Major transcriptome re-organisation and abrupt changes in signalling, cell cycle and chromatin regulation at neural differentiation in vivo.

Here, we exploit the spatial separation of temporal events of neural differentiation in the elongating chick body axis to provide the first analysis of transcriptome change in progressively more differentiated neural cell populations in vivo. Microarray data, validated against direct RNA sequencing, identified: (1) a gene cohort characteristic of the multi-potent stem zone epiblast, which contains neuro-mesodermal progenitors that progressively generate the spinal cord; (2) a major transcriptome re-organisation as cells then adopt a neural fate; and (3) increasing diversity as neural patterning and neuron production begin. Focussing on the transition from multi-potent to neural state cells, we capture changes in major signalling pathways, uncover novel Wnt and Notch signalling dynamics, and implicate new pathways (mevalonate pathway/steroid biogenesis and TGFß). This analysis further predicts changes in cellular processes, cell cycle, RNA-processing and protein turnover as cells acquire neural fate. We show that these changes are conserved across species and provide biological evidence for reduced proteasome efficiency and a novel lengthening of S phase. This latter step may provide time for epigenetic events to mediate large-scale transcriptome re-organisation; consistent with this, we uncover simultaneous downregulation of major chromatin modifiers as the neural programme is established. We further demonstrate that transcription of one such gene, HDAC1, is dependent on FGF signalling, making a novel link between signals that control neural differentiation and transcription of a core regulator of chromatin organisation. Our work implicates new signalling pathways and dynamics, cellular processes and epigenetic modifiers in neural differentiation in vivo, identifying multiple new potential cellular and molecular mechanisms that direct differentiation.

Transcriptomic analysis of mouse limb tendon cells during development.

The molecular signals driving tendon development are not fully identified. We have undertaken a transcriptome analysis of mouse limb tendon cells that were isolated at different stages of development based on scleraxis (Scx) expression. Microarray comparisons allowed us to establish a list of genes regulated in tendon cells during mouse limb development. Bioinformatics analysis of the tendon transcriptome showed that the two most strongly modified signalling pathways were TGF-ß and MAPK. TGF-ß/SMAD2/3 gain- and loss-of-function experiments in mouse limb explants and mesenchymal stem cells showed that TGF-ß signalling was sufficient and required via SMAD2/3 to drive mouse mesodermal stem cells towards the tendon lineage ex vivo and in vitro. TGF-ß was also sufficient for tendon gene expression in late limb explants during tendon differentiation. FGF does not have a tenogenic effect and the inhibition of the ERK MAPK signalling pathway was sufficient to activate Scx in mouse limb mesodermal progenitors and mesenchymal stem cells.

Loss of FGF-dependent mesoderm identity and rise of endogenous retinoid signalling determine cessation of body axis elongation.

The endogenous mechanism that determines vertebrate body length is unknown but must involve loss of chordo-neural-hinge (CNH)/axial stem cells and mesoderm progenitors in the tailbud. In early embryos, Fibroblast growth factor (FGF) maintains a cell pool that progressively generates the body and differentiation onset is driven by retinoid repression of FGF signalling. This raises the possibility that FGF maintains key tailbud cell populations and that rising retinoid activity underlies cessation of body axis elongation. Here we show that sudden loss of the mesodermal gene (Brachyury) from CNH and the mesoderm progenitor domain correlates with FGF signalling decline in the late chick tailbud. This is accompanied by expansion of neural gene expression and a similar change in cell fate markers is apparent in the human tailbud. Fate mapping of chick tailbud further revealed that spread of neural gene expression results from continued ingression of CNH-derived cells into the position of the mesoderm progenitor domain. Using gain and loss of function approaches in vitro and in vivo, we then show that attenuation of FGF/Erk signalling mediates this loss of Brachyury upstream of Wnt signalling, while high-level FGF maintains Brachyury and can induce ectopic CNH-like cell foci. We further demonstrate a rise in endogenous retinoid signalling in the tailbud and show that here FGF no longer opposes retinoid synthesis and activity. Furthermore, reduction of retinoid signalling at late stages elevated FGF activity and ectopically maintained mesodermal gene expression, implicating endogenous retinoid signalling in loss of mesoderm identity. Finally, axis termination is concluded by local cell death, which is reduced by blocking retinoid signalling, but involves an FGFR-independent mechanism. We propose that cessation of body elongation involves loss of FGF-dependent mesoderm identity in late stage tailbud and provide evidence that rising endogenous retinoid activity mediates this step and ultimately promotes cell death in chick tailbud.

Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension.

Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however, molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore, somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. Here we identify retinoic acid (RA) as this somitic signal and show that retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation, including ventral neural patterning, while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm, where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. Our data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension.

Molecular mechanisms controlling dorsal dermis generation from the somitic dermomyotome.

The initiation of the development of skin appendages (hair/feathers/scales) requires a signal from the competent dense dermis to the epidermis (Dhouailly, 1977). It is therefore essential to understand how to make a competent dermis. In recent years, a few studies have focused on the development of the dorsal dermis from the somitic dermomyotome. Our first aim in this review is to attempt to reconcile the available data on the origin of the dorsal dermis and summarize the present knowledge on the molecular mechanisms implicated in dermal lineage induction. Secondly, we open the discussion on the formation of a loose pre-dermal mesenchyme and more importantly of a dense dermis capable of participating in appendage development. To go further we draw a comparison between the chick and mouse systems to gain a new insight into how to initiate appendage morphogenesis and regulate the extent of hair/feather fields.

Skin field formation: morphogenetic events.

This chapter is mostly a review of the pioneering work of the Philippe Sengel school in Grenoble carried out in the late sixties and the seventies. The questions raised concerning the morphogenesis of feather tracts were approached by means of microsurgery on chick embryos. P. Sengel and his wife M. Kieny had the feeling that proteins synthesized by the neural tube were required for the formation of feather fields. It was my pleasure to carry on the story from the beginning. Although some clarifications concerning this morphogenesis have been contributed by my group and by a few other laboratories interested in this subject, the most important contributions to recent research have been the elucidation of the nature of the required messages, which will be explored further in other papers in this Issue.

The different steps of skin formation in vertebrates.

Skin morphogenesis occurs following a continuous series of cell-cell interactions which can be subdivided into three main stages: 1- the formation of a dense dermis and its overlying epidermis in the future appendage fields (macropattern); 2- the organization of these primary homogeneous fields into heterogeneous ones by the appearance of cutaneous appendage primordia (micropattern) and 3- cutaneous appendage organogenesis itself. In this review, we will first show, by synthesizing novel and previously published data from our laboratory, how heterogenetic and heterospecific dermal/epidermal recombinations have allowed us to distinguish between the respective roles of the dermis and the epidermis. We will then summarize what is known from the work of many different research groups about the molecular signaling which mediates these interactions in order to introduce the following articles of this Special Issue and to highlight what remains to done.

Transcription factor EGR1 directs tendon differentiation and promotes tendon repair.

Tendon formation and repair rely on specific combinations of transcription factors, growth factors, and mechanical parameters that regulate the production and spatial organization of type I collagen. Here, we investigated the function of the zinc finger transcription factor EGR1 in tendon formation, healing, and repair using rodent animal models and mesenchymal stem cells (MSCs). Adult tendons of Egr1-/- mice displayed a deficiency in the expression of tendon genes, including Scx, Col1a1, and Col1a2, and were mechanically weaker compared with their WT littermates. EGR1 was recruited to the Col1a1 and Col2a1 promoters in postnatal mouse tendons in vivo. Egr1 was required for the normal gene response following tendon injury in a mouse model of Achilles tendon healing. Forced Egr1 expression programmed MSCs toward the tendon lineage and promoted the formation of in vitro-engineered tendons from MSCs. The application of EGR1-producing MSCs increased the formation of tendon-like tissues in a rat model of Achilles tendon injury. We provide evidence that the ability of EGR1 to promote tendon differentiation is partially mediated by TGF-ß2. This study demonstrates EGR1 involvement in adult tendon formation, healing, and repair and identifies Egr1 as a putative target in tendon repair strategies.

FGF and retinoic acid activity gradients control the timing of neural crest cell emigration in the trunk.

