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In mammalian development, oscillatory activation of Notch signaling is required for segmentation clock function during somitogenesis. Notch activity oscillations are synchronized between neighboring cells in the presomitic mesoderm (PSM) and have a period that matches the rate of somite formation. Normal clock function requires cyclic expression of the Lunatic fringe (LFNG) glycosyltransferase, as well as expression of the inhibitory Notch ligand Delta-like 3 (DLL3). How these factors coordinate Notch activation in the clock is not well understood. Recent evidence suggests that LFNG can act in a signal-sending cell to influence Notch activity in the clock, raising the possibility that in this context, glycosylation of Notch pathway proteins by LFNG may affect ligand activity. Here we dissect the genetic interactions of Lfng and Dll3 specifically in the segmentation clock and observe distinctions in the skeletal and clock phenotypes of mutant embryos showing that paradoxically, loss of Dll3 is associated with strong reductions in Notch activity in the caudal PSM. The patterns of Notch activity in the PSM suggest that the loss of Dll3 is epistatic to the loss of Lfng in the segmentation clock, and we present direct evidence for the modification of several DLL1 and DLL3 EGF-repeats by LFNG. We further demonstrate that DLL3 expression in cells co-expressing DLL1 and NOTCH1 can potentiate a cell's signal-sending activity and that this effect is modulated by LFNG, suggesting a mechanism for coordinated regulation of oscillatory Notch activation in the clock by glycosylation and cis -inhibition. [Display omitted] • Axial skeletal phenotypes after loss of DLL3 or LFNG are similar except in the sacrum. • Loss of DLL3 in the mouse PSM counterintuitively leads to a reduction in Notch signaling. • Both EGF repeats 2 and 5 of DLL3 are efficiently modified by LFNG. • When cells co-express NOTCH1 and DLL1, induction of DLL3 expression potentiates signal-sending activities. • LFNG blocks DLL3's effect on signal-sending activity, providing cyclic regulation of cis -interactions during somitogenesis. [ABSTRACT FROM AUTHOR]

T is the founding member of the T-box family of transcription factors; family members are critical for cell fate decisions and tissue morphogenesis throughout the animal kingdom. T is expressed in the primitive streak and notochord with mouse mutant studies revealing its critical role in mesoderm formation in the primitive streak and notochord integrity. We previously demonstrated that misexpression of Tbx6 in the paraxial and lateral plate mesoderm results in embryos resembling Tbx15 and Tbx18 nulls. This, together with results from in vitro transcriptional assays, suggested that ectopically expressed Tbx6 can compete with endogenously expressed Tbx15 and Tbx18 at the binding sites of target genes. Since T-box proteins share a similar DNA binding domain, we hypothesized that misexpressing T in the paraxial and lateral plate mesoderm would also interfere with the endogenous Tbx15 and Tbx18, causing embryonic phenotypes resembling those seen upon Tbx6 expression in the somites and limbs. Interestingly, ectopic T expression led to distinct embryonic phenotypes, specifically, reduced-sized somites in embryos expressing the highest levels of T , which ultimately affects axis length and neural tube morphogenesis. We further demonstrate that ectopic T leads to ectopic expression of Tbx6 and Mesogenin 1 , known targets of T. These results suggests that ectopic T expression contributes to the phenotype by activating its own targets rather than via a straight competition with endogenous T-box factors. [Display omitted] • Generation of 3-component transgenic mouse system to inducibly express T under Cre recombinase and Doxycycline control. • Ectopic T expression in the mouse paraxial mesoderm affects somite maturation. • Somites can differentiate into dermomyotome and sclerotome but these are often irregular. • Secondary effects include undulating neural tube, compaction of the axis, and fusion of fore- and hindlimb buds. • Ectopic expression of a subset of T target genes potentially contributes to the phenotype. [ABSTRACT FROM AUTHOR]

During vertebrate somitogenesis an inherent segmentation clock coordinates the spatiotemporal signaling to generate segmented structures that pattern the body axis. Using our experimental and quantitative approach, we study the cell movements and the genetic oscillations of her1 expression level at single-cell resolution simultaneously and scale up to the entire pre-somitic mesoderm (PSM) tissue. From the experimentally determined phases of PSM cellular oscillators, we deduced an in vivo frequency profile gradient along the anterior-posterior PSM axis and inferred precise mathematical relations between spatial cell–level period and tissue-level somitogenesis period. We also confirmed a gradient in the relative velocities of cellular oscillators along the axis. The phase order parameter within an ensemble of oscillators revealed the degree of synchronization in the tailbud and the posterior PSM being only partial, whereas synchronization can be almost complete in the presumptive somite region but with temporal oscillations. Collectively, the degree of synchronization itself, possibly regulated by cell movement and the synchronized temporal phase of the transiently expressed clock protein Her1, can be an additional control mechanism for making precise somite boundaries. • Somite formation in context of moving cellular oscillators being phase synchronized. • Precise mathematical relation between spatial cell periods and somitogenesis period. • Her1 synchronization levels temporally oscillate only in the forming somite. • Low synchronization strength in tailbud/posterior cells. • Clock components are dynamically tuned along the anterior-posterior axis. [ABSTRACT FROM AUTHOR]

The two somite compartments, dorso-lateral dermomyotome and medio-ventral sclerotome are major vertebrate novelties, but little is known about their evolutionary origin. We determined that sclerotome cells in Xenopus come from lateral somitic frontier (LSF) by lineage tracing, ablation experiments and histological analysis. We identified Twist1 as marker of migrating sclerotome progenitors in two amphibians, Xenopus and axolotl. From these results, three conclusions can be drawn. First, LSF is made up of multipotent somitic cells (MSCs) since LSF gives rise to sclerotome but also to dermomytome as already shown in Xenopus. Second, the basic scheme of somite compartmentalization is conserved from cephalochordates to anamniotes since in both cases, lateral cells envelop dorsally and ventrally the ancestral myotome, suggesting that lateral MSCs should already exist in cephalochordates. Third, the transition from anamniote to amniote vertebrates is characterized by extension of the MSCs domain to the entire somite at the expense of ancestral myotome since amniote somite is a naive tissue that subdivides into sclerotome and dermomyotome. Like neural crest pluripotent cells, MSCs are at the origin of major vertebrate novelties, namely hypaxial region of the somite, dermomyotome and sclerotome compartments. Hence, change in MSCs properties and location is involved in somite evolution. • Sclerotome progenitors originate from lateral somitic frontier. • Lateral somitic frontier is made of multipotent somitic cells. • Somite compartmentalization is conserved between cephalochordates and anamniotes. • Multipotent somitic cells domain is expended to entire somite in amniote vertebrates. [ABSTRACT FROM AUTHOR]

