The formation of the vertebrate body involves the coordinated and progressive production of trunk tissues from progenitors located in the posterior of the embryo. In vitro models based on pluripotent stem cells (PSCs) replicate aspects of this process, but they lack some tissue components normally present in the trunk. Most strikingly, the notochord, a hallmark of chordates and the source of midline signals that pattern surrounding tissues, is absent from current models of human trunk formation. To investigate how trunk tissue is formed, we performed single-cell transcriptomic analysis of chick embryos. This delineated molecularly discrete progenitor populations, which we spatially locate in the embryo, compare across species, and relate to signalling activity. Guided by this map, we determined how differentiating human PSCs develop a stereotypical spatial organization of tissue types. We found that LATS1/2 repression of YAP activity, in conjunction with FGF-mediated MAPK activation, induced the transcription factor Bra/TBXT and facilitated WNT signaling. In addition, inhibiting a WNT-induced NODAL and BMP signaling cascade at the appropriate time regulated the proportions of different tissue types produced, including notochordal cells. We used this information to create an integrated 3D model of human gastrulation undergoing morphogenetic movements to produce elongated structures with a notochord and spatially patterned neural tissue formation. Together the data provide insight into the mechanisms responsible for the formation of the tissues that comprise the vertebrate trunk and pave the way for future studies of patterning in a tissue-like environment.
The extensive array of basic helix-loop-helix (bHLH) transcription factors and their combinations as dimers underpin the diversity of molecular function required for cell type specification during embryogenesis. The bHLH factor TWIST1 plays pleiotropic roles during development. However, which combinations of TWIST1 dimers are involved and what impact each dimer imposes on the gene regulation network controlled by TWIST1 remain elusive. In this work, proteomic profiling of human TWIST1-expressing cell lines and transcriptome analysis of mouse cranial mesenchyme have revealed that TWIST1 homodimers and heterodimers with TCF3, TCF4, and TCF12 E-proteins are the predominant dimer combinations. Disease-causing mutations in TWIST1 can impact dimer formation or shift the balance of different types of TWIST1 dimers in the cell, which may underpin the defective differentiation of the craniofacial mesenchyme. Functional analyses of the loss and gain of TWIST1–E-protein dimer activity have revealed previously unappreciated roles in guiding lineage differentiation of embryonic stem cells: TWIST1–E-protein heterodimers activate the differentiation of mesoderm and neural crest cells, which is accompanied by the epithelial-to-mesenchymal transition. At the same time, TWIST1 homodimers maintain the stem cells in a progenitor state and block entry to the endoderm lineage.
Circular RNAs (circRNA) are a unique class of transcripts that can only be identified from sequence alignments spanning discordant junctions, commonly referred to as backsplice junctions (BSJ). Canonical splicing is also linked with circRNA biogenesis either from the parental transcript or internal to the circRNA, and is not fully utilized in circRNA software. Here we present Ularcirc, a software tool that integrates the visualization of both BSJ and forward splicing junctions and provides downstream analysis of selected circRNA candidates. Ularcirc utilizes the output of CIRI, circExplorer, or raw chimeric output of the STAR aligner and assembles BSJ count table to allow multi-sample analysis. We used Ularcirc to identify and characterize circRNA from public and in-house generated data sets and demonstrate how it can be used to (i) discover novel splicing patterns of parental transcripts, (ii) detect internal splicing patterns of circRNA, and (iii) reveal the complexity of BSJ formation. Furthermore, we identify circRNA that have potential open reading frames longer than their linear sequence. Finally, we detected and validated the presence of a novel class of circRNA generated from ApoA4 transcripts whose BSJ derive from multiple non-canonical splicing sites within coding exons. Ularcirc is accessed via https://github.com/VCCRI/Ularcirc.
Combinations of bHLH factors dimers generate great functional diversity required for cell type specification in development. The bHLH factor TWIST1 plays pleiotropic roles in craniofacial development. However, which combination of TWIST1 dimers are involved in craniofacial development, and what impact each dimer impose on gene regulation network remained elusive. Proteome profiling of human-TWIST1 expressing cell lines and transcriptome analysis of mouse cranial mesenchyme revealed TWIST1 homodimer and heterodimers with TCF3, TCF4 and TCF12 E-proteins as preferred combinations. We found that balance of homo-and heterodimers were impaired by human disease mutations in TWIST1 Helix domains, which may underpin the developmental defects in haploinsufficiency. Further, loss or gain of function analysis of TWIST1-E-protein dimers in differentiating embryonic stem cells revealed their previously unappreciated roles in lineage specification. TWIST1-E-protein heterodimers activate mesoderm and neural crest differentiation through epithelial-to-mesenchymal transition, whilst TWIST1 homodimers maintained a progenitor-like state and blocked entry to endoderm lineages. Short title: TWIST1 dimers in lineage differentiation
In many developing tissues the patterns of gene expression that assign cell fate are organised by secreted signals functioning in a graded manner over multiple cell diameters. Cis Regulatory Elements (CREs) interpret these graded inputs to control gene expression. How this is accomplished remains poorly understood. In the neural tube, a gradient of the morphogen Sonic hedgehog allocates neural progenitor identity. Here, we uncover two distinct ways in which CREs translate graded Shh signaling into differential gene expression. In the majority of ventral neural progenitors a common set of CREs are used to control gene activity. These CREs integrate cell type specific inputs to control gene expression. By contrast, the most ventral progenitors use a unique set of CREs. These are established by the pioneer factor FOXA2, paralleling the role of FOXA2 in endoderm. Moreover, FOXA2 binds a subset of the same sites in neural and endoderm cells. Together the data identify distinct cis regulatory strategies for the interpretation of morphogen signaling and raise the possibility of an evolutionarily conserved role for FOXA2-mediated regulatory strategy across tissues.
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