The nephric mesenchyme lineage of intermediate mesoderm is derived from Tbx6-expressing derivatives of neuro-mesodermal progenitors via BMP-dependent Osr1 function.

In vertebrate embryos, the kidney primordium metanephros is formed from two distinct cell lineages, Wolffian duct and metanephric mesenchyme, which were classically grouped as intermediate mesoderm. Whereas the reciprocal interactions between these two cell populations in kidney development have been studied extensively, the mechanisms generating them remain elusive. Here, we show that the mouse cell lineage that forms nephric mesenchyme develops as a subpopulation of Tbx6-expressing mesodermal precursor derivatives of neuro-mesodermal progenitors (NMPs) under the condition of bone morphogenetic protein (BMP)-signal-dependent Osr1 expression. The Osr1-expressing nephric mesenchyme precursors were confirmed as descendants of NMPs because they were labeled by Sox2 N1 enhancer-EGFP. In Tbx6 mutant embryos, nephric mesenchyme changed its fate into neural tissues, which reflected its NMP origin. In Osr1 mutant embryos, the specific region of the Tbx6-expressing mesoderm precursor, which normally expresses Osr1 and develops into the nephric mesenchyme, instead expressed the somite marker FoxC2. BMP signaling activated Osr1 expression in a region of TBX6-expressing mesoderm and elicited nephric mesenchyme development. This study suggested a new model of cell lineage segregation during gastrulation.

Development of myofibres and associated connective tissues in fish axial muscle: Recent insights and future perspectives.

Fish axial muscle consists of a series of W-shaped muscle blocks, called myomeres, that are composed primarily of multinucleated contractile muscle cells (myofibres) gathered together by an intricate network of connective tissue that transmits forces generated by myofibre contraction to the axial skeleton. This review summarises current knowledge on the successive and overlapping myogenic waves contributing to axial musculature formation and growth in fish. Additionally, this review presents recent insights into muscle connective tissue development in fish, focusing on the early formation of collagenous myosepta separating adjacent myomeres and the late formation of intramuscular connective sheaths (i.e. endomysium and perimysium) that is completed only at the fry stage when connective fibroblasts expressing collagens arise inside myomeres. Finally, this review considers the possibility that somites produce not only myogenic, chondrogenic and myoseptal progenitor cells as previously reported, but also mesenchymal cells giving rise to muscle resident fibroblasts.

Corepressor SMRT is required to maintain Hox transcriptional memory during somitogenesis.

Nuclear hormone receptors (NRs), such as retinoic acid receptors (RARs), play critical roles in vertebrate development and homeostasis by regulating target gene transcription. Their activity is controlled by ligand-dependent release of corepressors and subsequent recruitment of coactivators, but how these individual receptor modes contribute to development are unknown. Here, we show that mice carrying targeted knockin mutations in the corepressor Silencing Mediator of Retinoid and Thyroid hormone receptor (SMRT) that specifically disable SMRT function in NR signaling (SMRTmRID), display defects in cranial neural crest cell-derived structures and posterior homeotic transformations of axial vertebrae. SMRTmRID embryos show enhanced transcription of RAR targets including Hox loci, resulting in respecification of vertebral identities. Up-regulated histone acetylation and decreased H3K27 methylation are evident in the Hox loci whose somitic expression boundaries are rostrally shifted. Furthermore, enhanced recruitment of super elongation complex is evident in rapidly induced non-Pol II-paused targets in SMRTmRID embryonic stem cells. These results demonstrate that SMRT-dependent repression of RAR is critical to establish and maintain the somitic Hox code and segmental identity during fetal development via epigenetic marking of target loci.

Discrete Notch signaling requirements in the specification of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.

Migrating cells mediate long-range WNT signaling.

In amniotes, it is widely accepted that WNTs secreted by the dorsal neural tube form a concentration gradient that regulates early somite patterning and myotome organization. Here we demonstrate in the chicken embryo that WNT protein is not secreted to act at a distance, but rather loaded onto migrating neural crest cells that deliver it to somites. Inhibiting neural crest migration or ablating their population has a profound impact on the WNT response in somites. Furthermore, we show that a central player in the efficient delivery of WNT to somites is the heparan sulfate proteoglycan GPC4, expressed by neural crest. Together, our data describe a novel mode of signaling whereby WNT proteins hitch a ride on migratory neural crest cells to pattern the somites at a distance from its source.

Aspp2 negatively regulates body growth but not developmental timing by modulating IRS signaling in zebrafish embryos.