Abstract The enteric nervous system is mostly derived from vagal neural crest (NC) cells adjacent to somites (s)1–7. We used in ovo focal fluorescent vital dyes and focal electroporation of fluorophore-encoding plasmids in quail embryos to investigate NC cell migration to the foregut initially and later throughout the entire gut. NC cells of different somite-level origins were largely separate until reaching the foregut at about QE2.5, when all routes converged. By QE3.5, NC cells of different somite-levels became mixed, although s1-s2 NC cells were mainly confined to rostral foregut. Mid-vagal NC-derived cells (s3 and s4 level) arrived earliest at the foregut, and occurred in greatest number. By QE6.5 ENS was present from foregut to hindgut. Mid-vagal NC-derived cells occurred in greatest numbers from foregut to distal hindgut. NC-derived cells of s2, s5, and s6 levels were fewer and were widely distributed but were never observed in the distal hindgut. Rostro-vagal (s1) and caudo-vagal (s7) levels were few and restricted to the foregut. Single somite levels of quail neural tube/NC from s1 to s8 were combined with chick aneural ChE4.5 midgut and hindgut and the ensemble was grown on the chorio-allantoic membrane for 6 days. This tests ENS-forming competence in the absence of intra-segmental competition between NC cells, of differential influences of segmental paraxial tissues, and of positional advantage. All vagal NC-levels, but not s8 level, furnished enteric plexuses in the recipient gut, but the density of both ENS cells in total and neurons was highest from mid-vagal level donors, as was the length colonised. We conclude that the fate and competence for ENS formation of vagal NC sub-levels is not uniform over the vagal level but is biased to favour mid-vagal levels. Overviewing this and prior studies suggests the vagal region is, as in its traditional sense, a natural unit but with complex sub-divisions. Highlights • Neural crest cells of somite level s2 to s6 are fated to contribute to the ENS throughout the gut of avian embryos. • Neural crest cells of s3 and s4 levels have the most extensive and numerically largest contribution to the ENS. • Neural crest cells of somite levels s1 and s7 contribute ENS only to the foregut. • The capability of isolated somite levels of neural crest to form ENS in experiments exactly parallels the fate pattern. • The vagal neural crest level with somites s1 to s7 is a natural unit with complex axial pre-specification. [ABSTRACT FROM AUTHOR]

The diaphragm is a mammalian skeletal muscle essential for respiration and for separating the thoracic and abdominal cavities. Development of the diaphragm requires the coordinated development of muscle, muscle connective tissue, tendon, nerves, and vasculature that derive from different embryonic sources. However, defects in diaphragm development are common and the cause of an often deadly birth defect, Congenital Diaphragmatic Hernia (CDH). Here we comprehensively describe the normal developmental origin and complex spatial-temporal relationship between the different developing tissues to form a functional diaphragm using a developmental series of mouse embryos genetically and immunofluorescently labeled and analyzed in whole mount. We find that the earliest developmental events are the emigration of muscle progenitors from cervical somites followed by the projection of phrenic nerve axons from the cervical neural tube. Muscle progenitors and phrenic nerve target the pleuroperitoneal folds (PPFs), transient pyramidal-shaped structures that form between the thoracic and abdominal cavities. Subsequently, the PPFs expand across the surface of the liver to give rise to the muscle connective tissue and central tendon, and the leading edge of their expansion precedes muscle morphogenesis, formation of the vascular network, and outgrowth and branching of the phrenic nerve. Thus development and morphogenesis of the PPFs is critical for diaphragm formation. In addition, our data indicate that the earliest events in diaphragm development are critical for the etiology of CDH and instrumental to the evolution of the diaphragm. CDH initiates prior to E12.5 in mouse and suggests that defects in the early PPF formation or their ability to recruit muscle are an important source of CDH. Also, the recruitment of muscle progenitors from cervical somites to the nascent PPFs is uniquely mammalian and a key developmental innovation essential for the evolution of the muscularized diaphragm. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are highly conserved small non-coding RNA molecules that post-transcriptionally regulate gene expression in multicellular organisms. Within the set of muscle-specific miRNAs, miR-206 expression is largely restricted to skeletal muscle and is found exclusively within the bony fish lineage. Although many studies have implicated miR-206 in muscle maintenance and disease, its role in skeletal muscle development remains largely unknown. Here, we examine the role of miR-206 during Xenopus laevis somitogenesis. In Xenopus laevis, miR-206 expression coincides with the onset of somitogenesis. We show that both knockdown and over-expression of miR-206 result in abnormal somite formation affecting muscle cell rotation, attachment, and elongation. In particular, our data suggests that miR-206 regulates changes in cell adhesion that affect the ability of newly formed somites to adhere to the notochord as well as to the intersomitic boundaries. Additionally, we show that β-dystroglycan and F-actin expression levels are significantly reduced, suggesting that knockdown of miR-206 levels affects cellular mechanics necessary for cell shape changes and attachments that are required for proper muscle formation. [ABSTRACT FROM AUTHOR]

Paxillin (Pxn) is a key adapter protein and signaling regulator at sites of cell-extracellular matrix (ECM) adhesion. Here, we investigated the role of Pxn during vertebrate development using the zebrafish embryo as a model system. We have characterized two Pxn genes, pxna and pxnb , in zebrafish that are maternally supplied and expressed in multiple tissues. Gene editing and antisense gene knockdown approaches were used to uncover Pxn functions during zebrafish development. While mutation of either pxna or pxnb alone did not cause gross embryonic phenotypes, double mutants lacking maternally supplied pxna or pxnb displayed defects in cardiovascular, axial, and skeletal muscle development. Transient knockdown of Pxn proteins resulted in similar defects. Irregular myotome shape and ECM composition were observed, suggesting an “inside-out” signaling role for Paxillin genes in the development of myotendinous junctions. Inhibiting non-muscle Myosin-II during somitogenesis altered the subcellular localization of Pxn protein and phenocopied pxn gene loss-of-function. This indicates that Paxillin genes are effectors of actomyosin contractility-driven morphogenesis of trunk musculature in zebrafish. Together, these results reveal new functions for Pxn during muscle development and provide novel genetic models to elucidate Pxn functions. [ABSTRACT FROM AUTHOR]

Somitogenesis and subsequent axial skeletal development is regulated by the interaction of pathways that determine the periodicity of somite formation, rostrocaudal somite polarity and segment identity. Here we use a hypomorphic mutant mouse line to demonstrate that Supt20 ( Suppressor of Ty20 ) is required for development of the axial skeleton. Supt20 hypomorphs display fusions of the ribs and vertebrae at lower thoracic levels along with anterior homeotic transformation of L1 to T14. These defects are preceded by reduction of the rostral somite and posterior shifts in Hox gene expression. While cycling of Notch target genes in the posterior presomitic mesoderm (PSM) appeared normal, expression of Lfng was reduced. In the anterior PSM, Mesp2 expression levels and cycling were unaffected; yet, expression of downstream targets such as Lfng, Ripply2 , Mesp1 and Dll3 in the prospective rostral somite was reduced accompanied by expansion of caudal somite markers such as EphrinB2 and Hes7. Supt20 interacts with the Gcn5-containing SAGA histone acetylation complex. Gcn5 hypomorphic mutant embryos show similar defects in axial skeletal development preceded by posterior shift of Hoxc8 and Hoxc9 gene expression. We demonstrate that Gcn5 and Supt20 hypomorphs show similar defects in rostral-caudal somite patterning potentially suggesting shared mechanisms. [ABSTRACT FROM AUTHOR]

