass culture as an in vitro model for chondrogenesis and cartilage maturation. , 2000, Methods in molecular biology.

[39]  H. Kondoh,et al.  DeltaEF1, a zinc finger and homeodomain transcription factor, is required for skeleton patterning in multiple lineages. , 1998, Development.