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HOX transcription factors are members of an evolutionarily conserved family of proteins required for the establishment of the anteroposterior body axis during bilaterian development. Although they are often deregulated in cancers, the molecular mechanisms by which they act as oncogenes or tumor suppressor genes are only partially understood. Since the MAPK/ERK signaling pathway is deregulated in most cancers, we aimed at apprehending if and how the Hox proteins interact with ERK oncogenicity. Using an in vivo neoplasia model in the chicken embryo consisting in the overactivation of the ERK1/2 kinases in the trunk neural tube, we analyzed the consequences of the HOXB8 gain of function at the morphological and transcriptional levels. We found that HOXB8 acts as a tumor suppressor, counteracting ERK-induced neoplasia. The HOXB8 tumor suppressor function relies on a large reversion of the oncogenic transcriptome induced by ERK. In addition to showing that the HOXB8 protein controls the transcriptional responsiveness to ERK oncogenic signaling, our study identified new downstream targets of ERK oncogenic activation in an in vivo context that could provide clues for therapeutic strategies.

Most human cancers demonstrate activated MAPK/ERK pathway signaling as a key tumor initiation step, but the immediate steps of further oncogenic progression are poorly understood due to a lack of appropriate models. Spinal cord differentiation follows caudal elongation in vertebrate embryos; both processes are regulated by a FGF8 gradient highest in neuromesodermal progenitors (NMP), where kinase effectors ERK1/2 maintain an undifferentiated state. FGF8/ERK signal attenuation is necessary for NMPs to progress to differentiation. We show that sustained ERK1/2 activity, using a constitutively active form of the kinase MEK1 (MEK1ca) in the chicken embryo, reproducibly provokes neopasia in the developing spinal cord. Transcriptomic data show that neoplasia not only relies on the maintenance of NMP gene expression, and the inhibition of genes expressed in the differentiating spinal cord, but also on a profound change in the transcriptional signature of the spinal cord cells leading to a complete loss of cell-type identity. MEK1ca expression in the developing spinal cord of the chicken embryo is therefore a tractable in vivo model to identify the critical factors fostering malignancy in ERK-induced tumorigenesis.

Differential Hox gene expression is central for specification of axial neuronal diversity in the spinal cord. Here, we uncover an additional function of Hox proteins in the developing spinal cord, restricted to B cluster Hox genes. We found that members of the HoxB cluster are expressed in the trunk neural tube of chicken embryo earlier than Hox from the other clusters, with poor antero-posterior axial specificity and with overlapping expression in the intermediate zone (IZ). Gain-of-function experiments of HoxB4, HoxB8 and HoxB9, respectively, representative of anterior, central and posterior HoxB genes, resulted in ectopic progenitor cells in the mantle zone. The search for HoxB8 downstream targets in the early neural tube identified the leucine zipper tumor suppressor 1 gene (Lzts1), the expression of which is also activated by HoxB4 and HoxB9. Gain- and loss-of-function experiments showed that Lzts1, which is expressed endogenously in the IZ, controls neuronal delamination. These data collectively indicate that HoxB genes have a generic function in the developing spinal cord, controlling the expression of Lzts1 and neuronal delamination.

Differential Hox gene expression is central for specification of axial neuronal diversity in the spinal cord. Here, we uncover an additional function of Hox proteins in the developing spinal cord, restricted to B cluster Hox genes. We found that members of the HoxB cluster are expressed in the trunk neural tube of chicken embryo earlier than Hox from the other clusters, with poor antero-posterior axial specificity and with overlapping expression in the intermediate zone (IZ). Gain-of-function experiments of HoxB4, HoxB8 and HoxB9, respectively, representative of anterior, central and posterior HoxB genes, resulted in ectopic progenitor cells in the mantle zone. The search for HoxB8 downstream targets in the early neural tube identified the leucine zipper tumor suppressor 1 gene (