In vertebrates, the periodic formation of somites from the presomitic mesoderm (PSM) is driven by the molecular oscillator known as the segmentation clock. The hairy -related gene, hes6/her13.2, functions as a hub by dimerizing with other oscillators of the segmentation clock in zebrafish embryos. Although hes6 exhibits a posterior–anterior expression gradient in the posterior PSM with a peak at the tailbud, the detailed mechanisms underlying this unique expression pattern have not yet been clarified. By establishing several transgenic lines, we found that the transcriptional regulatory region downstream of hes6 in combination with the hes6 3′UTR recapitulates the endogenous gradient of hes6 mRNA expression. This downstream region, which we termed the PT enhancer, possessed several putative binding sites for the T-box and Ets transcription factors that were required for the regulatory activity. Indeed, the T-box transcription factor (Tbx16) and Ets transcription factor (Pea3) bound specifically to the putative binding sites and regulated the enhancer activity in zebrafish embryos. In addition, the 3′UTR of hes6 is required for recapitulation of the endogenous mRNA expression. Furthermore, the PT enhancer with the 3′UTR of hes6 responded to the inhibition of retinoic acid synthesis and fibroblast growth factor signaling in a manner similar to endogenous hes6 . The results showed that transcriptional regulation by the T-box and Ets transcription factors, combined with the mRNA stability given by the 3′UTR, is responsible for the unique expression gradient of hes6 mRNA in the posterior PSM of zebrafish embryos. [ABSTRACT FROM AUTHOR]

Klhl31 is a member of the Kelch-like family in vertebrates, which are characterized by an amino-terminal b road complex t ram-track, b ric-a-brac/ po xvirus and z inc finger (BTB/POZ) domain, carboxy-terminal Kelch repeats and a central linker region (Back domain). In developing somites Klhl31 is highly expressed in the myotome downstream of myogenic regulators (MRF), and it remains expressed in differentiated skeletal muscle. In vivo gain- and loss-of-function approaches in chick embryos reveal a role of Klhl31 in skeletal myogenesis. Targeted mis-expression of Klhl31 led to a reduced size of dermomyotome and myotome as indicated by detection of relevant myogenic markers, Pax3, Myf5, myogenin and myosin heavy chain (MF20). The knock-down of Klhl31 in developing somites, using antisense morpholinos (MO), led to an expansion of Pax3, Myf5, MyoD and myogenin expression domains and an increase in the number of mitotic cells in the dermomyotome and myotome. The mechanism underlying this phenotype was examined using complementary approaches, which show that Klhl31 interferes with β-catenin dependent Wnt signaling. Klhl31 reduced the Wnt-mediated activation of a luciferase reporter in cultured cells. Furthermore, Klhl31 attenuated secondary axis formation in Xenopus embryos in response to Wnt1 or β-catenin. Klhl31 mis-expression in the developing neural tube affected its dorso-ventral patterning and led to reduced dermomyotome and myotome size. Co-transfection of a Wnt3a expression vector with Klhl31 in somites or in the neural tube rescued the phenotype and restored the size of dermomyotome and myotome. Thus, Klhl31 is a novel modulator of canonical Wnt signaling, important for vertebrate myogenesis. We propose that Klhl31 acts in the myotome to support cell cycle withdrawal and differentiation. [ABSTRACT FROM AUTHOR]

The precise border of somites formed during mouse somitogenesis is defined by a Tbx6 expression domain, which is established by Mesp2-mediated Tbx6 suppression in the anterior part of the presomitic mesoderm (PSM). Ripply2 , a target of Mesp2, is proposed to be involved in this down-regulation because Ripply2 deficiency causes an anterior expansion of the Tbx6 domain, resembling the Mesp2 -null phenotype. However, it is unclear whether Ripply2 acts on Tbx6 independently or in association with Mesp2. To address this question, we generated three sets of transgenic mice with the following Ripply2 expression patterns: (1) overexpression in the endogenous expression domain, (2) expression instead of Mesp2 ( Ripply2 -knockin), and (3) ectopic expression in the entire PSM. We found accelerated Tbx6 degradation in the embryos showing Ripply2 overexpression. In the Ripply2 -knockin embryos, the anterior limit of Tbx6 domain was generated by Ripply2 even in the absence of Mesp2. Ectopic Ripply2 expression along the entire PSM suppressed Tbx6 and induced Sox2-positive neural tube formation at the bilateral domain, resembling the Tbx6 -null phenotype. This phenotype resulted from Tbx6 protein and not mRNA elimination, suggesting the post-translational down-regulation of Tbx6 by Ripply2. Taken together, our results demonstrate that Ripply2 represses Tbx6 in a Mesp2-independent manner, which contributes to the accurate segmental border formation. [ABSTRACT FROM AUTHOR]

During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb level somites and migrate into the limb bud where they form the dorsal and ventral muscle masses. Only then do they begin to differentiate and express markers of myogenic commitment and determination such as Myf5 and MyoD . However the signals regulating this process remain poorly characterised. We show that FGF18, which is expressed in the distal mesenchyme of the limb bud, induces premature expression of both Myf5 and MyoD and that blocking FGF signalling also inhibits endogenous MyoD expression. This expression is mediated by ERK MAP kinase but not PI3K signalling. We also show that retinoic acid (RA) can inhibit the myogenic activity of FGF18 and that blocking RA signalling allows premature induction of MyoD by FGF18 at HH19. We propose a model where interactions between FGF18 in the distal limb and retinoic acid in the proximal limb regulate the timing of myogenic gene expression during limb bud development. [ABSTRACT FROM AUTHOR]

Abstract: The segmental structure of the axial skeleton is formed during somitogenesis. During this process, paired somites bud from the presomitic mesoderm (PSM), in a process regulated by a genetic clock called the segmentation clock. The Notch pathway and the Notch modulator Lunatic fringe (Lfng) play multiple roles during segmentation. Lfng oscillates in the posterior PSM as part of the segmentation clock, but is stably expressed in the anterior PSM during presomite patterning. We previously found that mice lacking overt oscillatory Lfng expression in the posterior PSM (Lfng ∆FCE) exhibit abnormal anterior development but relatively normal posterior development. This suggests distinct requirements for segmentation clock activity during the formation of the anterior skeleton (primary body formation), compared to the posterior skeleton and tail (secondary body formation). To build on these findings, we created an allelic series that progressively lowers Lfng levels in the PSM. Interestingly, we find that further reduction of Lfng expression levels in the PSM does not increase disruption of anterior development. However tail development is increasingly compromised as Lfng levels are reduced, suggesting that primary body formation is more sensitive to Lfng dosage than is secondary body formation. Further, we find that while low levels of oscillatory Lfng in the posterior PSM are sufficient to support relatively normal posterior development, the period of the segmentation clock is increased when the amplitude of Lfng oscillations is low. These data support the hypothesis that there are differential requirements for oscillatory Lfng during primary and secondary body formation and that posterior development is less sensitive to overall Lfng levels. Further, they suggest that modulation of the Notch signaling by Lfng affects the clock period during development. [Copyright &y& Elsevier]

