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The location and regulation of fusion events within skeletal muscles during development remain unknown. Using the fusion marker myomaker (Mymk), named TMEM8C in chicken, as a readout of fusion, we identified a co-segregation of TMEM8C-positive cells and MYOG-positive cells in single-cell RNA-sequencing datasets of limbs from chicken embryos. We found that TMEM8C transcripts, MYOG transcripts and the fusion-competent MYOG-positive cells were preferentially regionalized in central regions of foetal muscles. We also identified a similar regionalization for the gene encoding the NOTCH ligand JAG2 along with an absence of NOTCH activity in TMEM8C+ fusion-competent myocytes. NOTCH function in myoblast fusion had not been addressed so far. We analysed the consequences of NOTCH inhibition for TMEM8C expression and myoblast fusion during foetal myogenesis in chicken embryos. NOTCH inhibition increased myoblast fusion and TMEM8C expression and released the transcriptional repressor HEYL from the TMEM8C regulatory regions. These results identify a regionalization of TMEM8C-dependent fusion and a molecular mechanism underlying the fusion-inhibiting effect of NOTCH in foetal myogenesis. The modulation of NOTCH activity in the fusion zone could regulate the flux of fusion events.

International audience; The elaboration of scaffolds able to efficiently promote cell differentiation toward a given cell type remains challenging. Here, we engineered dense type I collagen threads with the aim of providing scaffolds with specific morphological and mechanical properties for C3H10T1/2 mesenchymal stem cells. Extrusion of pure collagen solutions at different concentrations (15, 30, and 60 mg/mL) in a PBS 5× buffer generated dense fibrillated collagen threads. For the two highest concentrations, threads displayed a core-shell structure with a marked fibril orientation of the outer layer along the longitudinal axis of the threads. Young's modulus and ultimate tensile stress as high as 1 and 0.3 MPa, respectively, were obtained for the most concentrated collagen threads without addition of any cross-linkers. C3H10T1/2 cells oriented themselves with a mean angle of 15-24° with respect to the longitudinal axis of the threads. Cells penetrated the 30 mg/mL scaffolds but remained on the surface of the 60 mg/mL ones. After three weeks of culture, cells displayed strong expression of the tendon differentiation marker Tnmd, especially for the 30 mg/mL threads. These results suggest that both the morphological and mechanical characteristics of collagen threads are key factors in promoting C3H10T1/2 differentiation into tenocytes, offering promising levers to optimize tissue engineering scaffolds for tendon regeneration.

Positional information driving limb muscle patterning is contained in lateral plate mesoderm-derived tissues, such as tendon or muscle connective tissue but not in myogenic cells themselves. The long-standing consensus is that myogenic cells originate from the somitic mesoderm, while connective tissue fibroblasts originate from the lateral plate mesoderm. We challenged this model using cell and genetic lineage tracing experiments in birds and mice, respectively, and identified a subpopulation of myogenic cells at the muscle tips close to tendons originating from the lateral plate mesoderm and derived from connective tissue gene lineages. Analysis of single-cell RNA-sequencing data obtained from limb cells at successive developmental stages revealed a subpopulation of cells displaying a dual muscle and connective tissue signature, in addition to independent muscle and connective tissue populations. Active BMP signalling was detected in this junctional cell sub-population and at the tendon/muscle interface in developing limbs. BMP gain- and loss-of-function experiments performedin vivoandin vitroshowed that this signalling pathway regulated a fibroblast-to-myoblast conversion. We propose that localised BMP signalling converts a subset of lateral plate mesoderm-derived fibroblasts to a myogenic fate and establishes a boundary of fibroblast-derived myonuclei at the muscle/tendon interface to control the muscle pattern during limb development.

Differentiation and fusion are two intricate processes involved in skeletal muscle development. The close association of differentiation and fusion makes it difficult to address the process of fusion independently of differentiation. Using the fusion markermyomaker, namedTMEM8Cin chicken, we found that bothTMEM8Ctranscripts and the differentiated and fusion-competent MYOG+ cells are preferentially regionalized in the central regions of limb foetal muscles in chicken embryos. Because the NOTCH signalling pathway is a potent inhibitor of muscle differentiation during developmental myogenesis, NOTCH function in myoblast fusion was not addressed so far. We analysed the consequences of NOTCH inhibition for myoblast fusion andTMEM8Cexpression during foetal myogenesis using in vitro and in vivo chicken systems. NOTCH inhibition following chicken embryo immobilisation or in myoblast cultures increasedTMEM8Cexpression and myoblast fusion. Moreover, we showed that NOTCH inhibition induced the un-binding of the HEYL transcriptional repressor from theTMEM8Cregulatory regions in limb muscles and myoblast cultures. These results identify a molecular mechanism underlying the fusion-promoting effect of NOTCH-inhibition during foetal myogenesis.