The growth and developmental rate of developing embryos and fetus are tightly controlled and coordinated to maintain proper body shape and size. The insulin receptor substrate (IRS) proteins, key intracellular transducers of insulin and insulin-like growth factor signaling, play essential roles in the regulation of growth and development. A short isoform of apoptosis-stimulating protein of p53 2 (ASPP2) was recently identified as a binding partner of IRS-1 and IRS-2 in mammalian cells in vitro. However, it is unclear whether ASPP2 plays any role in vertebrate embryonic growth and development. Here, we show that zebrafish Aspp2a and Aspp2b negatively regulate embryonic growth without affecting developmental rate. Human ASPP2 had similar effects on body growth in zebrafish embryos. Aspp2a and 2b inhibit Akt signaling. This inhibition was reversed by coinjection of myr-Akt1, a constitutively active form of Akt1. Zebrafish Aspp2a and Aspp2b physically bound with Irs-1, and the growth inhibitory effects of ASPP2/Aspp2 depend on the presence of their ankyrin repeats and SH3 domains. These findings uncover a novel role of Aspp2 in regulating vertebrate embryonic growth.

Osmoregulatory response to low salinities in the European sea bass embryos: a multi-site approach.

Embryonic osmoregulation effected by embryonic ionocytes in the European sea bass Dicentrarchus labrax has been studied at several sites, including the yolk sac membrane, the first gill slits and the gut ionocytes. D. labrax embryos, spawned in seawater (SW) (39 ‰), were exposed to dilute seawater (DSW) (5 ‰) during 48 h, from stage 10 pairs of somites (10S) to hatching time (HT). Control embryos originating from the same spawn were maintained in SW. Both SW and DSW embryos were examined after 24- and 48-h exposure. Nanoosmometric measurements of the embryonic fluids osmolality suggest that late embryos are confronted with the variations in external salinity and that they were able to slightly regulate their osmolality. Immunolocalization of Na⁺/K⁺ ATPase, NKCC and CFTR has shown that DSW-exposed embryos can limit ion losses due to compensatory physiological mechanisms. CFTR and NKCC were not observed in DSW embryos in the yolk sac ionocytes and in the tegumentary ionocytes of the gill slits. The quantification of mRNA indicated that NKA, NKCC1 and CFTR transcript levels increased from stage 10S to stage HT. At stage HT, following 48 h of DSW- or SW-exposure, different responses were observed according to salinity. These results, when compared to those obtained in D. labrax juveniles and adults long-term exposed to fresh water (FW), show that in embryos the physiological response following a short-term DSW exposure is different. The mechanisms of hyper-osmoregulation observed in D. labrax embryos, although not fully efficient, allow their survival for several days in DSW.

Ccdc80-l1 Is involved in axon pathfinding of zebrafish motoneurons.

Axon pathfinding is a subfield of neural development by which neurons send out axons to reach the correct targets. In particular, motoneurons extend their axons toward skeletal muscles, leading to spontaneous motor activity. In this study, we identified the zebrafish Ccdc80 and Ccdc80-like1 (Ccdc80-l1) proteins in silico on the basis of their high aminoacidic sequence identity with the human CCDC80 (Coiled-Coil Domain Containing 80). We focused on ccdc80-l1 gene that is expressed in nervous and non-nervous tissues, in particular in territories correlated with axonal migration, such as adaxial cells and muscle pioneers. Loss of ccdc80-l1 in zebrafish embryos induced motility issues, although somitogenesis and myogenesis were not impaired. Our results strongly suggest that ccdc80-l1 is involved in axon guidance of primary and secondary motoneurons populations, but not in their proper formation. ccdc80-l1 has a differential role as regards the development of ventral and dorsal motoneurons, and this is consistent with the asymmetric distribution of the transcript. The axonal migration defects observed in ccdc80-l1 loss-of-function embryos are similar to the phenotype of several mutants with altered Hedgehog activity. Indeed, we reported that ccdc80-l1 expression is positively regulated by the Hedgehog pathway in adaxial cells and muscle pioneers. These findings strongly indicate ccdc80-l1 as a down-stream effector of the Hedgehog pathway.

[A dermomyotome in fish?]. / Un dermomyotome chez les poissons?

The dermomyotome is a transient epithelial sheet that forms from the dorsal aspect of the somite. The dermomyotome gives rise to a variety of tissues, most importantly myotomal muscle and dermis. Despite the central importance of the dermomyotome in the development of amniotes, the question of its existence in lower vertebrates has been lastingly eluded. The combination of single-cell lineage tracing and gene expression analysis has recently led to the identification in fish of a somitic sub-domain that exhibits structural and functional features of the amniote dermomyotome.

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function.