Abstract: Both Glis, the downstream effectors of hedgehog signaling, and Zic transcription factors are required for Myf5 expression in the epaxial somite. Here we demonstrate a novel synergistic interaction between members of both families and Pax3, a paired-domain transcription factor that is essential for both myogenesis and neural crest development. We show that Pax3 synergizes with both Gli2 and Zic1 in transactivating the Myf5 epaxial somite (ES) enhancer in concert with the Myf5 promoter. This synergy is dependent on conserved functional domains of the proteins, as well as on a novel homeodomain motif in the Myf5 promoter and the essential Gli motif in the ES enhancer. Importantly, overexpression of Zic1 and Pax3 in the 10T1/2 mesodermal cell model results in enrichment of these factors at the endogenous Myf5 locus and induction of Myf5 expression. In our previous work, we showed that by enhancing nuclear translocation of Gli factors, Zics provide spatiotemporal patterning for Gli family members in the epaxial induction of Myf5 expression. Our current study indicates a complementary mechanism in which association with DNA-bound Pax3 strengthens the ability of both Zic1 and Gli2 to transactivate Myf5 in the epaxial somite. [Copyright &y& Elsevier]

Vertebrate segmentation is manifested during embryonic development as serially repeated units termed somites that give rise to vertebrae, fibs, skeletal muscle and dermis. Many theoretical models including the 'clock and wavefront' model have been proposed. There is compelling genetic evidence showing that Notch--Delta signaling is indispensable for somitogenesis. Notch receptor and its target genes, Hairy/E(spl) homologues, are known to be crucial for the ticking of the segmentation clock. Through the work done in mouse, chick, Xenopus and zebrafish, an oscillator operated by cyclical transcriptional activation and delayed negative feedback regulation is emerging as the fundamental mechanism underlying the segmentation clock. Ubiquitin-dependent protein degradation and probably other posttranslational regulations are also required. Fgf8 and Wnt3a gradients are important in positioning somite boundaries and, probably, in coordinating tail growth and segmentation. The circadian clock is another biochemical oscillator, which, similar to the segmentation clock, is operated with a negative transcription-regulated feedback mechanism. While the circadian clock uses a more complicated network of pathways to achieve homeostasis, it appears that the segmentation clock exploits the Notch pathway to achieve both signal generation and synchronization. We also discuss mathematical modeling and future directions in the end. [c] 2003 Elsevier Inc. All rights reserved. Keywords: Notch; Delta: Fgf; Wnt: Hesl; Hes7; Herl; Her7; Lfng; Somitogenesis; Segmentation clock; Circadian clock; Gradient; Negative feedback; Modeling

Abstract: Heparan sulfate proteoglycans (HSPGs) are glycosylated extracellular or membrane-associated proteins. Their unbranched heparan sulfate (HS) disaccharide chains interact with many growth factors and receptors, modifying their activity or diffusion. The pattern of HS sulfation can be altered by the enzymes Sulf1 and Sulf2, secreted extracellular 6-O endosulfatases, which remove specific sulfate groups from HS. Modification by Sulf enzymes changes the binding affinity of HS for protein such as ligands and receptors, affecting growth factor gradients and activities. The precise expression of these sulfatases are thought to be necessary for normal development. We have examined the role of the sulf1 gene in trunk development of zebrafish embryos. sulf1 is expressed in the developing trunk musculature and as well as in midline structures such as the notochord, floorplate and hypochord. Knockdown of sulf1 with antisense morpholinos results in poor differentiation of the somitic trunk muscle, loss of the horizontal myoseptum, lack of pigmentation along the mediolateral stripe, and improper migration of the lateral line primordium. sulf1 knockdown results in a decrease in the number of Pax7-expressing dermomyotome cells, particularly along the midline where the horizontal myoseptum develops. It also leads to decreased sdf1/cxcl12 expression along the mediolateral trunk musculature. Both the Pax7 and cxcl12 expression can be restored by inhibition pharmacological inhibition of BMP signaling, which also restores formation of the myoseptum, fast muscle development, and pigmentation patterning. Lateral line migration and neuromast deposition depend on sdf1/cxcl12 and FGF signaling respectively, both of which are disrupted in sulf1 morphants. Pharmacological activation of FGF signaling can rescue the spacing of neuromast deposition in these fish. Together this data indicate that sulf1 plays a crucial role in modulating both BMP and FGF signaling along the developing myoseptum to coordinate the morphogenesis of trunk musculature, associated pigment cells, and lateral line neuromasts. [Copyright &y& Elsevier]

Abstract: The Myf5 gene plays an important role in myogenic determination during mouse embryo development. Multiple genomic regions of the Mrf4–Myf5 locus have been characterised as enhancer sequences responsible for the complex spatiotemporal expression of the Myf5 gene at the onset of myogenesis. These include an enhancer sequence, located at −111kb upstream of the Myf5 transcription start site, which is responsible of Myf5 activation in ventral somitic domains (Ribas et al., 2011. Dev. Biol. 355, 372–380). We show that the −111kb-Myf5 enhancer also directs transgene expression in some limb muscles, and is active at foetal as well as embryonic stages. We have carried out further characterisation of the regulation of this enhancer and show that the paired-box Pax3 transcription factor binds to it in vitro as in vivo, and that Pax binding sites are essential for its activity. This requirement is independent of the previously reported regulation by TEAD transcription factors. Six1/4 which, like Pax3, are important upstream regulators of myogenesis, also bind in vivo to sites in the −111 kb-Myf5 enhancer and modulate its activity. The −111 kb-Myf5 enhancer therefore shares common functional characteristics with another Myf5 regulatory sequence, the hypaxial and limb 145bp-Myf5 enhancer, both being directly regulated in vivo by Pax3 and Six1/4 proteins. However, in the case of the −111 kb-Myf5 enhancer, Six has less effect and we conclude that Pax regulation plays a major role in controlling this aspect of the Myf5 gene expression at the onset of myogenesis in the embryo. [Copyright &y& Elsevier]