One of the main challenges relating to tendons is to understand the regulators of the tendon differentiation program. The optimum culture conditions that favor tendon cell differentiation have not been identified. Mesenchymal stem cells present the ability to differentiate into multiple lineages in cultures under different cues ranging from chemical treatment to physical constraints. We analyzed the tendon differentiation potential of C3H10T1/2 cells, a murine cell line of mesenchymal stem cells, upon different 2D- and 3D-culture conditions. We observed that C3H10T1/2 cells cultured in 2D conditions on silicone substrate were more prone to tendon differentiation, assessed with the expression of the tendon markers Scx, Col1a1 and Tnmd as compared to cells cultured on plastic substrate. The 3D-fibrin environment was more favorable for Scx and Col1a1 expression compared to 2D cultures. We also identified TGFβ2 as a negative regulator of Tnmd expression in C3H10T1/2 cells in 2D and 3D cultures. Altogether, our results provide us with a better understanding of the culture conditions that promote tendon gene expression and identify mechanical and molecular parameters upon which we could act to define the optimum culture conditions that favor tenogenic differentiation in mesenchymal stem cells., Summary: Our results provide a better understanding of the culture conditions upon which we could act to trigger tendon cell differentiation from undifferentiated stem cells.

International audience; Connective tissues support organs and play crucial roles in development, homeostasis and fibrosis, yet our understanding of their formation is still limited. To gain insight into the molecular mechanisms of connective tissue specification, we selected five zinc-finger transcription factors-OSR1, OSR2, EGR1, KLF2 and KLF4-based on their expression patterns and/or known involvement in connective tissue subtype differentiation. RNA-seq and ChIP-seq profiling of chick limb micromass cultures revealed a set of common genes regulated by all five transcription factors, which we describe as a connective tissue core expression set. This common core was enriched with genes associated with axon guidance and myofibroblast signature, including fibrosis-related genes. In addition, each transcription factor regulated a specific set of signalling molecules and extracellular matrix components. This suggests a concept whereby local molecular niches can be created by the expression of specific transcription factors impinging on the specification of local microenvironments. The regulatory network established here identifies common and distinct molecular signatures of limb connective tissue subtypes, provides novel insight into the signalling pathways governing connective tissue specification, and serves as a resource for connective tissue development.

BackgroundConnective tissues support, connect and separate tissues and organs, playing crucial roles in development, homeostasis and fibrosis. Cell specification and differentiation is triggered by the activity of specific transcription factors. While key transcription factors have been identified for differentiation processes of most tissues, connective tissue differentiation remains largely unstudied.ResultsTo gain insight into the regulatory cascades involved in connective tissue differentiation, we selected five zinc finger transcription factors - OSR1, OSR2, EGR1, KLF2 and KLF4 - based on their expression patterns and/or known involvement in the differentiation of mesenchymal cells into connective tissue subtypes. We combined RNA-seq with ChIP-seq profiling in chick limb cells following overexpression of individual transcription factors. We identified a set of common genes regulated by all five transcription factors, which constitutes a connective tissue core expression set. This common core was enriched in genes associated with axon guidance and myofibroblast signature. In addition, each of the transcription factors regulated a different set of extracellular matrix components and signalling molecules, which define local molecular niches important for connective tissue development and function.ConclusionsThe established regulatory network identifies common and distinct molecular signatures downstream of five connective tissue-associated transcription factors and provides insight into the signalling pathways governing limb connective tissue differentiation. It also suggests a concept whereby local molecular niches can be created via the expression of specific transcription factors impinging on the specification of microenvironments.

