Expression of the mouse α1(II) collagen gene is not restricted to cartilage during development

The mouse alpha 1(II) collagen gene has been isolated and a 5' portion of the gene which has low homology to other collagen genes was used to study the pattern of expression during mouse embryogenesis. In situ hybridization studies show that in the mouse, like the chick, alpha 1(II) collagen is expressed in chondrogenic tissues in advance of chondrocyte differentiation. The gene is expressed early in embryogenesis at 9.5 days both in the cranial mesenchyme destined for the chondrocranium, and the sclerotome of the somites, and at 12.5 days in the primordia of the hyoid and the laryngeal cartilage. Type II collagen gene transcripts were found in all the chondrogenic tissues of the axial and appendicular skeleton until the onset of endochondral ossification. Expression of alpha 1(II) collagen mRNA was also observed in non-chondrogenic tissues such as the notochord which may be responsible for inducing chondrogenesis in somitic mesoderm, neural retina, the corneal and conjunctival epithelia and sclera of the developing eye. Expression in the tail tendon was late, at 16.5-18.5 days. Transient expression was also found in the heart at 9.5-12.5 days, the epidermis at 10.5-14.5 days, the calvarial mesenchyme at 12.5-16.5 days, the inner ear at 14.5 days and the fetal brain from 9.5-14.5 days. Within the neural tube, alpha 1(II) collagen mRNA was localized in the proliferative ventricular cells of the forebrain and midbrain of 9.5- to 10.5-day embryos. Subsequently, transcription of the alpha 1(II) collagen gene was confined to restricted areas of the rhombencephalic basal plate, the ventricular layer of the hindbrain and the cervical spinal cord. These examples of expression of the type II collagen gene in the developing nervous system seem to suggest that active transcription of this gene might be associated with early stages of neuroblast differentiation. Type II collagen may therefore have additional roles in development unrelated to chondrogenesis.published_or_final_versio

Different cis-regulatory DNA elements mediate developmental stage- and tissue-specific expression of the human COL2A1 gene in transgenic mice

Expression of the type II collagen gene (human COL2A1, mouse Col2a1) heralds the differentiation of chondrocytes. It is also expressed in progenitor cells of some nonchondrogenic tissues during embryogenesis. DNA sequences in the 5' flanking region and intron 1 are known to control tissue- specific expression in vitro, but the regulation of COL2A1 expression in vivo is not clearly understood. We have tested the regulatory activity of DNA sequences from COL2A1 on the expression of a lacZ reporter gene in transgenic mice. We have found that type II collagen characteristic expression of the transgene requires the enhancer activity of a 309-bp fragment (+2,388 to +2,696) in intron 1 in conjunction with 6.1-kb 5' sequences. Different regulatory elements were found in the 1.6-kb region (+701 to +2,387) of intron 1 which only needs 90-bp 5' sequences for tissue-specific expression in different components of the developing cartilaginous skeleton. Distinct positive and negative regulatory elements act together to control tissue- specific transgene expression in the developing midbrain neuroepithelium. Positive elements affecting expression in the midbrain were found in the region from -90 to -1,500 and from +701 to +2,387, whereas negatively acting elements were detected in the regions from -1,500 to -6,100 and +2,388 to +2,855.published_or_final_versio

Initiating head development in mouse embryos: integrating signalling and transcriptional activity

The generation of an embryonic body plan is the outcome of inductive interactions between the progenitor tissues that underpin their specification, regionalization and morphogenesis. The intercellular signalling activity driving these processes is deployed in a time- and site-specific manner, and the signal strength must be precisely controlled. Receptor and ligand functions are modulated by secreted antagonists to impose a dynamic pattern of globally controlled and locally graded signals onto the tissues of early post-implantation mouse embryo. In response to the WNT, Nodal and Bone Morphogenetic Protein (BMP) signalling cascades, the embryo acquires its body plan, which manifests as differences in the developmental fate of cells located at different positions in the anterior–posterior body axis. The initial formation of the anterior (head) structures in the mouse embryo is critically dependent on the morphogenetic activity emanating from two signalling centres that are juxtaposed with the progenitor tissues of the head. A common property of these centres is that they are the source of antagonistic factors and the hub of transcriptional activities that negatively modulate the function of WNT, Nodal and BMP signalling cascades. These events generate the scaffold of the embryonic head by the early-somite stage of development. Beyond this, additional tissue interactions continue to support the growth, regionalization, differentiation and morphogenesis required for the elaboration of the structure recognizable as the embryonic head

Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development

The ability of SV40 T antigen to cause abnormalities in cartilage development in transgenic mice and chimeras has been tested. The cis- regulatory elements of the COL2A1 gene were used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice and chimeras derived from embryonal stem (ES) cells bearing the same transgene. The major phenotypic consequences of transgenic (pAL21) expression are malformed skeleton, disproportionate dwarfism, and perinatal/neonatal death. Expression of T antigen was tissue specific and in the main characteristic of the mouse α1(II) collagen gene. Chondrocyte densities and levels of α1(II) collagen mRNAs were reduced in the transgenic mice. Islands of cells which express cartilage characteristic genes such as type IIB procollagen, long form α1(IX) collagen, α2(XI) collagen, and aggrecan were found in the articular and growth cartilages of pAL21 chimeric fetuses and neonates. But these cells, which were expressing T antigen, were not properly organized into columns of proliferating chondrocytes. Levels of α1(II) collagen mRNA were reduced in these chondrocytes. In addition, these cells did not express type X collagen, a marker for hypertrophic chondrocytes. The skeletal abnormality in pAL21 mice may therefore be due to a retardation of chondrocyte maturation or an impaired ability of chondrocytes to complete terminal differentiation and an associated paucity of some cartilage matrix components.published_or_final_versio

PTBP1 Is Required for Embryonic Development before Gastrulation

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures

Mapping Differentiation under Mixed Culture Conditions Reveals a Tunable Continuum of T Cell Fates

Cell differentiation is typically directed by external signals that drive opposing regulatory pathways. Studying differentiation under polarizing conditions, with only one input signal provided, is limited in its ability to resolve the logic of interactions between opposing pathways. Dissection of this logic can be facilitated by mapping the system's response to mixtures of input signals, which are expected to occur in vivo, where cells are simultaneously exposed to various signals with potentially opposing effects. Here, we systematically map the response of naïve T cells to mixtures of signals driving differentiation into the Th1 and Th2 lineages. We characterize cell state at the single cell level by measuring levels of the two lineage-specific transcription factors (T-bet and GATA3) and two lineage characteristic cytokines (IFN-γ and IL-4) that are driven by these transcription regulators. We find a continuum of mixed phenotypes in which individual cells co-express the two lineage-specific master regulators at levels that gradually depend on levels of the two input signals. Using mathematical modeling we show that such tunable mixed phenotype arises if autoregulatory positive feedback loops in the gene network regulating this process are gradual and dominant over cross-pathway inhibition. We also find that expression of the lineage-specific cytokines follows two independent stochastic processes that are biased by expression levels of the master regulators. Thus, cytokine expression is highly heterogeneous under mixed conditions, with subpopulations of cells expressing only IFN-γ, only IL-4, both cytokines, or neither. The fraction of cells in each of these subpopulations changes gradually with input conditions, reproducing the continuous internal state at the cell population level. These results suggest a differentiation scheme in which cells reflect uncertainty through a continuously tuneable mixed phenotype combined with a biased stochastic decision rather than a binary phenotype with a deterministic decision

Resolving early mesoderm diversification through single-cell expression profiling.

In mammals, specification of the three major germ layers occurs during gastrulation, when cells ingressing through the primitive streak differentiate into the precursor cells of major organ systems. However, the molecular mechanisms underlying this process remain unclear, as numbers of gastrulating cells are very limited. In the mouse embryo at embryonic day 6.5, cells located at the junction between the extra-embryonic region and the epiblast on the posterior side of the embryo undergo an epithelial-to-mesenchymal transition and ingress through the primitive streak. Subsequently, cells migrate, either surrounding the prospective ectoderm contributing to the embryo proper, or into the extra-embryonic region to form the yolk sac, umbilical cord and placenta. Fate mapping has shown that mature tissues such as blood and heart originate from specific regions of the pre-gastrula epiblast, but the plasticity of cells within the embryo and the function of key cell-type-specific transcription factors remain unclear. Here we analyse 1,205 cells from the epiblast and nascent Flk1(+) mesoderm of gastrulating mouse embryos using single-cell RNA sequencing, representing the first transcriptome-wide in vivo view of early mesoderm formation during mammalian gastrulation. Additionally, using knockout mice, we study the function of Tal1, a key haematopoietic transcription factor, and demonstrate, contrary to previous studies performed using retrospective assays, that Tal1 knockout does not immediately bias precursor cells towards a cardiac fate.We thank M. de Bruijn, A. Martinez-Arias, J. Nichols and C. Mulas for discussion, the Cambridge Institute for Medical Research Flow Cytometry facility for their expertise in single-cell index sorting, and S. Lorenz from the Sanger Single Cell Genomics Core for supervising purification of Tal1−/− sequencing libraries. ChIP-seq reads were processed by R. Hannah. Research in the authors’ laboratories is supported by the Medical Research Council, Cancer Research UK, the Biotechnology and Biological Sciences Research Council, Bloodwise, the Leukemia and Lymphoma Society, and the Sanger-EBI Single Cell Centre, and by core support grants from the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust - MRC Cambridge Stem Cell Institute and by core funding from Cancer Research UK and the European Molecular Biology Laboratory. Y.T. was supported by a fellowship from the Japan Society for the Promotion of Science. W.J. is a Wellcome Trust Clinical Research Fellow. A.S. is supported by the Sanger-EBI Single Cell Centre. This work was funded as part of Wellcome Trust Strategic Award 105031/D/14/Z ‘Tracing early mammalian lineage decisions by single-cell genomics’ awarded to W. Reik, S. Teichmann, J. Nichols, B. Simons, T. Voet, S. Srinivas, L. Vallier, B. Göttgens and J. Marioni.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nature1863