Abstract: Somites form by an iterative process from unsegmented, presomitic mesoderm (PSM). Notch pathway components, such as deltaC (dlc) have been shown to play a role in this process, while the T-box transcription factors Ntla and Tbx16 regulate somite formation upstream of this by controlling supply and movement of cells into the PSM during gastrulation and tailbud outgrowth. In this work, we report that Ntla and Tbx16 play a more explicit role in segmentation by directly regulating dlc expression. In addition we describe a cis-regulatory module (CRM) upstream of dlc that drives expression of a reporter in the tailbud, PSM and somites during somitogenesis. This CRM is bound by both Ntla and Tbx16 at a cluster of T-box binding sites, which are required in combination for activation of the CRM. [Copyright &y& Elsevier]

Abstract: The molecular mechanism underlying somite development differs along the embryonic antero-posterior axis. In zebrafish, cell lineage tracing and genetic analysis have revealed a difference in somite development between the trunk and tail. For instance, spadetail/tbx16 (spt) mutant embryos lack trunk somites but not tail ones. Trunk and tail somites are developed from mesodermal progenitor cells (MPCs) located in the tailbud. While the undifferentiated state of MPCs is maintained by mutual activation between Wnt and Brachyury/Ntl, the mechanism by which the MPCs differentiate into presomitic mesoderm (PSM) cells remains largely unclear. Especially, the molecules that promote PSM differentiation during tail development should be clarified. Here, we show that zebrafish embryos defective in mesogenin1 (msgn1) and spt failed to differentiate into PSM cells in tail development and show increased expression of wnt8 and ntl. Msgn1 acted in a cell-autonomous manner and as a transcriptional activator in PSM differentiation. The expression of msgn1 initially overlapped with that of ntl in the ventral tailbud, as previously reported; and its mis-expression caused ectopic expression of tbx24, a PSM marker gene, only in the tailbud and posterior notochord, both of which expressed ntl in zebrafish embryos. Furthermore, the PSM-inducing activity of misexpressed msgn1 was enhanced by co-expression with ntl. Thus, Msgn1 exercised its PSM-inducing activity in cells expressing ntl. Based on these results, we speculate that msgn1 expression in association with that of ntl may allow the differentiation of progenitor cells to proceed during development of somites in the tail. [Copyright &y& Elsevier]

Abstract: It is generally thought that vertebral patterning and identity are globally determined prior to somite formation. Relatively little is known about the regulators of vertebral specification after somite segmentation. Here, we demonstrated that Ndrg2, a tumor suppressor gene, was dynamically expressed in the presomitic mesoderm (PSM) and at early stage of differentiating somites. Loss of Ndrg2 in mice resulted in vertebral homeotic transformations in thoracic/lumbar and lumbar/sacral transitional regions in a dose-dependent manner. Interestingly, the inactivation of Ndrg2 in osteoblasts or chondrocytes caused defects resembling those observed in Ndrg2 −/− mice, with a lower penetrance. In addition, forced overexpression of Ndrg2 in osteoblasts or chondrocytes also conferred vertebral defects, which were distinct from those in Ndrg2 −/− mice. These genetic analyses revealed that Ndrg2 modulates vertebral identity in segmented somites rather than in the PSM. At the molecular level, combinatory alterations of the amount of Hoxc8-11 gene transcripts were detected in the differentiating somites of Ndrg2 −/− embryos, which may partially account for the vertebral defects in Ndrg2 mutants. Nevertheless, Bmp/Smad signaling activity was elevated in the differentiating somites of Ndrg2 −/− embryos. Collectively, our findings unveiled Ndrg2 as a novel regulator of vertebral specification in differentiating somites. [Copyright &y& Elsevier]

Abstract: Paraxial mesoderm is the tissue which gives rise to the skeletal muscles and vertebral column of the body. A gene regulatory network operating in the formation of paraxial mesoderm has been described. This network hinges on three key factors, Wnt3a, Msgn1 and Tbx6, each of which is critical for paraxial mesoderm formation, since absence of any one of these factors results in complete absence of posterior somites. In this study we determined and compared the spatial and temporal patterns of expression of Wnt3a, Msgn1 and Tbx6 at a time when paraxial mesoderm is being formed. Then, we performed a comparative characterization of mutants in Wnt3a, Msgn1 and Tbx6. To determine the epistatic relationship between these three genes, and begin to decipher the complex interplay between them, we analyzed double mutant embryos and compared their phenotypes to the single mutants. Through the analysis of molecular markers in mutants, our data support the bipotential nature of the progenitor cells for paraxial mesoderm and establish regulatory relationships between genes involved in the choice between neural and mesoderm fates. [Copyright &y& Elsevier]

Abstract: In Drosophila, trunk metamerization is established by a cascade of segmentation gene activities: the gap genes, the pair rule genes, and the segment polarity genes. In the anterior head, metamerization requires also gap-like genes and segment polarity genes. However, because the pair rule genes are not active in this part of the embryo, the question on which gene activities are fulfilling the role of the second order regulator genes still remains to be solved. Here we provide first molecular evidence that the Helix–Loop–Helix–COE transcription factor Collier fulfills this role by directly activating the expression of the segment polarity gene hedgehog in the posterior part of the intercalary segment. Collier thereby occupies a newly identified binding site within an intercalary-specific cis-regulatory element. Moreover, we identified a direct physical association between Collier and the basic-leucine-zipper transcription factor Cap‘n''collar B, which seems to restrict the activating input of Collier to the posterior part of the intercalary segment and to lead to the attenuation of hedgehog expression in the intercalary lobes at later stages. [Copyright &y& Elsevier]

Abstract: The transcriptional regulation of the Mrf4/Myf5 locus depends on a multitude of enhancers that, in equilibria with transcription balancing sequences and the promoters, regulate the expression of the two genes throughout embryonic development and in the adult. Transcription in a particular set of muscle progenitors can be driven by the combined outputs of several enhancers that are not able to recapitulate the entire expression pattern in isolation, or by the action of a single enhancer the activity of which in isolation is equivalent to that within the context of the locus. We identified a new enhancer element of this second class, ECR111, which is highly conserved in all vertebrate species and is necessary and sufficient to drive Myf5 expression in ventro-caudal and ventro-rostral somitic compartments in the mouse embryo. EMSA analyses and data obtained from binding-site mutations in transgenic embryos show that a binding site for a TEA Domain (TEAD) transcription factor is essential for the function of this new enhancer, while ChIP assays show that at least two members of the family of transcription factors bind to it in vivo. [Copyright &y& Elsevier]