Cell division, differentiation and morphogenesis are coordinated during embryonic development, and frequently are in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but that undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and use the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is driven substantially by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression, and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1(-/-). The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize, as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for the normal segmental arrangement of epithelial borders and internal mesenchymal cells.

Identification of oscillatory genes in somitogenesis from functional genomic analysis of a human mesenchymal stem cell model.

During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 h period consistent with available data on the rate of somitogenesis in humans. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 h, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Expression patterns of these genes were disrupted in Wnt3a(tm1Amc) mutants but not in Dll3(pu) mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro.

Wnt/beta-catenin signaling controls Mespo expression to regulate segmentation during Xenopus somitogenesis.

The vertebral column is derived from somites, which are transient segments of the paraxial mesoderm that are present in developing vertebrates. The strict spatial and temporal regulation of somitogenesis is of crucial developmental importance. Signals such as Wnt and FGF play roles in somitogenesis, but details regarding how Wnt signaling functions in this process remain unclear. In this study, we report that Wnt/beta-catenin signaling regulates the expression of Mespo, a basic-helix-loop-helix (bHLH) gene critical for segmental patterning in Xenopus somitogenesis. Transgenic analysis of the Mespo promoter identifies Mespo as a direct downstream target of Wnt/beta-catenin signaling pathway. We also demonstrate that activity of Wnt/beta-catenin signaling in somitogenesis can be enhanced by the PI3-K/AKT pathway. Our results illustrate that Wnt/beta-catenin signaling in conjunction with PI3-K/AKT pathway plays a key role in controlling development of the paraxial mesoderm.

MAPping out arteries and veins.

Growing evidence suggests that a genetic program specifies the identity of arteries and veins before the onset of circulation. A signaling cascade involving sonic hedgehog (Shh), vascular endothelial growth factor (VEGF), the VEGF receptor 2 (VEGFR2), homeobox proteins Foxc1 and Foxc2, the Notch receptor, and the downstream transcription factor gridlock is required for expression of arterial markers, whereas only a single transcription factor, COUP-TFII (chicken ovalbumin upstream promoter-transcription factor II), has previously been implicated in maintaining venous fate. Recent work has now implicated two competing pathways downstream of VEGFR2 in arterial versus venous specification: Activation of the phospholipase C-gamma (PLC-gamma)-mitogen-activated protein kinase (MAPK) pathway acts in arterial specification, whereas the phosphoinositide 3-kinase (PI3K)-Akt pathway acts to allow a venous fate by inhibition of the PLC-gamma-MAPK pathway. Here, we review this work and discuss how activation of the MAPK signaling cascade could stimulate an arterial fate.

Two endothelial cell lines derived from the somite.

Somites are sequentially formed, metameric units of the paraxial mesoderm of vertebrate embryos. They are the most obvious correlative of the segmental patterning along the cranio-caudal axis and transfer segmentation to other tissues such as the spinal nerves and dorsal aortic branches. Furthermore, somites are the source of numerous mesodermal cell types such as smooth and striated muscle, cartilage and tendon cells, and soft connective tissue. They also give rise to endothelial cells. Here we focus on the finding that two lineages of endothelial cells, blood vascular endothelial cells and lymphatic endothelial cells are derived from the somite. Their precursors, angioblasts, and lymphangioblasts, respectively, are born in the somite at different time points. Angioblasts are characterized by the expression of vascular endothelial growth factor receptor-2, whereas lymphangioblasts express the homeobox transcription factor Prox1. There seem to be two types of lymphangioblasts. Type 1 is derived from venous endothelium, while type 2 originates from mesenchymal precursor cells. The molecular networks of angioblast and lymphangioblast development and the relation between the two cell types and hematopoietic cells are discussed.

Somite formation and expression of MyoD, myogenin and myosin in Atlantic halibut (Hippoglossus hippoglossus L.) embryos incubated at different temperatures: transient asymmetric expression of MyoD.

Genes encoding the myogenic regulating factors MyoD and myogenin and the structural muscle proteins myosin light chain 2 (MyLC2) and myosin heavy chain (MyHC) were isolated from juvenile Atlantic halibut (Hippoglossus hippoglossus L.). The impact of temperature on their temporal and spatial expression during somitogenesis were examined by incubating halibut embryos at 4, 6 and 8 degrees C, and regularly sampling for whole-mount in situ hybridisation and reverse transcription (RT)-PCR. There were no significant effects of temperature on the onset of somitogenesis or number of somites at hatching. The rate of somite formation increased with increasing temperature, and the expression of MyoD, myogenin and MyHC followed the cranial-to-caudal somite formation. Hence, no significant effect of temperature on the spatial and temporal expression of the genes studied was found in relation to somite stage. MyoD, which has subsequently been shown to encode the MyoD2 isoform, displayed a novel bilaterally asymmetric expression pattern only in white muscle precursor cells during early halibut somitogenesis. The expression of myogenin resembled that previously described for other fish species, and preceded the MyHC expression by approximately five somites. Two MyLC2 cDNA sequences were for the first time described for a flatfish, probably representing embryonic (MyLC2a) and larval/juvenile (MyLC2b) isoforms. Factors regulating muscle determination, differentiation and development have so far mostly been studied in vertebrates with external bilateral symmetry. The findings of the present study suggest that more such investigations of flatfish species could provide valuable information on how muscle-regulating mechanisms work in species with different anatomical, physiological and ecological traits.

