Concepción Rodríguez-Esteban

Dickkopf1 Is Required for Embryonic Head Induction and Limb Morphogenesis in the Mouse

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Here, we show that Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. Our data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning.

Pitx2 determines left-right asymmetry of internal organs in vertebrates

The handedness of visceral organs is conserved among vertebrates and is regulated by asymmetric signals relayed by molecules such as Shh, Nodal and activin. The gene Pitx2 is expressed in the left lateral plate mesoderm and, subsequently, in the left heart and gut of mouse, chick and Xenopus embryos. Misexpression of Shh and Nodal induces Pitx2 expression, whereas inhibition of activin signalling blocks it. Misexpression of Pitx2 alters the relative position of organs and the direction of body rotation in chick and Xenopus embryos. Changes in Pitx2 expression are evident in mouse mutants with laterality defects. Thus, Pitx2 seems to serve as a critical downstream transcription target that mediates left–right asymmetry in vertebrates.

The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity

During embryonic development, initially similar fields can develop into distinct structures, such as the vertebrate fore- and hindlimbs. Although considerable progress has been made in our understanding of the genetic control underlying the establishment of the different limb axes,,, the molecular cues that specify the differential development of the fore- and hindlimbs are unknown. Possible candidates for genes determining limb identity are Pitx1, a gene whose transcripts are detected in the early hind- but not forelimb bud, and two members of the T-box (Tbx) gene family, Tbx4 and Tbx5, which are specifically expressed in the hindlimb and forelimb buds, respectively,,. Here we show that Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the activity of three signalling proteins that are required for limb outgrowth and patterning: fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and Wnt. In addition, we provide evidence that Tbx4 and Tbx5 are involved in controlling limb identity. Our findings provide insight into how similar developmental fields can evolve into homologous but distinct structures.

Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells

ES cells represent a valuable model for investigating early embryo development and hold promise for future regenerative medicine strategies. The self-renewal of pluripotent mouse ES cells has been shown to require extrinsic stimulation by the bone morphogenetic protein (BMP) and leukemia inhibitory factor signaling pathways and the expression of the transcription factors Oct4 and Nanog. However, the network of interactions among extrinsic and intrinsic determinants of ES cell pluripotency is currently poorly understood. Here, we show that Nanog expression is up-regulated in mouse ES cells by the binding of T (Brachyury) and STAT3 to an enhancer element in the mouse Nanog gene. We further show that Nanog blocks BMP-induced mesoderm differentiation of ES cells by physically interacting with Smad1 and interfering with the recruitment of coactivators to the active Smad transcriptional complexes. Taken together, our findings illustrate the existence of ES cell-specific regulatory networks that underlie the maintenance of ES cell pluripotency and provide mechanistic insights into the role of Nanog in this process.

Retinoic acid signalling links left-right asymmetric patterning and bilaterally symmetric somitogenesis in the zebrafish embryo.

During embryogenesis, cells are spatially patterned as a result of highly coordinated and stereotyped morphogenetic events. In the vertebrate embryo, information on laterality is conveyed to the node, and subsequently to the lateral plate mesoderm, by a complex cascade of epigenetic and genetic events, eventually leading to a left–right asymmetric body plan. At the same time, the paraxial mesoderm is patterned along the anterior–posterior axis in metameric units, or somites, in a bilaterally symmetric fashion. Here we characterize a cascade of laterality information in the zebrafish embryo and show that blocking the early steps of this cascade (before it reaches the lateral plate mesoderm) results in random left–right asymmetric somitogenesis. We also uncover a mechanism mediated by retinoic acid signalling that is crucial in buffering the influence of the flow of laterality information on the left–right progression of somite formation, and thus in ensuring bilaterally symmetric somitogenesis.

Notch activity acts as a sensor for extracellular calcium during vertebrate left-right determination

During vertebrate embryo development, the breaking of the initial bilateral symmetry is translated into asymmetric gene expression around the node and/or in the lateral plate mesoderm. The earliest conserved feature of this asymmetric gene expression cascade is the left-sided expression of Nodal, which depends on the activity of the Notch signalling pathway. Here we present a mathematical model describing the dynamics of the Notch signalling pathway during chick embryo gastrulation, which reveals a complex and highly robust genetic network that locally activates Notch on the left side of Hensens node. We identify the source of the asymmetric activation of Notch as a transient accumulation of extracellular calcium, which in turn depends on left–right differences in H+/K+-ATPase activity. Our results uncover a mechanism by which the Notch signalling pathway translates asymmetry in epigenetic factors into asymmetric gene expression around the node.

