Control of Tissue Development by Morphogens.

Intercellular signaling molecules, known as morphogens, act at a long range in developing tissues to provide spatial information and control properties such as cell fate and tissue growth. The production, transport, and removal of morphogens shape their concentration profiles in time and space. Downstream signaling cascades and gene regulatory networks within cells then convert the spatiotemporal morphogen profiles into distinct cellular responses. Current challenges are to understand the diverse molecular and cellular mechanisms underlying morphogen gradient formation, as well as the logic of downstream regulatory circuits involved in morphogen interpretation. This knowledge, combining experimental and theoretical results, is essential to understand emerging properties of morphogen-controlled systems, such as robustness and scaling.

Blimp-1 and c-Maf regulate immune gene networks to protect against distinct pathways of pathobiont-induced colitis.

Intestinal immune responses to microbes are controlled by the cytokine IL-10 to avoid immune pathology. Here, we use single-cell RNA sequencing of colon lamina propria leukocytes (LPLs) along with RNA-seq and ATAC-seq of purified CD4+ T cells to show that the transcription factors Blimp-1 (encoded by Prdm1) and c-Maf co-dominantly regulate Il10 while negatively regulating proinflammatory cytokines in effector T cells. Double-deficient Prdm1fl/flMaffl/flCd4Cre mice infected with Helicobacter hepaticus developed severe colitis with an increase in TH1/NK/ILC1 effector genes in LPLs, while Prdm1fl/flCd4Cre and Maffl/flCd4Cre mice exhibited moderate pathology and a less-marked type 1 effector response. LPLs from infected Maffl/flCd4Cre mice had increased type 17 responses with increased Il17a and Il22 expression and an increase in granulocytes and myeloid cell numbers, resulting in increased T cell-myeloid-neutrophil interactions. Genes over-expressed in human inflammatory bowel disease showed differential expression in LPLs from infected mice in the absence of Prdm1 or Maf, revealing potential mechanisms of human disease.

Nervous System Regionalization Entails Axial Allocation before Neural Differentiation.

Neural induction in vertebrates generates a CNS that extends the rostral-caudal length of the body. The prevailing view is that neural cells are initially induced with anterior (forebrain) identity; caudalizing signals then convert a proportion to posterior fates (spinal cord). To test this model, we used chromatin accessibility to define how cells adopt region-specific neural fates. Together with genetic and biochemical perturbations, this identified a developmental time window in which genome-wide chromatin-remodeling events preconfigure epiblast cells for neural induction. Contrary to the established model, this revealed that cells commit to a regional identity before acquiring neural identity. This "primary regionalization" allocates cells to anterior or posterior regions of the nervous system, explaining how cranial and spinal neurons are generated at appropriate axial positions. These findings prompt a revision to models of neural induction and support the proposed dual evolutionary origin of the vertebrate CNS.

Publisher Correction: c-Maf controls immune responses by regulating disease-specific gene networks and repressing IL-2 in CD4+ T cells.

In the version of this article initially published, the Supplementary Data file was an incorrect version. The correct version is now provided. The error has been corrected in the HTML and PDF version of the article.

Publisher Correction: A call for a better understanding of causation in cell biology.

In the above article, the name of the first author was spelled incorrectly. This has been corrected in the HTML and PDF versions of the article.

c-Maf controls immune responses by regulating disease-specific gene networks and repressing IL-2 in CD4+ T cells.

The transcription factor c-Maf induces the anti-inflammatory cytokine IL-10 in CD4+ T cells in vitro. However, the global effects of c-Maf on diverse immune responses in vivo are unknown. Here we found that c-Maf regulated IL-10 production in CD4+ T cells in disease models involving the TH1 subset of helper T cells (malaria), TH2 cells (allergy) and TH17 cells (autoimmunity) in vivo. Although mice with c-Maf deficiency targeted to T cells showed greater pathology in TH1 and TH2 responses, TH17 cell-mediated pathology was reduced in this context, with an accompanying decrease in TH17 cells and increase in Foxp3+ regulatory T cells. Bivariate genomic footprinting elucidated the c-Maf transcription-factor network, including enhanced activity of NFAT; this led to the identification and validation of c-Maf as a negative regulator of IL-2. The decreased expression of the gene encoding the transcription factor RORγt (Rorc) that resulted from c-Maf deficiency was dependent on IL-2, which explained the in vivo observations. Thus, c-Maf is a positive and negative regulator of the expression of cytokine-encoding genes, with context-specific effects that allow each immune response to occur in a controlled yet effective manner.