Coordination between functionally related adjacent tissues is essential during development. For example, formation of trunk neural crest cells (NCCs) is highly influenced by the adjacent mesoderm, but the molecular mechanism involved is not well understood. As part of this mechanism, fibroblast growth factor (FGF) and retinoic acid (RA) mesodermal gradients control the onset of neurogenesis in the extending neural tube. In this paper, using gain- and loss-of-function experiments, we show that caudal FGF signaling prevents premature specification of NCCs and, consequently, premature epithelial-mesenchymal transition (EMT) to allow cell emigration. In contrast, rostrally generated RA promotes EMT of NCCs at somitic levels. Furthermore, we show that FGF and RA signaling control EMT in part through the modulation of elements of the bone morphogenetic protein and Wnt signaling pathways. These data establish a clear role for opposition of FGF and RA signaling in control of the timing of NCC EMT and emigration and, consequently, coordination of the development of the central and peripheral nervous system during vertebrate trunk elongation.

Stem cells, signals and vertebrate body axis extension.

The progressive generation of chick and mouse axial tissues - the spinal cord, skeleton and musculature of the body - has long been proposed to depend on the activity of multipotent stem cells. Here, we evaluate evidence for the existence and multipotency of axial stem cells. We show that although the data strongly support their existence, there is little definitive information about their multipotency or extent of contribution to the axis. We also review the location and molecular characteristics of these putative stem cells, along with their evolutionary conservation in vertebrates and the signalling mechanisms that regulate and arrest axis extension.

Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension.

Differentiation onset in the vertebrate body axis is controlled by a conserved switch from fibroblast growth factor (FGF) to retinoid signalling, which is also apparent in the extending limb and aberrant in many cancer cell lines. FGF protects tail-end stem zone cells from precocious differentiation by inhibiting retinoid synthesis, whereas later-produced retinoic acid (RA) attenuates FGF signalling and drives differentiation. The timing of RA production is therefore crucial for the preservation of stem zone cells and the continued extension of the body axis. Here we show that canonical Wnt signalling mediates the transition from FGF to retinoid signalling in the newly generated chick body axis. FGF promotes Wnt8c expression, which persists in the neuroepithelium as FGF signalling declines. Wnt signals then act here to repress neuronal differentiation. Furthermore, although FGF inhibition of neuronal differentiation involves repression of the RA-responsive gene, retinoic acid receptor beta (RARbeta), Wnt signals are weaker repressors of neuron production and do not interfere with RA signal transduction. Strikingly, as FGF signals decline in the extending axis, Wnt signals now elicit RA synthesis in neighbouring presomitic mesoderm. This study identifies a directional signalling relay that leads from FGF to retinoid signalling and demonstrates that Wnt signals serve, as cells leave the stem zone, to permit and promote RA activity, providing a mechanism to control the timing of the FGF-RA differentiation switch.

Differential regulation of the chick dorsal thoracic dermal progenitors from the medial dermomyotome.

The chick dorsal feather-forming dermis originates from the dorsomedial somite and its formation depends primarily on Wnt1 from the dorsal neural tube. We investigate further the origin and specification of dermal progenitors from the medial dermomyotome. This comprises two distinct domains: the dorsomedial lip and a more central region (or intervening zone) that derives from it. We confirm that Wnt1 induces Wnt11 expression in the dorsomedial lip as previously shown, and show using DiI injections that some of these cells, which continue to express Wnt11 migrate under the ectoderm, towards the midline, to form most of the dorsal dermis. Transplantation of left somites to the right side to reverse the mediolateral axis confirms this finding and moreover suggests the presence of an attractive or permissive environment produced by the midline tissues or/and a repellent or inadequate environment by the lateral tissues. By contrast, the dorsolateral dermal cells just delaminate from the surface of the intervening space, which expresses En1. Excision of the axial organs or the ectoderm, and grafting of Wnt1-secreting cells, shows that, although the two populations of dermal progenitors both requires Wnt1 for their survival, the signalling required for their specification differs. Indeed Wnt11 expression relies on dorsal neural tube-derived Wnt1, while En1 expression depends on the presence of the ectoderm. The dorsal feather-forming dermal progenitors thus appear to be differentially regulated by dorsal signals from the neural tube and the ectoderm, and derive directly and indirectly from the dorsomedial lip. As these two dermomyotomal populations are well known to also give rise to epaxial muscles, an isolated domain of the dermomyotome that contains only dermal precursors does not exist and none of the dermomyotomal domains can be considered uniquely as a dermatome.