Abstract: Limbed vertebrates have functionally integrated postcranial axial and appendicular systems derived from two distinct populations of embryonic mesoderm. The axial skeletal elements arise from the paraxial somites, the appendicular skeleton and sternum arise from the somatic lateral plate mesoderm, and all of the muscles for both systems arise from the somites. Recent studies in amniotes demonstrate that the scapula has a mixed mesodermal origin. Here we determine the relative contribution of somitic and lateral plate mesoderm to the avian scapula from quail-chick chimeras. We generate 3D reconstructions of the grafted tissue in the host revealing a very different distribution of somitic cells in the scapula than previously reported. This novel 3D visualization of the cryptic border between somitic and lateral plate populations reveals the dynamics of musculoskeletal morphogenesis and demonstrates the importance of 3D visualization of chimera data. Reconstructions of chimeras make clear three significant contrasts with existing models of scapular development. First, the majority of the avian scapula is lateral plate derived and the somitic contribution to the scapular blade is significantly smaller than in previous models. Second, the segmentation of the somitic component of the blade is partially lost; and third, there are striking differences in growth rates between different tissues derived from the same somites that contribute to the structures of the cervical thoracic transition, including the scapula. These data call for the reassessment of theories on the development, homology, and evolution of the vertebrate scapula. [Copyright &y& Elsevier]

Abstract: Cells anchor to substrates by binding to extracellular matrix (ECM). In addition to this anchoring function however, cell–ECM binding is a mechanism for cells to sense their surroundings and to communicate and coordinate behaviour amongst themselves. Several ECM molecules and their receptors play essential roles in muscle development and maintenance. Defects in these proteins are responsible for some of the most severe muscle dystrophies at every stage of life from neonates to adults. However, recent studies have also revealed a role of cell–ECM interactions at much earlier stages of development as skeletal muscle forms. Here we review which ECM molecules are present during the early phases of myogenesis, how myogenic cells interact with the ECM that surrounds them and the potential consequences of those interactions. We conclude that cell–ECM interactions play significant roles during all stages of skeletal muscle development in the embryo and suggest that this “extracellular matrix dimension” should be added to our conceptual network of factors contributing to skeletal myogenesis. [Copyright &y& Elsevier]

Abstract: Dicer is an enzyme that processes microRNAs (miRNAs) to their mature forms. As miRNAs were first discovered for their role in the control of developmental timing, we investigated their potential requirement in mouse somitogenesis, an event with precise temporal periodicity. To address the collective role of miRNAs in mesoderm development including somite formation, we used T (Brachyury)-Cre mouse line to inactivate Dicer in most cells of the mesoderm lineage. This Dicer mutant exhibits a reduced anterior–posterior axis. Somite number remains normal in mutant embryos up until the death of the embryos more than two days after Dicer inactivation. Consistent with this, the molecular machineries required for establishing segmentation, including clock and wave front, are not perturbed. However, somite size is reduced and later-formed somites are caudalized, coincident with increased cell death. Outside of the paraxial mesoderm and prior to apparent reduction of the axis in the mutant, the position of the hindlimb bud, a lateral plate mesoderm-derived structure, is posteriorly shifted and the timing of hindlimb bud initiation is delayed accordingly. We observed changes in the expression of genes critical for limb positioning, which include a shifted and delayed downregulation of Hand2 and Tbx3, and shifted and delayed upregulation of Gli3 in the prospective limb bud field. The 3′ UTRs of both Hand2 and Tbx3 harbor target sites for a seed sequence-sharing family of miRNAs mir-25/32/92/363/367. As an example of the family we show that mir-363, a miRNA with elevated expression in the prospective limb bud field, is capable of inhibiting Hand2/Tbx3 expression in vitro in a binding site-dependent manner. Together, our findings provide the first demonstration that in mouse embryonic mesoderm, while Dicer is dispensable for somite segmentation, it is essential for proper limb bud positioning. [Copyright &y& Elsevier]

Abstract: In the avian embryo, precursor cells of the paraxial mesoderm that reside in the epiblast ingress through the primitive streak and migrate bilaterally in an anterolateral direction. Herein, we report on the roles of Protogenin (PRTG), an immunoglobulin superfamily protein expressed on the surface of the ingressing and migrating cells that give rise to the paraxial mesoderm, in paraxial mesoderm development. An aggregation assay using L-cells showed that PRTG mediates homophilic cell adhesion. Overexpression of PRTG in the presumptive paraxial mesoderm delayed mesodermal cell migration due to augmented adhesiveness. In contrast, siRNA knockdown of PRTG impaired successive ingression of epiblast cells and disorganized the epithelial structure of the somites. These results suggest that PRTG mediates cell adhesion to regulate continuous ingression of cells giving rise to the paraxial mesodermal lineage, as well as tissue integrity. [Copyright &y& Elsevier]

Abstract: Zic genes encode a conserved family of zinc finger proteins with essential functions in neural development and axial skeletal patterning in the vertebrate embryo. Zic proteins also function as Gli co-factors in Hedgehog signaling. Here, we report that Zic genes have a role in Myf5 regulation for epaxial somite myogenesis in the mouse embryo. In situ hybridization studies show that Zic1, 2, and 3 transcripts are expressed in Myf5-expressing epaxial myogenic progenitors in the dorsal medial dermomyotome of newly forming somites, and immunohistological studies show that Zic2 protein is co-localized with Myf5 and Pax3 in the dorsal medial lip of the dermomyotome, but is not expressed in the forming myotome. In functional reporter assays, Zic1 and Zic2, but not Zic3, potentiate the transactivation of Gli-dependent Myf5 epaxial somite-specific (ES) enhancer activity in 3T3 cells, and Zic1 activates endogenous Myf5 expression in 10T1/2 cells and in presomitic mesoderm explants. Zic2 also co-immunoprecipitates with Gli2, indicating that Zic2 forms complexes with Gli2 to promote Myf5 expression. Genetic studies show that, although Zic2 and Zic1 are activated normally in sonic hedgehog−/− mutant embryos, Myf5 expression in newly forming somites is deficient in both sonic hedgehog−/− and in Zic2 kd/kd mutant mouse embryos, providing further evidence that these Zic genes are upstream regulators of Hedgehog-mediated Myf5 activation. Myf5 activation in newly forming somites is delayed in Zic2 mutant embryos until the time of Zic1 activation, and both Zic2 and Myf5 require noggin for their activation. [Copyright &y& Elsevier]

Abstract: Differentiation often requires conversion of analogue signals to a stable binary output through positive feedback. Hedgehog (Hh) signalling promotes myogenesis in the vertebrate somite, in part by raising the activity of muscle regulatory factors (MRFs) of the Myod family above a threshold. Hh is known to enhance MRF expression. Here we show that Hh is also essential at a second step that increases Myod protein activity, permitting it to promote Myogenin expression. Hh acts by inducing expression of cdkn1c (p57 Kip2 ) in slow muscle precursor cells, but neither Hh nor Cdkn1c is required for their cell cycle exit. Cdkn1c co-operates with Myod to drive differentiation of several early zebrafish muscle fibre types. Myod in turn up-regulates cdkn1c, thereby providing a positive feedback loop that switches myogenic cells to terminal differentiation. [Copyright &y& Elsevier]