International audience; Tight regulation of cell proliferation and differentiation is required to ensure proper growth during development and post-natal life. The source and nature of signals regulating cell proliferation are not well identified in vivo. We investigated the specific pattern of proliferating cells in mouse limbs, using the Fluorescent ubiquitynation-based cell-cycle indicator (Fucci) system, which allowed the visualization of the G1, G1/S transition and S/G2/M phases of the cell cycle in red, yellow or green fluorescent colors, respectively. We also used the retroviral RCAS system to express a Fucci cassette in chick embryos. We performed a comprehensive analysis of the cell cycle state of myogenic cells in fetal limb muscles, adult myoblast primary cultures and isolated muscle fiber cultures using the Fucci transgenic mice. We found that myonuclei of terminally differentiated muscle fibers displayed Fucci red fluorescence during mouse and chick fetal development, in adult isolated muscle fiber (ex vivo) and adult myoblast (in vitro) mouse cultures. This indicated that myonuclei exited from the cell cycle in the G1 phase and are maintained in a blocked G1-like state. We also found that cycling muscle progenitors and myoblasts in G1 phase were not completely covered by the Fucci system. During mouse fetal myogenesis, Pax7+ cells labeled with the Fucci system were observed mostly in S/G2/M phases. Proliferating cells in S/G2/M phases displayed a specific pattern in mouse fetal limbs, delineating individualized muscles. In addition, we observed more Pax7+ cells in S/G2/M phases at muscle tips, compared to the middle of muscles. These results highlight a specific spatial regionalization of cycling cells at the muscle borders and muscle-tendon interface during fetal development.

International audience; The molecular programme underlying tendon development has not been fully identified. Interactions with components of the musculoskeletal system are important for limb tendon formation. Limb tendons initiate their development independently of muscles; however, muscles are required for further tendon differentiation. We show that both FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are required and sufficient for SCX expression in chick undifferentiated limb cells, whereas the FGF/ERK MAPK pathway inhibits Scx expression in mouse undifferentiated limb mesodermal cells. During differentiation, muscle contraction is required to maintain SCX, TNMD and THBS2 expression in chick limbs. The activities of FGF/ERK MAPK and TGFβ/SMAD2/3 signalling pathways are decreased in tendons under immobilisation conditions. Application of FGF4 or TGFβ2 ligands prevents SCX downregulation in immobilised limbs. TGFβ2 but not FGF4 prevent TNMD and THBS2 downregulation under immobilisation conditions. We did not identify any intracellular crosstalk between both signalling pathways in their positive effect on SCX expression. Independently of each other, both FGF and TGFβ promote tendon commitment of limb mesodermal cells and act downstream of mechanical forces to regulate tendon differentiation during chick limb development.

The importance of mechanical activity in the regulation of muscle progenitors during chick development has not been investigated. We show that immobilization decreases NOTCH activity and mimics a NOTCH loss-of-function phenotype, a reduction in the number of muscle progenitors and increased differentiation. Ligand-induced NOTCH activation prevents the reduction of muscle progenitors and the increase of differentiation upon immobilization. Inhibition of NOTCH ligand activity in muscle fibers suffices to reduce the progenitor pool. Furthermore, immobilization reduces the activity of the transcriptional co-activator YAP and the expression of the NOTCH ligand JAG2 in muscle fibers. YAP forced-activity in muscle fibers prevents the decrease of JAG2 expression and the number of PAX7+ cells in immobilization conditions. Our results identify a novel mechanism acting downstream of muscle contraction, where YAP activates JAG2 expression in muscle fibers, which in turn regulates the pool of fetal muscle progenitors via NOTCH in a non-cell-autonomous manner. DOI: http://dx.doi.org/10.7554/eLife.15593.001, eLife digest Skeletal muscle is attached to the skeleton and allows the body to move. Making a new muscle, or repairing an existing one, relies on stem cells that are present inside muscles. A major goal of skeletal muscle research is to understand the signals that regulate the abilities of muscle stem cells to divide and give rise to more stem cells or to become muscle cells. Molecular signals are known to regulate the numbers of stem cells in the muscle. Skeletal muscles become larger if they are exercised, but it is not clear if mechanical forces generated by muscle contractions directly affect the number of muscle stem cells. The NOTCH signaling pathway contributes to maintaining the population of stem cells in muscles by forcing the stem cells to divide and preventing them from becoming muscle cells. Here, Esteves de Lima et al. investigated whether muscle contraction regulates NOTCH signaling during muscle formation in chick fetuses. The experiments show that muscle contraction stimulates the activity of a protein called YAP in muscle cells, which in turn, activates a gene in the NOTCH signaling pathway known as JAG2. This increases NOTCH signaling activity in the neighboring stem cells and maintains the number of stem cells in the muscle. The next step following this work will be to establish if this mechanism also operates during muscle formation and regeneration in other animals such as mice and zebrafish. DOI: http://dx.doi.org/10.7554/eLife.15593.002