Posterior Hox genes (Hox9-13) are critical for patterning the limb skeleton along the proximodistal axis during embryonic development. Here we show that Hox11 paralogous genes, which developmentally pattern the zeugopod (radius/ulna and tibia/fibula), remain regionally expressed in the adult skeleton. Using Hoxa11eGFP reporter mice, we demonstrate expression exclusively in multi-potent mesenchymal stromal cells (MSCs) in the bone marrow of the adult zeugopod. Hox-positive cells express PDGFRα and CD51, are marked by LepR-Cre, exhibit CFU-F activity and tri-lineage differentiate in vitro. Loss of Hox11 function leads to fracture repair defects, including reduced cartilage formation and delayed ossification. Hox mutant cells are defective in osteoblastic and chondrogenic differentiation in tri-lineage differentiation experiments and these defects are zeugopod-specific. In the stylopod (humerus and femur) and sternum, BM-MSCs express other regionally restricted Hox genes and femur fractures heal normally in Hox11 mutants. Together, our data supports regional Hox expression and function in skeletal MSCs.

Limb muscle formation is spread out over time and, consequently, muscle cells are not easy to target at any particular stages. We aimed to design a technique to study gene function in the different steps of limb muscle formation. We have associated transposon-mediated gene transfer and a tetracycline-dependent activation method with forelimb somite electroporation. In addition, we have established a new vector combining a differentiated-muscle-cell-specific promoter and the transposon system, which allows stable gene expression in limb differentiated muscle cells and not in muscle progenitors. Using these methods, we observed that conditional Fgf4 expression in muscle cells at the onset of fetal muscle differentiation and specific Fgf4 expression in differentiated muscle cells alter muscle fiber formation in chick limbs. Limb somite electroporation with these set of vectors allowing stable, conditional, and differentiated-muscle-specific expression of transgenes opens new perspectives for investigating gene function at various steps of limb muscle formation.

In amniotes, the dermomyotome is the source of all skeletal muscles of the trunk and the limbs. Trunk skeletal muscles form in two sequential stages: in the first stage, cells located at the four borders of the epithelial dermomyotome delaminate to generate the primary myotome, composed of post-mitotic, mononucleated myocytes. The epithelio-mesenchymal transition (EMT) of the central dermomyotome initiates the second stage of muscle formation, characterised by a massive entry of mitotic muscle progenitors from the central region of the dermomyotome into the primary myotome. The signals that regulate the timing of the dermomyotome EMT are unknown. Here, we propose that this process is regulated by an FGF signal emanating from the primary myotome, a known source of FGF. The over-expression of FGF results in a precocious EMT of the dermomyotome, while on the contrary, the inhibition of FGF signalling by the electoporation of a dominant-negative form of FGFR4 delays this process. Within the dermomyotome, FGF signalling triggers a MAPK/ERK pathway that leads to the activation of the transcription factor Snail1, a known regulator of EMT in a number of cellular contexts. The activation or the inhibition of the MAPK/ERK pathway and of Snail1 mimics that of FGF signalling and leads to an early or delayed EMT of the dermomyotome, respectively. Altogether, our results indicate that in amniotes, the primary myotome is an organizing center that regulates the timely entry of embryonic muscle progenitors within the muscle masses, thus initiating the growth phase of the trunk skeletal muscles.

The regular spacing of somites during vertebrate embryogenesis involves a dynamic gradient of FGF signaling that controls the timing of maturation of cells in the presomitic mesoderm (PSM). How the FGF signal is transduced by PSM cells is unclear. Here, we first show that the FGF gradient is translated into graded activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway along the PSM in the chicken embryo. Usingin ovoelectroporation of PSM cells, we demonstrate that constitutive activation of ERK signaling in the PSM blocks segmentation by preventing maturation of PSM cells, thus phenocopying the overexpression of FGF8. Conversely, inhibition of ERK phosphorylation mimics a loss of function of FGF signaling in the PSM. Interestingly, video microscopy analysis of cell movements shows that ERK regulates the motility of PSM cells, suggesting that the decrease of cell movements along the PSM enables mesenchymal PSM cells to undergo proper segmentation. Together, our data demonstrate that ERK is the effector of the gradient of FGF in the PSM that controls the segmentation process.

