Analysis of intronless genes involved in oscillation and differentiation

The genomes of higher eukaryotes are replete with intron-containing genes. Transcription of these genes produces precursor mRNAs containing intervening sequences, which are subsequently removed and the exons spliced together to form the mature mRNA. However, a small proportion of eukaryotic protein-coding genes are intronless and therefore bypass post-transcriptional splicing events. Although a large proportion of intronless genes are known to code for certain types of proteins, their specific role in the genome of higher organism is perplexing. This research set out to elucidate the functions of intronless genes in humans by studying their involvement in the expression pattern of oscillatory gene that occurs in the pre-somitic mesoderm of developing embryo. Twenty-seven (27) human homologs of mouse oscillatory genes were analysed to determine the number of exons present in them using various bioinformatics databases. The result obtained identified two intronless genes –NRARP and ID1 – which are associated with the Notch signalling pathway of the segmentation clock. This represented 7.4% of the total oscillatory genes analysed. No intronless gene was found in the Wnt and FGF signalling pathways – two other pathways famous for oscillatory gene expression. The proteins encoded by the intronless genes are involved in several important biological processes including angiogenesis, cell cycle control and in the regulation of cellular senescence. Although oscillatory genes had fewer numbers of introns compared to the non-oscillatory genes, the intronless genes were not implicated in the regulation of the precise timing events of the segmentation clock. This result may also point to the fact that the rapid expression rate of the oscillatory genes in the PSM may favour the reduced intron length of the oscillatory genes.

From Bipotent Neuromesodermal Progenitors to Neural-Mesodermal Interactions during Embryonic Development

To ensure the formation of a properly patterned embryo, multiple processes must operate harmoniously at sequential phases of development. This is implemented by mutual interactions between cells and tissues that together regulate the segregation and specification of cells, their growth and morphogenesis. The formation of the spinal cord and paraxial mesoderm derivatives exquisitely illustrate these processes. Following early gastrulation, while the vertebrate body elongates, a population of bipotent neuromesodermal progenitors resident in the posterior region of the embryo generate both neural and mesodermal lineages. At later stages, the somitic mesoderm regulates aspects of neural patterning and differentiation of both central and peripheral neural progenitors. Reciprocally, neural precursors influence the paraxial mesoderm to regulate somite-derived myogenesis and additional processes by distinct mechanisms. Central to this crosstalk is the activity of the axial notochord, which, via sonic hedgehog signaling, plays pivotal roles in neural, skeletal muscle and cartilage ontogeny. Here, we discuss the cellular and molecular basis underlying this complex developmental plan, with a focus on the logic of sonic hedgehog activities in the coordination of the neural-mesodermal axis.

Role of Retinoic Acid Signaling, FGF Signaling and Meis Genes in Control of Limb Development

The function of retinoic acid (RA) during limb development is still debated, as loss and gain of function studies led to opposite conclusions. With regard to limb initiation, genetic studies demonstrated that activation of FGF10 signaling is required for the emergence of limb buds from the trunk, with Tbx5 and RA signaling acting upstream in the forelimb field, whereas Tbx4 and Pitx1 act upstream in the hindlimb field. Early studies in chick embryos suggested that RA as well as Meis1 and Meis2 (Meis1/2) are required for subsequent proximodistal patterning of both forelimbs and hindlimbs, with RA diffusing from the trunk, functioning to activate Meis1/2 specifically in the proximal limb bud mesoderm. However, genetic loss of RA signaling does not result in loss of limb Meis1/2 expression and limb patterning is normal, although Meis1/2 expression is reduced in trunk somitic mesoderm. More recent studies demonstrated that global genetic loss of Meis1/2 results in a somite defect and failure of limb bud initiation. Other new studies reported that conditional genetic loss of Meis1/2 in the limb results in proximodistal patterning defects, and distal FGF8 signaling represses Meis1/2 to constrain its expression to the proximal limb. In this review, we hypothesize that RA and Meis1/2 both function in the trunk to initiate forelimb bud initiation, but that limb Meis1/2 expression is activated proximally by a factor other than RA and repressed distally by FGF8 to generate proximodistal patterning.

Stepwise in vitro induction of human somitic mesoderm and its derivatives

Our understanding of human somitogenesis is limited and largely based on insights gained from model organisms. Pluripotent stem cell-based in vitro approaches aiming to recapitulate distinct aspects of this core developmental process have recently been reported, including our recent paper on the in vitro recapitulation of the human segmentation clock1. Here we describe in detail our stepwise induction protocol of presomitic mesoderm (PSM), somitic mesoderm (SM), and its two major derivatives, sclerotome (SCL) and dermomyotome (DM) from human induced pluripotent stem cells (iPSCs). We further briefly address the subsequent molecular and functional analysis of these in vitro induced human mesodermal lineages and cell-types.

Stepwise in vitro induction of human somitic mesoderm and its derivatives

Our understanding of human somitogenesis is limited and largely based on insights gained from model organisms. Pluripotent stem cell-based in vitro approaches aiming to recapitulate distinct aspects of this core developmental process have recently been reported, including our recent paper on the in vitro recapitulation of the human segmentation clock 1 . Here we describe in detail our stepwise induction protocol of presomitic mesoderm (PSM), somitic mesoderm (SM), and its two major derivatives, sclerotome (SCL) and dermomyotome (DM) from human induced pluripotent stem cells (iPSCs). We further briefly address the subsequent molecular and functional analysis of these in vitro induced human mesodermal lineages and cell-types.