International audience; The basic helix-loop-helix transcription factor MyoD is a central actor that triggers the skeletal myogenic program. Cell-autonomous and non-cell-autonomous regulatory pathways must tightly control MyoD expression to ensure correct initiation of the muscle program at different places in the embryo and at different developmental times. In the present study, we have addressed the involvement of Sim2 (single-minded 2) in limb embryonic myogenesis. Sim2 is a bHLH-PAS transcription factor that inhibits transcription by active repression and displays enhanced expression in ventral limb muscle masses during chick and mouse embryonic myogenesis. We have demonstrated that Sim2 is expressed in muscle progenitors that have not entered the myogenic program, in different experimental conditions. MyoD expression is transiently upregulated in limb muscle masses of Sim2(-/-) mice. Conversely, Sim2 gain-of-function experiments in chick and Xenopus embryos showed that Sim2 represses MyoD expression. In addition, we show that Sim2 represses the activity of the mouse MyoD promoter in primary myoblasts and is recruited to the MyoD core enhancer in embryonic mouse limbs. Sim2 expression is non-autonomously and negatively regulated by the dorsalising factor Lmx1b. We propose that Sim2 represses MyoD transcription in limb muscle masses, through Sim2 recruitment to the MyoD core enhancer, in order to prevent premature entry into the myogenic program. This MyoD repression is predominant in ventral limb regions and is likely to contribute to the differential increase of the global mass of ventral muscles versus dorsal muscles.

Mice deficient for the homeobox gene Six1 display defects in limb muscles consistent with the Six1 expression in myogenic cells. In addition to its myogenic expression domain, Six1 has been described as being located in digit tendons and as being associated with connective tissue patterning in mouse limbs. With the aim of determining a possible involvement of Six1 in tendon development, we have carefully characterised the non-myogenic expression domain of the Six1 gene in mouse and chick limbs. In contrast to previous reports, we found that this non-myogenic domain is distinct from tendon primordia and from tendons defined by scleraxis expression. The non-myogenic domain of Six1 expression establishes normally in the absence of muscle, in Pax3−/− mutant limbs. Moreover, the expression of scleraxis is not affected in early Six1−/− mutant limbs. We conclude that the expression of the Six1 gene is not related to tendons and that Six1, at least on its own, is not involved in limb tendon formation in vertebrates. Finally, we found that the posterior domain of Six1 in connective tissue is adjacent to that of the secreted factor Sonic hedgehog and that Sonic hedgehog is necessary and sufficient for Six1 expression in posterior limb regions.

Members of the cysteine-rich protein (CRP) family are evolutionary conserved proteins that have been implicated in the processes of cell proliferation and differentiation via the cytoskeletal proteins. In this paper, we present the dynamic expression pattern of CPR2 transcripts during chick limb bud development. CRP2 transcripts are located in various tissues, including muscle, arteries, cartilage, ligaments and digit tendons and also in the apical ectodermal ridge and feather buds.

In vertebrates, tendons connect muscles to skeletal elements. Surgical experiments in the chick have underlined developmental interactions between tendons and muscles. Initial formation of tendons occurs autonomously with respect to muscle. However, further tendon development requires the presence of muscle. The molecular signals involved in these interactions remain unknown. In the chick limb, Fgf4 transcripts are located at the extremities of muscles, where the future tendons will attach. In this paper, we analyse the putative role of muscle-Fgf4 on tendon development. We have used three general tendon markers, scleraxis, tenascin, and Fgf8 to analyse the regulation of these tendon-associated molecules by Fgf4 under different experimental conditions. In the absence of Fgf4, in muscleless and aneural limbs, the expression of the three tendon-associated molecules, scleraxis, tenascin, and Fgf8, is down-regulated. Exogenous implantation of Fgf4 in normal, aneural, and muscleless limbs induces scleraxis and tenascin expression but not that of Fgf8. These results indicate that Fgf4 expressed in muscle is required for the maintenance of scleraxis and tenascin but not Fgf8 expression in tendons.