Abstract: Gnathostomes (jawed vertebrates) possess skeletal muscles with unique functional and developmental features that are absent from cyclostomes—i.e., lamprey and hagfish. These gnathostome-specific traits include the epaxial and hypaxial division of myotomes, paired fin/limb muscles, shoulder girdle muscles, and the muscle associated with the tongue and the neck. Many of these muscles are derived from several rostral somites, specifically from their hypaxial myotomic domains. However, it has not been clarified how the complicated morphology of these muscles was acquired in the evolution of vertebrates. Here we describe the expression of lamprey homologs of transcription factor genes, including a myogenic regulatory factor of the Myod family (MRF), Pax3/7, Lbx, and Zic, which play important roles in the development of ep-/hypaxial somitic muscles in gnathostomes, and show that the ventral portion of lamprey somites is comparable to the ventral dermomyotome in gnathostomes. The supra- and infraoptic muscles, derived from the two anterior somites in the lamprey, are molecularly specified before their extensive invasion into the head region. Of these, the infraoptic myotomes are suggested to represent the cucullaris homologue in the lamprey based on their topographical position in the embryonic pattern. Slightly caudal myotomes in the lamprey give rise to the hypobranchial muscle, the developmental homologue of the gnathostome hypobranchial musculature. The dorsal moieties of the lamprey somites express a Zic gene, which in teleosts specifies the epaxial identities of the somites. These evidences suggest that, although the myotomes in the ancestral jawless vertebrates do not exhibit ep-/hypaxial distinction at the morphological level, their dorsoventral specification would have already been present at gene regulatory levels, prior to the cyclostome–gnathostome divergence, which may have functioned as the key innovation to establish the ep-/hypaxial distinction in gnathostomes. [Copyright &y& Elsevier]

Abstract: We previously reported that Tbx6, a T-box transcription factor, is required for the differentiation of ventral body wall muscle and for segment formation and somitic muscle differentiation. Here, we show that Tbx6 is also involved, at later stages, in cartilage differentiation from the cranial neural crest and head muscle development. In Tbx6 knockdown embryos, the cranial neural crest was shown to be correctly induced at the border of the neural plate and migrated in a slightly delayed manner, but finally reached positions in the pharyngeal arches nearly similar to those in the normal embryos as revealed by in situ hybridization and the neural crest-transplantation experiments. However, the neural crest cells failed to maintain Sox9 expression. Tbx6 knockdown also reduced the expression of Tbx1, another T-box gene expressed in more anterior paraxial structures. Tbx1 knockdown caused phenotypes milder but similar to those of Tbx6 morphants, including reduced formation of head muscles and cartilages, and attenuated Sox9 expression. Furthermore, the phenotypes caused by Tbx6 knockdown were partially rescued by Tbx1 plasmid injection. These results suggest that Tbx6 is involved in the cranial cartilage and head muscle development by regulating anterior paraxial genes such as Tbx1 and Sox9. [ABSTRACT FROM AUTHOR]

Abstract: The rostro-caudal patterning within a somite is periodically established in the presomitic mesoderm (PSM). In the mouse, Mesp2 is required for the rostral property whereas Notch signaling and Ripply2, a Mesp2-induced protein that suppresses Mesp2 transcription, are required for the caudal property. Here, we examined the mechanism behind rostro-caudal patterning by comparing the spatial movement of Notch activity with Mesp2 protein localization in wild-type embryos and those defective in Ripply1 and 2, both of which are expressed in the PSM. Mesp2 protein appears first as a thin band in the middle of the traveling Notch active domain in both wild-type and Ripply1/2-deficient embryos. In wild-type embryos, the Mesp2 band expands anteriorly to the expression front of Tbx6, an activator of Mesp2 transcription. Notch activity becomes localized further anteriorly to this Mesp2 domain, but does not pass over the anterior Mesp2 domain generated in the previous segmentation cycle. As a result, the Notch active domain appears to be restricted between these two Mesp2 domains. In Ripply1/2-deficient embryos, the Mesp2 band becomes more expanded and the Notch domain is finally diminished. Interestingly, Ripply1/2-deficient embryos exhibit anterior expansion of the Tbx6 protein domain, suggesting that Ripply1/2 regulates Mesp2 expression by modulating elimination of Tbx6 proteins. We propose that the rostro-caudal pattern is established by dynamic interaction of Notch activity with two Mesp2 domains, which are defined in successive segmentation cycles by Notch, Tbx6 and Ripply1/2. [Copyright &y& Elsevier]

Abstract: Wnt signaling has been implicated in somite, limb, and branchial arch myogenesis but the mechanisms and roles are not clear. We now show that Wnt signaling via Lef1 acts to regulate the number of premyogenic cells in somites but does not regulate myogenic initiation in the limb bud or maintenance in the first or second branchial arch. We have also analysed the function and regulation of a putative downstream transcriptional target of canonical Wnt signaling, Pitx2. We show that loss-of-function of Pitx2 decreases the number of myogenic cells in the somite, whereas overexpression increases myocyte number particularly in the epaxial region of the myotome. Increased numbers of mitotic cells were observed following overexpression of Pitx2 or an activated form of Lef1, suggesting an effect on cell proliferation. In addition, we show that Pitx2 expression is regulated by canonical Wnt signaling in the epaxial somite and second branchial arch, but not in the limb or the first branchial arch. These results suggest that Wnt/Lef1 signaling regulates epaxial myogenesis via Pitx2 but that this link is uncoupled in other regions of the body, emphasizing the unique molecular networks that control the development of various muscles in vertebrates. [Copyright &y& Elsevier]

Abstract: Skeletal muscles of body and limb are derived from somites, but most head muscles originate from cranial mesoderm. The resident stem cells of muscle are satellite cells, which have the same embryonic origin as the muscle in which they reside. Here, we analysed satellite cells with a different ontology, comparing those of the extensor digitorum longus (EDL) of the limb with satellite cells from the masseter of the head. Satellite cell-derived myoblasts from MAS and EDL muscles had distinct gene expression profiles and masseter cells usually proliferated more and differentiated later than those from EDL. When transplanted, however, masseter-derived satellite cells regenerated limb muscles as efficiently as those from EDL. Clonal analysis showed that functional properties differed markedly between satellite cells: ranging from clones that proliferated extensively and gave rise to both differentiated and self-renewed progeny, to others that divided minimally before differentiating completely. Generally, masseter-derived clones were larger and took longer to differentiate than those from EDL. This distribution in cell properties was preserved in both EDL-derived and masseter-derived satellite cells from old mice, although clones were generally less proliferative. Satellite cells, therefore, are a functionally heterogeneous population, with many occupants of the niche exhibiting stem cell characteristics in both somite-derived and branchiomeric muscles. [Copyright &y& Elsevier]