Gene regulatory logic for reading the Sonic Hedgehog signaling gradient in the vertebrate neural tube.

Secreted signals, known as morphogens, provide the positional information that organizes gene expression and cellular differentiation in many developing tissues. In the vertebrate neural tube, Sonic Hedgehog (Shh) acts as a morphogen to control the pattern of neuronal subtype specification. Using an in vivo reporter of Shh signaling, mouse genetics, and systems modeling, we show that a spatially and temporally changing gradient of Shh signaling is interpreted by the regulatory logic of a downstream transcriptional network. The design of the network, which links three transcription factors to Shh signaling, is responsible for differential spatial and temporal gene expression. In addition, the network renders cells insensitive to fluctuations in signaling and confers hysteresis--memory of the signal. Our findings reveal that morphogen interpretation is an emergent property of the architecture of a transcriptional network that provides robustness and reliability to tissue patterning.

TRF2-independent chromosome end protection during pluripotency.

Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3-9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.

ERK1/2 signalling dynamics promote neural differentiation by regulating chromatin accessibility and the polycomb repressive complex.

Fibroblast growth factor (FGF) is a neural inducer in many vertebrate embryos, but how it regulates chromatin organization to coordinate the activation of neural genes is unclear. Moreover, for differentiation to progress, FGF signalling must decline. Why these signalling dynamics are required has not been determined. Here, we show that dephosphorylation of the FGF effector kinase ERK1/2 rapidly increases chromatin accessibility at neural genes in mouse embryos, and, using ATAC-seq in human embryonic stem cell derived spinal cord precursors, we demonstrate that this occurs genome-wide across neural genes. Importantly, ERK1/2 inhibition induces precocious neural gene transcription, and this involves dissociation of the polycomb repressive complex from key gene loci. This takes place independently of subsequent loss of the repressive histone mark H3K27me3 and transcriptional onset. Transient ERK1/2 inhibition is sufficient for the dissociation of the repressive complex, and this is not reversed on resumption of ERK1/2 signalling. Moreover, genomic footprinting of sites identified by ATAC-seq together with ChIP-seq for polycomb protein Ring1B revealed that ERK1/2 inhibition promotes the occupancy of neural transcription factors (TFs) at non-polycomb as well as polycomb associated sites. Together, these findings indicate that ERK1/2 signalling decline promotes global changes in chromatin accessibility and TF binding at neural genes by directing polycomb and other regulators and appears to serve as a gating mechanism that provides directionality to the process of differentiation.

The mechanisms of Hedgehog signalling and its roles in development and disease.

The cloning of the founding member of the Hedgehog (HH) family of secreted proteins two decades ago inaugurated a field that has diversified to encompass embryonic development, stem cell biology and tissue homeostasis. Interest in HH signalling increased when the pathway was implicated in several cancers and congenital syndromes. The mechanism of HH signalling is complex and remains incompletely understood. Nevertheless, studies have revealed novel biological insights into this system, including the function of HH lipidation in the secretion and transport of this ligand and details of the signal transduction pathway, which involves Patched 1, Smoothened and GLI proteins (Cubitus interruptus in Drosophila melanogaster), as well as, in vertebrates, primary cilia.

Single-cell transcriptome profiling of the human developing spinal cord reveals a conserved genetic programme with human-specific features.