Fibroblast growth factor 8 (Fgf8) is a secreted growth factor involved in the initiation, outgrowth and patterning of vertebrate limbs (Genes Dev. 12 (1998) 1571). In this paper, we present a new site of expression of Fgf8 in the chick limb. Fgf8 transcripts are localised close to the muscle fibres, at the same level as the tendon-associated molecules, tenascin and scleraxis. Fgf8 is expressed in a sub-region of the tendons during limb development; its location being restricted to the area near the muscle. In addition, the restricted Fgf8 expression in the tendons allowed us to observe that the myogenic determination factor (MyoD) is not detected at the myotendinous junction.

Skeletal muscle development involves an initial period of myoblast replication followed by a phase in which some myoblasts continue to proliferate while others undergo terminal differentiation. The latter process involves the permanent cessation of DNA synthesis, activation of muscle-specific gene expression, and fusion of single cells to generate multinucleated muscle fibres. The in vivo signals regulating the progression through all these steps remain unknown. Fibroblast growth factors (Fgfs) and Fgf receptors comprise a large family whose members have been shown to play multiple roles in the development of skeletal muscle in vitro. Exogenously applied Fgfs are able to stimulate proliferation and suppress myogenic differentiation in cell culture. We sought to determine the role played by Fgf-4 during limb myogenesis in vivo. Fgf-4 transcripts are located at both extremities of myotubes whereas the mRNAs of one of the Fgf receptors, Frek, are detected in mononucleated proliferating myoblasts surrounding the multinucleated fibres. Overexpression of mouse Fgf-4 (mFgf-4) using a replication-competent retrovirus, RCAS, leads to a down-regulation of muscle markers followed by an inhibition of terminal differentiation in limb muscles. Using quail/chick transplantations we were able to follow the muscle cells and found a dramatic decrease in their number after exposure to mFgf-4. Interestingly ectopic mFgf-4 down-regulates Frek transcripts in limb muscle areas. We conclude that overexpression of mFgf-4 inhibits myoblast proliferation, probably by down-regulating Frek mRNAs. This suggests a role for Fgf-4, located at the extremities of the myotubes, where it could be responsible for the absence of Frek mRNA in the muscle fibre.

We cloned the chick homolog of Xenopus and mouse Frzb-1, a secreted Wnt antagonist and performed in situ hybridisations to determine the pattern of cFrzb-1 expression in the developing chick embryo. At early stages, cFrzb-1 transcripts are located exclusively in the ectodermal layer corresponding to the neural plate. The labelling continues in the neural tube, but is always excluded from the floor plate. cFrzb-1 mRNA is expressed by migrating cephalic and truncal neural crest cells. Later, cFrzb-1 transcripts are found in a subset of neural crest derivatives such as cephalic cartilage, nerves and spinal ganglia. In addition to ectodermal derivatives, cFrzb-1 transcripts were also observed in mesodermal derivatives such as vertebral and limb cartilage, the adrenal cortex, the gonads, and a subpopulation of blood cells.

International audience; 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, Colla1, and Colla2, and were mechanically weaker compared with their WT littermates. EGR1 was recruited to the Colla1 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-beta 2. This study demonstrates EGR1 involvement in adult tendon formation, healing, and repair and identifies Egr1 as a putative target in tendon repair strategies.

International audience; Muscle formation and vascular assembly during embryonic development are usually considered separately. In this paper, we investigate the relationship between the vasculature and muscles during limb bud development. We show that endothelial cells are detected in limb regions before muscle cells and can organize themselves in space in the absence of muscles. In chick limbs,endothelial cells are detected in the future zones of muscle cleavage,delineating the cleavage pattern of muscle masses. We therefore perturbed vascular assembly in chick limbs by overexpressing VEGFA and demonstrated that ectopic blood vessels inhibit muscle formation, while promoting connective tissue. Conversely, local inhibition of vessel formation using a soluble form of VEGFR1 leads to muscle fusion. The endogenous location of endothelial cells in the future muscle cleavage zones and the inverse correlation between blood vessels and muscle suggests that vessels are involved in the muscle splitting process. We also identify the secreted factor PDGFB (expressed in endothelial cells) as a putative molecular candidate mediating the muscle-inhibiting and connective tissue-promoting functions of blood vessels. Finally, we propose that PDGFB promotes the production of extracellular matrix and attracts connective tissue cells to the future splitting site, allowing separation of the muscle masses during the splitting process.