A unified mechanism for spatiotemporal patterns in somitogenesis

Somitogenesis, the process of body segmentation during embryonic development, exhibits a key set of features that is conserved across all vertebrate species despite differences in the detailed mechanisms. Prior to the formation of somites along the pre-somitic mesoderm (PSM), periodic expression of clock genes is observed in its constituent cells. As the PSM expands through the addition of new cells at its posterior, the oscillations in the cells closer to the anterior cease and eventually lead to formation of rostral and caudal halves of the somites. This pattern formation is believed to be coordinated by interactions between neighboring cells via receptor-ligand coupling. However, the mechanism underlying the transition from synchronized oscillations to traveling waves and subsequent arrest of activity, followed by the appearance of polarized somites, has not yet been established. In this paper we have proposed a unified mechanism that reproduces the sequence of dynamical transitions observed during somitogenesis by combining the local interactions mediated via Notch-Delta intercellular coupling with global spatial heterogeneity introduced through a morphogen gradient that is known to occur along the anteroposterior axis of the growing PSM. Our model provides a framework that integrates a boundary-organized pattern formation mechanism, which uses positional information provided by a morphogen gradient, with the coupling-mediated self-organized emergence of collective dynamics, to explain the processes that lead to segmentation.

A Mechanical Model of Early Somite Segmentation

The clock-and-wavefront model (CW) hypothesizes that the formation of somites in vertebrate embryos results from the interplay of molecular oscillations with a wave traveling along the body axis. This model however does not explain how molecular information is interpreted by cells to modulate their rearrangement into somites. Here we performed Scanning Electron Microscopy (SEM) on the pre-somitic mesoderm (PSM) of chicken embryos at stages 11-12 to describe in detail the cell shape changes occurring along the axis of the PSM. This reveals a wave of epithelialization of the dorsal PSM that precedes somite segmentation. Signs of spatially periodic apical constriction appear in this layer starting at least 3-4 somite lengths caudal to the most recently formed somite. The sizes of these clusters correspond to the typical diameter of chicken somites. We propose that a mechanical instability process leads to the separation of cells into these structures and positions the future inter-somite boundaries. We present a model in which a wave of apical constriction leads to increasing tension and periodic failure of adhesion junctions within the dorsal epithelial layer of the PSM, thus positioning somite boundaries. This model can produce spatially periodic segments whose size depends on the speed of the contraction wave (W) and the rate of increase of apical contractility (Λ). The Λ/W ratio determines whether this mechanism produces spatially and temporally regular or irregular segments, and whether segment sizes increase with the wave speed (scaling) as in the CW model. We discuss the limitations of a purely mechanical model of somite segmentation and the role of biomechanics along with CW during somitogenesis.

Cell Fate Determining Molecular Switches and Signaling Pathway in Pax7-expressing Somitic Mesoderm

Pax7-expressing progenitor cells in the somitic mesoderm differentiate into multiple lineages, such as brown adipose tissue, dorsal dermis, as well as muscle in the dorsal trunk and the diaphragm; however, the key molecular switches that determine and control the process of lineage commitment and cell fate are unknown. To probe the mechanisms behind mesoderm development, Pax7creER/R26-stop-EYFP embryos were tamoxifen-induced at E9.5 to label Pax7+ cells for lineage tracing and collected at later time points for analysis. The YFP-labelled cells which belonged to the Pax7 lineage were enriched by fluorescence-activated cell sorting (FACS) and subject to single-cell RNA profiling. We observed that a subpopulation of cells differentiated into the myogenic lineage, showing Myf5 expression as early as E12.5, whereas the rest of the population was fibroblast-like and appeared to be the early stage of the adipogenic and dermal lineages. Cells at E14.5 had distinct myogenic populations that expressed Myod1 and Myog; we also identified other populations with Ebf2 or Twist2 expression, which could belong to adipogenic or dermal lineages, respectively. Cell surface markers were also found for each specific lineage, providing insights in sorting strategy for lineage-of-interest for further functional evaluation. Adipogenic lineage was successfully sorted with a combination of Pdgfra and Thy1 antibodies. In addition, we found that upregulation of Wnt signaling pathway activity is dynamically regulated in dermal lineage. Finally, transcription factors that could potentially drive, or reprogram cell fate, were identified at different developmental time points.Summary statementInvestigation of Pax7 lineage transcriptomic profile at single-cell level identified multiple cell types, fate commitment time point, surface markers, transcription factors and signaling pathways that determine cell fate.

Quantitative study of the somitogenetic wavefront in zebrafish

ABSTRACTA quantitative description of the molecular networks that sustain morphogenesis is one of the challenges of developmental biology. Specifically, a molecular understanding of the segmentation of the antero-posterior axis in vertebrates has yet to be achieved. This process known as somitogenesis is believed to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. Here we quantitatively study and perturb the network in zebrafish that sustains this wavefront and compare our observations to a model whereby the wavefront is due to a switch between stable states resulting from reciprocal negative feedbacks of Retinoic Acid (RA) on the activation of ERK and of ERK on RA synthesis. This model quantitatively accounts for the near linear shortening of the post-somitic mesoderm (PSM) in response to the observed exponential decrease during somitogenesis of the mRNA concentration of a morphogen (Fgf8). It also accounts for the observed dynamics of the PSM when the molecular components of the network are perturbed. The generality of our model and its robustness allows for its test in other model organisms.