RLIM inhibits functional activity of LIM homeodomain transcription factors via recruitment of the histone deacetylase complex

LIM domains are required for both inhibitory effects on LIM homeodomain transcription factors and synergistic transcriptional activation events. The inhibitory actions of the LIM domain can often be overcome by the LIM co-regulator known as CLIM2, LDB1 and NLI (referred to hereafter as CLIM2; refs 2, 3, 4). The association of the CLIM cofactors with LIM domains does not, however, improve the DNA-binding ability of LIM homeodomain proteins, suggesting the action of a LIM-associated inhibitor factor. Here we present evidence that LIM domains are capable of binding a novel RING-H2 zinc-finger protein, Rlim (for RING finger LIM domain-binding protein), which acts as a negative co-regulator via the recruitment of the Sin3A/histone deacetylase corepressor complex. A corepressor function of RLIM is also suggested by in vivo studies of chick wing development. Overexpression of the gene Rnf12, encoding Rlim, results in phenotypes similar to those observed after inhibition of the LIM homeodomain factor LHX2, which is required for the formation of distal structures along the proximodistal axis, or by overexpression of dominant-negative CLIM1. We conclude that Rlim is a novel corepressor that recruits histone deacetylase-containing complexes to the LIM domain.

In Vivo Activation of a Conserved MicroRNA Program Induces Mammalian Heart Regeneration

Heart failure is a leading cause of mortality and morbidity in the developed world, partly because mammals lack the ability to regenerate heart tissue. Whether this is due to evolutionary loss of regenerative mechanisms present in other organisms or to an inability to activate such mechanisms is currently unclear. Here we decipher mechanisms underlying heart regeneration in adult zebrafish and show that the molecular regulators of this response are conserved in mammals. We identified miR-99/100 and Let-7a/c and their protein targets smarca5 and fntb as critical regulators of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. Although human and murine adult cardiomyocytes fail to elicit an endogenous regenerative response after myocardial infarction, we show that in vivo manipulation of this molecular machinery in mice results in cardiomyocyte dedifferentiation and improved heart functionality after injury. These data provide a proof of concept for identifying and activating conserved molecular programs to regenerate the damaged heart.

Maintenance of embryonic stem cell pluripotency by Nanog-mediated reversal of mesoderm specification

Embryonic stem cells (ESCs) can be propagated indefinitely in culture, while retaining the ability to differentiate into any cell type in the organism. The molecular and cellular mechanisms underlying ESC pluripotency are, however, poorly understood. We characterize a population of early mesoderm-specified (EM) progenitors that is generated from mouse ESCs by bone morphogenetic protein stimulation. We further show that pluripotent ESCs are actively regenerated from EM progenitors by the action of the divergent homeodomain-containing protein Nanog, which, in turn, is upregulated in EM progenitors by the combined action of leukemia inhibitory factor and the early mesoderm transcription factor T/Brachyury. These findings uncover specific roles of leukemia inhibitory factor, Nanog, and bone morphogenetic protein in the self-renewal of ESCs and provide novel insights into the cellular bases of ESC pluripotency.

Zebrafish IκB Kinase 1 Negatively Regulates NF-κB Activity

The IkappaB kinase (IKK) activity is critical for processing IkappaB inhibitory proteins and activating the NF-kappaB signaling, which is involved in a series of physiological and developmental steps in vertebrates. The IKK activity resides in two catalytic subunits, IKK1 and IKK2, and two regulatory subunits, NEMO and ELKS. IKK2 is the major cytokine-responsive IkappaB kinase because depletion of IKK1 does not interfere with the IKK activity. In fact, IKK1-/- mice display morphological abnormalities that are independent of its kinase activity and NF-kappaB activation. Hence, using zebrafish (Danio rerio) as a model, we examined the evolutionary role of IKK1 in modulating NF-kappaB. Ikk1-/- zebrafish embryos present head and tail malformations and, surprisingly, show upregulation of NF-kappaB-responsive genes and increased NF-kappaB-dependent apoptosis. Overexpression of ikk1 leads to midline structure defects that resemble NF-kappaB blockage in vivo. Zebrafish Ikk1 forms complexes with NEMO that represses NF-kappaB in vertebrate cells. Indeed, truncation of its NEMO binding domain (NBD) restores NF-kappaB-dependent transcriptional activity and, consequently, the ikk1-overexpressing phenotype. Here, we report that Ikk1 negatively regulates NF-kappaB by sequestering NEMO from active IKK complexes, indicating that IKK1 can function as a repressor of NF-kappaB.