Abstract: The transcription factor FoxD5 is expressed in the paraxial mesoderm of zebrafish. However, the roles of FoxD5 in anterior pre-somitic mesoderm (PSM) during somitogenesis are unknown. We knocked down FoxD5 in embryos, which resulted in defects of the newly formed somites, including loss of the striped patterns of anterior–posterior polarity genes deltaC, notch2, notch3 and EphB2a, as well as the absence of mespa expression in S-I. Also, the expression of mespb exhibited a ‘salt and pepper’ pattern, indicating that FoxD5 is necessary for somite patterning in anterior PSM. Embryos were treated with SU5402, an Fgf receptor (FGFR) inhibitor, resulting in reduction of FoxD5 expression. This finding was consistent with results obtained from Tg(hsp70l:dnfgfr1-EGFP)pd1 embryos, whose dominant-negative form of FGFR1 was produced by heat-induction. Loss of FoxD5 expression was observed in the embryos injected with fgf3-/fgf8-double-morpholinos (MOs). Excessive FoxD5 mRNA could rescue the defective expression levels of mespa and mespb in fgf3-/fgf8-double morphants, suggesting that Fgf signaling acts as an upstream modulator of FoxD5 during somitogenesis. We concluded that FoxD5 is required for maintaining anterior–posterior polarity within a somite and that the striped pattern of FoxD5 in anterior PSM is mainly regulated by Fgf. An Fgf-FoxD5-Mesps signaling network is therefore proposed. [Copyright &y& Elsevier]

Abstract: During the early formation of the dorsal aorta, the first-forming embryonic vessel in amniotes, a subset of somitic cells selected as presumptive angioblasts, migrates toward the dorsal aorta, where they eventually differentiate into endothelial cells. We have recently shown that these processes are controlled by Notch signals (Sato, Y., Watanabe, T., Saito, D., Takahashi, T., Yoshida, S., Kohyama, J., Ohata, E., Okano, H., and Takahashi, Y., 2008. Notch mediates the segmental specification of angioblasts in somites and their directed migration toward the dorsal aorta in avian embryos. Dev. Cell 14, 890–901.). Here, we studied a possible link between Notch and chemokine signals, SDF1/CXCR4, the latter found to be dominantly expressed in developing aorta/somites. Although CXCR4 overexpression caused a directed migration of somitic cells to the aortic region in a manner similar to Notch, no positive epistatic relationships between Notch and SDF1/CXCR4 were detected. After reaching the aortic region, the CXCR4-electroporated cells exhibited no endothelial character. Importantly, however, once provided with Notch activity, they could successfully be incorporated into developing vessels as endothelial cells. These findings were obtained combining the tetracycline-inducible gene expression method with the transposon-mediated stable gene transfer technique. We conclude that Notch activation is sufficient to direct naïve mesenchymal cells to differentiate into endothelial cells once the cells are conveyed to the aortic region. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Meox1 and Meox2 are two related homeodomain transcription factor genes that together are essential for the development of all somite compartments. Here we show that mice homozygous for Meox1 mutations alone have abnormalities that are restricted to the sclerotome and its derivatives. A prominent and consistent phenotype of these mutations is a remodeling of the cranio-cervical joints whose major feature is the assimilation of the atlas into the basioccipital bone so that the skull rests on the axis. These abnormalities can be traced back to changes in the relative rates of cell proliferation in the rostral and caudal sclerotome compartments, and they are associated with alterations in the expression of at least three transcription factor genes, Tbx18, Uncx, and Bapx1. As previously observed for Bapx1, MEOX1 protein occupies evolutionarily conserved promoter regions of Tbx18 and Uncx, suggesting that Meox1 regulates these genes at least in part directly. Hence, Meox1 is part of a regulatory circuit that serves an essential, non-redundant function in the maintenance of rostro-caudal sclerotome polarity and axial skeleton formation. [Copyright &y& Elsevier]

Abstract: Satellite cells are tissue-specific stem cells responsible for skeletal muscle growth and regeneration. Although satellite cells were identified almost 50 years ago, the identity of progenitor populations from which they derive remains controversial. We developed MyoD iCre knockin mice, and used Cre/lox lineage analysis to determine whether satellite cell progenitors express MyoD, a marker of myogenic commitment. Recombination status of satellite cells was determined by confocal microscopy of isolated muscle fibers and by electron microscopic observation of muscle tissue fixed immediately following isolation, using R26R-EYFP and R26R (β-gal) reporter mice, respectively. We show that essentially all adult satellite cells associated with limb and body wall musculature, as well as the diaphragm and extraocular muscles, originate from MyoD+ progenitors. Neonatal satellite cells were Cre-recombined, but only a small minority exhibited ongoing Cre expression, indicating that most satellite cells had expressed MyoD prenatally. We also show that satellite cell development in MyoD-null mice is not due to functional compensation by MyoD non-expressing lineages. The results suggest that satellite cells are derived from committed myogenic progenitors, irrespective of the anatomical location, embryological origin, or physiological properties of associated musculature. [Copyright &y& Elsevier]

Abstract: Somites are embryonic precursors of the ribs, vertebrae and certain dermis tissue. Somite formation is a periodic process regulated by a molecular clock which drives cyclic expression of a number of clock genes in the presomitic mesoderm. To date the mechanism regulating the period of clock gene oscillations is unknown. Here we show that chick homologues of the Wnt pathway genes that oscillate in mouse do not cycle across the chick presomitic mesoderm. Strikingly we find that modifying Wnt signalling changes the period of Notch driven oscillations in both mouse and chick but these oscillations continue. We propose that the Wnt pathway is a conserved mechanism that is involved in regulating the period of cyclic gene oscillations in the presomitic mesoderm. [Copyright &y& Elsevier]

Abstract: The medulla oblongata (or caudal hindbrain) is not overtly segmented, since it lacks observable interrhombomeric boundaries. However, quail–chick fate maps showed that it is formed by 5 pseudorhombomeres (r7–r11) which were empirically found to be delimited consistently at planes crossing through adjacent somites (). We aimed to reexamine the possible segmentation or rostrocaudal regionalisation of this brain region attending to molecular criteria. To this end, we studied the expression of Hox genes from groups 3 to 7 correlative to the differentiating nuclei of the medulla oblongata. Our results show that these genes are differentially expressed in the mature medulla oblongata, displaying instances of typical antero-posterior (3′ to 5′) Hox colinearity. The different sensory and motor columns, as well as the reticular formation, appear rostrocaudally regionalised according to spaced steps in their Hox expression pattern. The anterior limits of the respective expression domains largely fit boundaries defined between the experimental pseudorhombomeres. Therefore the medulla oblongata shows a Hox-related rostrocaudal molecular regionalisation comparable to that found among rhombomeres, and numerically consistent with the pseudorhombomere list. This suggests that medullary pseudorhombomeres share some AP patterning mechanisms with the rhombomeres present in the rostral, overtly-segmented hindbrain, irrespective of variant boundary properties. [Copyright &y& Elsevier]