International audience; C-low-threshold mechanoreceptors (C-LTMRs) are unique among C-unmyelinated primary sensory neu-rons. These neurons convey two opposite aspects of touch sensation: a sensation of pleasantness, and a sensation of injury-induced mechanical pain. Here, we show that TAFA4 is a specific marker of C-LTMRs. Genetic labeling in combination with electrophy-siological recordings show that TAFA4+ neurons have intrinsic properties of mechano-nociceptors. TAFA4-null mice exhibit enhanced mechanical and chemical hypersensitivity following inflammation and nerve injury as well as increased excitability of spinal cord lamina IIi neurons, which could be reversed by intrathecal or bath application of recom-binant TAFA4 protein. In wild-type C57/Bl6 mice, intrathecal administration of TAFA4 strongly reversed carrageenan-induced mechanical hypersensitivity, suggesting a potent analgesic role of TAFA4 in pain relief. Our data provide insights into how C-LTMR-derived TAFA4 modulates neuronal excitability and controls the threshold of somatic sensation.

Hox protein function during development and evolution relies on conserved multiple interaction modes with cofactors of the PBC and Meis families., Hox transcription factors control a number of developmental processes with the help of the PBC class proteins. In vitro analyses have established that the formation of Hox/PBC complexes relies on a short conserved Hox protein motif called the hexapeptide (HX). This paradigm is at the basis of the vast majority of experimental approaches dedicated to the study of Hox protein function. Here we questioned the unique and general use of the HX for PBC recruitment by using the Bimolecular Fluorescence Complementation (BiFC) assay. This method allows analyzing Hox-PBC interactions in vivo and at a genome-wide scale. We found that the HX is dispensable for PBC recruitment in the majority of investigated Drosophila and mouse Hox proteins. We showed that HX-independent interaction modes are uncovered by the presence of Meis class cofactors, a property which was also observed with Hox proteins of the cnidarian sea anemone Nematostella vectensis. Finally, we revealed that paralog-specific motifs convey major PBC-recruiting functions in Drosophila Hox proteins. Altogether, our results highlight that flexibility in Hox-PBC interactions is an ancestral and evolutionary conserved character, which has strong implications for the understanding of Hox protein functions during normal development and pathologic processes., Author Summary Hox proteins are key transcriptional regulators of animal development, famously helping to determine identity along the anterior-posterior body axis. Although their evolution and developmental roles are well established, the molecular mechanisms underlying their specific functions remain poorly characterized. The current dominant view is that interaction with different members of the PBC family of transcription factors confers specific DNA-binding properties on different Hox proteins. However, this idea conflicts with in vitro evidence that a short “hexapeptide” (HX) motif shared by most Hox proteins is solely responsible for generic PBC recruitment. Here we have used the BiFC (bimolecular fluorescence complementation) method to address the global importance of the HX motif for Hox-PBC interactions in living cells and living animals including fruit flies and chick embryos. We observe that most interactions between Hox and PBC proteins do not depend on HX, and that alternative protein motifs are widely used for PBC recruitment in vivo. We also show that DNA binding by a second family of cofactors, the Meis proteins, unmasks these alternative interaction modes and that this property is conserved not only across Bilateria, but also in the basal animal phylum Cnidaria. Taken together, our results demonstrate that Hox-PBC partnership relies on multiple interaction modes, which can be influenced by additional transcriptional partners. We propose that this ancestral feature has been essential for ensuring Hox functional plasticity during development and evolution.