The spinal cord receives input from peripheral sensory neurons and controls motor output by regulating muscle innervating motor neurons. These functions are carried out by neural circuits comprising molecularly distinct neuronal subtypes generated in a characteristic spatiotemporal arrangement from progenitors in the embryonic neural tube. To gain insight into the diversity and complexity of cells in the developing human neural tube, we used single-cell mRNA sequencing to profile cervical and thoracic regions in four human embryos of Carnegie stages (CS) CS12, CS14, CS17 and CS19 from gestational weeks 4-7. Analysis of progenitor and neuronal populations from the neural tube and dorsal root ganglia identified dozens of distinct cell types and facilitated the reconstruction of the differentiation pathways of specific neuronal subtypes. Comparison with mouse revealed overall similarity of mammalian neural tube development while highlighting some human-specific features. These data provide a catalogue of gene expression and cell type identity in the human neural tube that will support future studies of sensory and motor control systems. The data can be explored at https://shiny.crick.ac.uk/scviewer/neuraltube/.

Precision of tissue patterning is controlled by dynamical properties of gene regulatory networks.

During development, gene regulatory networks allocate cell fates by partitioning tissues into spatially organised domains of gene expression. How the sharp boundaries that delineate these gene expression patterns arise, despite the stochasticity associated with gene regulation, is poorly understood. We show, in the vertebrate neural tube, using perturbations of coding and regulatory regions, that the structure of the regulatory network contributes to boundary precision. This is achieved, not by reducing noise in individual genes, but by the configuration of the network modulating the ability of stochastic fluctuations to initiate gene expression changes. We use a computational screen to identify network properties that influence boundary precision, revealing two dynamical mechanisms by which small gene circuits attenuate the effect of noise in order to increase patterning precision. These results highlight design principles of gene regulatory networks that produce precise patterns of gene expression.

A shared transcriptional code orchestrates temporal patterning of the central nervous system.

The molecular mechanisms that produce the full array of neuronal subtypes in the vertebrate nervous system are incompletely understood. Here, we provide evidence of a global temporal patterning program comprising sets of transcription factors that stratifies neurons based on the developmental time at which they are generated. This transcriptional code acts throughout the central nervous system, in parallel to spatial patterning, thereby increasing the diversity of neurons generated along the neuraxis. We further demonstrate that this temporal program operates in stem cell-derived neurons and is under the control of the TGFß signaling pathway. Targeted perturbation of components of the temporal program, Nfia and Nfib, reveals their functional requirement for the generation of late-born neuronal subtypes. Together, our results provide evidence for the existence of a previously unappreciated global temporal transcriptional program of neuronal subtype identity and suggest that the integration of spatial and temporal patterning mechanisms diversifies and organizes neuronal subtypes in the vertebrate nervous system.

Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak.

The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.

Local retinoic acid signaling directs emergence of the extraocular muscle functional unit.

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.

The PAX-FOXO1s trigger fast trans-differentiation of chick embryonic neural cells into alveolar rhabdomyosarcoma with tissue invasive properties limited by S phase entry inhibition.

The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS.

Establishing neuronal diversity in the spinal cord: a time and a place.

The vertebrate spinal cord comprises multiple functionally distinct neuronal cell types arranged in characteristic positions. During development, these different types of neurons differentiate from transcriptionally distinct neural progenitors that are arrayed in discrete domains along the dorsal-ventral and anterior-posterior axes of the embryonic spinal cord. This organization arises in response to morphogen gradients acting upstream of a gene regulatory network, the architecture of which determines the spatial and temporal pattern of gene expression. In recent years, substantial progress has been made in deciphering the regulatory network that underlies the specification of distinct progenitor and neuronal cell identities. In this Review, we outline how distinct neuronal cell identities are established in response to spatial and temporal patterning systems, and outline novel experimental approaches to study the emergence and function of neuronal diversity in the spinal cord.

Thomas M. Jessell (1951-2019).

Thomas M. Jessell died on April 28, 2019. Tom revolutionized our understanding of the mechanisms through which neuronal cell type identities are programmed during development to dictate their function in the adult nervous system. Here, we (two former postdocs from his lab) remember some of his most important scientific contributions and how these changed the way we now understand and think about neuronal circuits controlling movement.