Background Secreted Hedgehog (Hh) signalling molecules have profound influences on many developing and regenerating tissues. Yet in most vertebrate tissues it is unclear which Hh-responses are the direct result of Hh action on a particular cell type because Hhs frequently elicit secondary signals. In developing skeletal muscle, Hhs promote slow myogenesis in zebrafish and are involved in specification of medial muscle cells in amniote somites. However, the extent to which non-myogenic cells, myoblasts or differentiating myocytes are direct or indirect targets of Hh signalling is not known. Results We show that Sonic hedgehog (Shh) can act directly on cultured C2 myoblasts, driving Gli1 expression, myogenin up-regulation and terminal differentiation, even in the presence of growth factors that normally prevent differentiation. Distinct myoblasts respond differently to Shh: in some slow myosin expression is increased, whereas in others Shh simply enhances terminal differentiation. Exposure of chick wing bud cells to Shh in culture increases numbers of both muscle and non-muscle cells, yet simultaneously enhances differentiation of myoblasts. The small proportion of differentiated muscle cells expressing definitive slow myosin can be doubled by Shh. Shh over-expression in chick limb bud reduces muscle mass at early developmental stages while inducing ectopic slow muscle fibre formation. Abundant later-differentiating fibres, however, do not express extra slow myosin. Conversely, Hh loss of function in the limb bud, caused by implanting hybridoma cells expressing a functionally blocking anti-Hh antibody, reduces early slow muscle formation and differentiation, but does not prevent later slow myogenesis. Analysis of Hh knockout mice indicates that Shh promotes early somitic slow myogenesis. Conclusions Taken together, the data show that Hh can have direct pro-differentiative effects on myoblasts and that early-developing muscle requires Hh for normal differentiation and slow myosin expression. We propose a simple model of how direct and indirect effects of Hh regulate early limb myogenesis.

The regionalization of the neural tube along the anteroposterior axis is established through the action of patterning signals from the endomesoderm including the organizer. These signals set up a pre-pattern which is subsequently refined through local patterning events. The midbrain-hindbrain junction, or isthmus, is endowed with such an organizing activity. It is able to induce graded expression of the Engrailed protein in the adjacent mesencephalon and rhombencephalon, and subsequently elicits the development of tectal and cerebellar structures. Ectopically grafted isthmus was also shown to induce Engrailed expression in diencephalon and otic and pre-otic rhombencephalon. Fgf8 is a signalling protein which is produced by the isthmus and which is able to mimic most isthmic properties. We show here that the isthmus, when transposed to the level of either rhombomere 8 or the spinal cord, loses its ability to induce Engrailed and cerebellar development in adjacent tissues. This is accompanied by the down-regulation of fgf8 expression in the grafted isthmus and by the up-regulation of a marker of the recipient site, Hoxb-4. Moreover, these changes in gene activity in the transplant are followed by a transformation of the fate of the grafted cells which adjust to their novel environment. These results show that the fate of the isthmus is not determined at 10-somite stage and that the molecular loop of isthmic maintenance can be disrupted by exogenous signals.

It has been previously shown that Hox gene expression in the rhombencephalon is controlled by environmental cues. Thus posterior transposition of anterior rhombomeres to the r7/8 level results in the activation of Hox genes of the four first paralog groups and in homeotic transformations of the neuroepithelial fate according to its position along the anteroposterior axis. We demonstrate here that although the anteroposterior levels of r2 to r6 express Hox genes they do not have inducing activity on more anterior territories. If transposed at the posterior rhombencephalon and trunk level, however, the same anterior regions are able to express Hox gene such as Hoxa-2, a-3 or b-4. We also provide evidence that these signals are transferred by two paths: one vertical, arising from the paraxial mesoderm, and one planar, travelling in the neural epithelium. The competence to express Hox genes extends up to the forebrain and midbrain but expression of Hox genes does not preclude Otx2 expression in these territories and results only in slight changes in their phenotypes. Similarly, rhombomeres transplanted to posterior truncal levels turned out to be able to express posterior genes of the first eight paralog groups to the exclusion of others located downstream in the Hox genes genomic clusters. This suggests that the neural tube is divided into large territories characterized by different Hox gene regulatory features.