Temporal control can be considered the fourth dimension in embryonic development. The identification of the somitogenesis molecular clock provided new insight into how embryonic cells measure time. We provide the first evidence of a molecular clock operating during chick fore-limb autopod outgrowth and patterning, by showing that the expression of the somitogenesis clock component hairy2 cycles in autopod chondrogenic precursor cells with a 6 h periodicity. We determined the length of time required to form an autopod skeletal limb element, and established a correlation between the latter and the periodicity of cyclic hairy2 gene expression. We suggest that temporal control exerted by cyclic gene expression can be a widespread mechanism providing cellular temporal information during vertebrate embryonic development., We thank Raquel P. Andrade, Fernanda Bajanca,Leonor Saúde and Fernando Rodrigues for fruitfuldiscussions, and Mónica Ferreira for technical sup-port.FinancialsupportwasprovidedbyFCT/FEDER(POCTI/BCI/42040/2001), the EU/FP6-Network ofExcellence-Cells into Organs, ICVS and by IGC. S.P.(SFRH/BD/8657/2002) and J.R-L. (SFRH/BPD/5511/2001) were supported by FCT. C.R.C. wassupported by the Calouste Gulbenkian Foundation.

Forced expression of the bHLH myogenic factors, Myf5 and MyoD, in various mammalian cell lines induces the full program of myogenic differentiation. However, this property has not been extensively explored in vivo. We have taken advantage of the chick model to investigate the effect of electroporation of the mouse Myf5 and MyoD genes in the embryonic neural tube. We found that misexpression of either mouse Myf5 or MyoD in the chick neural tube leads to ectopic skeletal muscle differentiation, assayed by the expression of the myosin heavy chains in the neural tube and neural crest derivatives. We also showed that the endogenous neuronal differentiation program is inhibited under the influence of either ectopic mouse Myf5 or MyoD . We used this new system to analyse, in vivo, the transcriptional regulation between the myogenic factors. We found that MyoD and Myogenin expression can be activated by ectopic mouse Myf5 or MyoD , while Myf5 expression cannot be activated either by mouse MyoD or by itself. We also analysed the transcriptional regulation between the myogenic factors and the different genes involved in myogenesis, such as Mef2c, Pax3, Paraxis, Six1, Mox1, Mox2 and FgfR4 . We established the existence of an unexpected regulatory loop between MyoD and FgfR4 . The consequences for myogenesis are discussed.

The Paraxis gene encodes a basic helix-loop-helix (bHLH) transcription factor expressed in paraxial mesoderm and whose mutant displays an inability to form epithelial somites. We analyzed the later expression pattern of Paraxis transcripts in the chick limb. Paraxis transcripts are located in the Pax3-expressing myoblasts migrating from the somites and persist in proliferating myoblasts in the limb bud. Moreover, the QH1 antibody, which recognizes quail endothelial cells, reacts with a subset of the Paraxis/Pax3 expressing cells in the quail limb. Paraxis is then down-regulated during muscle differentiation.

The myogenic basic helix-loop-helix (bHLH) transcription factors, Myf5, MyoD, myogenin and MRF4, are unique in their ability to direct a program of specific gene transcription leading to skeletal muscle phenotype. The observation that Myf5 and MyoD can force myogenic conversion in non-muscle cells in vitro does not imply that they are equivalent. In this paper, we show that Myf5 transcripts are detected before those of MyoD during chick limb development. The Myf5 expression domain resembles that of Pax3 and is larger than that of MyoD. Moreover, Myf5 and Pax3 expression is correlated with myoblast proliferation, while MyoD is detected in post-mitotic myoblasts. These data indicate that Myf5 and MyoD are involved in different steps during chick limb bud myogenesis, Myf5 acting upstream of MyoD. The progression of myoblasts through the differentiation steps must be carefully controlled to ensure myogenesis at the right place and time during wing development. Because Notch signalling is known to prevent differentiation in different systems and species, we sought to determine whether these molecules regulate the steps occurring during chick limb myogenesis. Notch1 transcripts are associated with immature myoblasts, while cells expressing the ligands Delta1 and Serrate2 are more advanced in myogenesis. Misexpression of Delta1 using a replication-competent retrovirus activates the Notch pathway. After activation of this pathway, myoblasts still express Myf5 and Pax3 but have downregulated MyoD, resulting in inhibition of terminal muscle differentiation. We conclude that activation of Notch signalling during chick limb myogenesis prevents Myf5-expressing myoblasts from progressing to the MyoD-expressing stage.