XHas2 activity is required during somitogenesis and precursor cell migration in Xenopus development.

In vertebrates, hyaluronan biosynthesis is regulated by three transmembrane catalytic enzymes denoted Has1, Has2 and Has3. We have previously cloned the Xenopus orthologues of the corresponding genes and defined their spatiotemporal distribution during development. During mammalian embryogenesis, Has2 activity is known to be crucial, as its abrogation in mice leads to early embryonic lethality. Here, we show that, in Xenopus, morpholino-mediated loss-of-function of XHas2 alters somitogenesis by causing a disruption of the metameric somitic pattern and leads to a defective myogenesis. In the absence of XHas2, early myoblasts underwent apoptosis, failing to complete their muscle differentiation programme. XHas2 activity is also required for migration of hypaxial muscle cells and trunk neural crest cells (NCC). To approach the mechanism whereby loss of HA, following XHas2 knockdown, could influence somitogenesis and precursor cell migration, we cloned the orthologue of the primary HA signalling receptor CD44 and addressed its function through an analogous knockdown approach. Loss of XCD44 did not disturb somitogenesis, but strongly impaired hypaxial muscle precursor cell migration and the subsequent formation of the ventral body wall musculature. In contrast to XHas2, loss of function of XCD44 did not seem to be essential for trunk NCC migration, suggesting that the HA dependence of NCC movement was rather associated with an altered macromolecular composition of the ECM structuring the cells' migratory pathways. The presented results, extend our knowledge on Has2 function and, for the first time, demonstrate a developmental role for CD44 in vertebrates. On the whole, these data underlie and confirm the emerging importance of cell-ECM interactions and modulation during embryonic development.

The zebrafish bcl-2 homologue Nrz controls development during somitogenesis and gastrulation via apoptosis-dependent and -independent mechanisms.

Although the role of the b-cell lymphoma (Bcl)-2 family of apoptosis inhibitors is well documented in tumor cells and tissue morphogenesis, their role during the early development of vertebrates is unknown. Here, we characterize Nrz, a new Bcl-2-related inhibitor of apoptosis in zebrafish. Nrz is a mitochondrial protein, antagonizing the death-accelerator Bax. The nrz gene is mainly expressed during gastrulation and somitogenesis. The knockdown of nrz with antisense morpholinos leads to alterations of the somites, correlated with an increase in apoptosis. In addition, earlier during development, in the zebrafish gastrula, nrz knockdown results in an increase of snail-1 expression at the margin and frequent gastrulation arrest at the shield stage, independently of apoptosis. Together these data suggest that Nrz, in addition to its effect on apoptosis, contributes to cell movements during gastrulation by negatively regulating the expression of Snail-1, a transcription factor that controls cell adhesion.

Generation of fertile and diploid fish, medaka (Oryzias latipes), from nuclear transplantation of blastula and four-somite-stage embryonic cells into nonenucleated unfertilized eggs.

In two experimental series of transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid nuclear transplants were successfully generated. In the first experiment, nuclei from blastula cells of a medaka stock with the wild-type body color were transplanted into 1722 eggs from the orange-red variety. Of 26 adult nuclear transplants with the wild-type body color, 22 were, as expected, triploid and sterile, but the other four were fertile. Three of the four were diploid, and the last one was tetraploid. They transmitted the wild-type body color to the F1 and F2 progenies in a Mendelian fashion. In the second experiment, cell nuclei from four-somite-stage embryos of the orangered variety carrying the green fluorescent protein (GFP) transgene were transplanted into 1688 recipients of the same strain. Three adult nuclear transplants expressing GFP were obtained. Two of them were triploid and sterile, but the remaining one was fertile and diploid. The transgene of the donor nuclei was transmitted to the F(1) and F(2) offspring in a Mendelian fashion. These observations that diploid and fertile nuclear transplants could be obtained without enucleation of the recipient eggs may have important implications for future nuclear transplantation in medaka.