It has been shown by using the quail/chick chimera system that Hox gene expression in the hindbrain is influenced by positional signals arising from the environment. In order to decipher the pathway that leads to Hox gene induction, we have investigated whether a Hox gene regulator, the leucine zipper transcription factor MafB/Kr, is itself transcriptionally regulated by the environmental signals. This gene is normally expressed in rhombomeres (r) 5 and 6 and their associated neural crest. MafB/Kr expression is maintained in r5/6 when grafted into the environment of r3/4. On the contrary, the environment of rhombomeres 7/8 represses MafB/Kr expression. Thus, as previously shown for the expression of Hox genes, MafB/Kr expression is regulated by a posterior-dominant signal, which in this case induces the loss of expression of this gene. We also show that the posterior signal can be transferred to the r5/6 neuroepithelium by posterior somites (somites 7 to 10) grafted laterally to r5/6. At the r4 level, the same somites induce MafB/Kr in r4, leading it to behave like r5/6. The posterior environment regulates MafB/Kr expression in the neural crest as it does in the corresponding hindbrain level, showing that some positional regulatory mechanisms are shared by neural tube and neural crest cells. Retinoic acid beads mimic the effect produced by the somites in repressing MafB/Kr in r5/6 and progressively inducing it more rostrally as its concentration increases. We therefore propose that the MafB/Kr expression domain is defined by a molecule unevenly distributed in the paraxial mesoderm. This molecule would allow the expression of the MafB/Kr gene in a narrow window of concentration by activating its expression at a definite threshold and repressing it at higher levels, accounting for its limited domain of expression in only two rhombomeres. It thus appears that the regulation of MafB/Kr expression in the rhombomeres could be controlled by the same posteriorizing factor(s) as Hox genes.

Homeobox genes play an important role in the regulation of early embryonic development. They represent a family of evolutionarily highly conserved transcription factors. In this work, several genes that belong to the four HOX gene clusters are assigned by in situ hybridization to four distinct chicken chromosomes. The four gene clusters are mapped to 2p2.1 (HOXA), 3q3.1 (HOXB), 1q3.1 (HOXC) and 7q1.3→ q1.4 (HOXD). We confirm partial homologies already detected by genetic mapping between chicken chromosomes 1, 2 and 7 and human chromosomes 12, 7 and 2 and we describe a new conserved segment between chicken chromosome 3 and human chromosome 17. These results represent the first data that confirm the physical linkage between chicken HOX genes and may improve our understanding of phylogenetic relationships and genome evolution.

In this study we have analysed the expression of Hoxb-4, Hoxb-1, Hoxa-3, Hoxb-3, Hoxa-4 and Hoxd-4 in the neural tube of chick and quail embryos after rhombomere (r) heterotopic transplantations within the rhombencephalic area. Grafting experiments were carried out at the 5-somite stage, i.e before rhombomere boundaries are visible. They were preceeded by the establishment of the precise fate map of the rhombencephalon in order to determine the presumptive territory corresponding to each rhombomere. When a rhombomere is transplanted from a caudal to a more rostral position it expresses the same set of Hox genes as in situ. By contrast in many cases, if rhombomeres are transplanted from rostral to caudal their Hox gene expression pattern is modified. They express genes normally activated at the new location of the explant, as evidenced by unilateral grafting. This induction occurs whether transplantation is carried out before or after rhombomere boundary formation. Moreover, the fate of the cells of caudally transplanted rhombomeres is modified: the rhombencephalic nuclei in the graft develop according to the new location as shown for an r5/6 to r8 transplantation. Transplantation of 5 consecutive rhombomeres (i.e. r2 to r6), to the r8 level leads to the induction of Hoxb-4 in the two posteriormost rhombomeres but not in r2,3,4. Transplantations to more caudal regions (posterior to somite 3) result in some cases in the induction of Hoxb-4 in the whole transplant. Neither the mesoderm lateral to the graft nor the notochord is responsible for the induction. Thus, the inductive signal emanates from the neural tube itself, suggesting that planar signalling and predominance of posterior properties are involved in the patterning of the neural primordium.