Miguel Torres

The development of the vertebrate inner ear

The inner ear is a complex sensory organ responsible for balance and sound detection in vertebrates. It originates from a transient embryonic structure, the otic vesicle, that contains all of the information to develop autonomously into the mature inner ear. We review here the development of the otic vesicle, bringing together classical embryological experiments and recent genetic and molecular data. The specification of the prospective ectoderm and its commitment to the otic fate are very early events and can be related to the expression of genes with restricted expression domains. A combinatorial gene expression model for placode specification and diversification, based on classical embryological evidence and gene expression patterns, is discussed. The formation of the otic vesicle is dependent on inducing signals from endoderm, mesoderm and neuroectoderm. Ear induction consists of a sequence of discrete instructions from those tissues that confer its final identity on the otic field, rather than a single all-or-none process. The important role of the neural tube in otic development is highlighted by the abnormalities observed in mouse mutants for the Hoxa1, kreisler and fgf3 genes and those reported in retinoic acid-deficient quails. Still, the nature of the relation between the neural tube and otic development remains unclear. Gene targeting experiments in the mouse have provided evidence for genes potentially involved in regional and cell-fate specification in the inner ear. The disruption of the mouse Brn3.1 gene identifies the first mutation affecting sensory hair-cell specification, and mutants for Pax2 and Nkx5.1 genes show their requirement for the development of specific regions of the otic vesicle. Several growth-factors contribute to the patterned cell proliferation of the otic vesicle. Among these, IGF-I and FGF-2 are expressed in the otic vesicle and may act in an autocrine manner. Finally, little is known about early mechanisms involved in guiding ear innervation. However, targeted disruption of genes coding for neurotrophins and Trk receptors have shown that once synaptic contacts are established, they depend on specific trophic interactions that involve these two gene families. The accessibility of new cellular and molecular approaches are opening new perspectives in vertebrate development and are also starting to be applied to ear development. This will allow this classical and attractive model system to see a rapid progress in the near future.

Conserved regulation of proximodistal limb axis development by Meis1/Hth.

Vertebrate limbs grow out from the flanks of embryos, with their main axis extending proximodistally from the trunk. Distinct limb domains, each with specific traits, are generated in a proximal-to-distal sequence during development. Diffusible factors expressed from signalling centres promote the outgrowth of limbs and specify their dorsoventral and anteroposterior axes. However, the molecular mechanism by which limb cells acquire their proximodistal (P–D) identity is unknown. Here we describe the role of the homeobox genes Meis1/2 and Pbx1 in the development of mouse, chicken and Drosophila limbs. We find that Meis1/2 expression is restricted to a proximal domain, coincident with the previously reported domain in which Pbx1 is localized to the nucleus, and resembling the distribution of the Drosophila homologues homothorax (hth) and extradenticle (exd); that Meis1 regulates Pbx1 activity by promoting nuclear import of the Pbx1 protein; and that ectopic expression of Meis1 in chicken and hth in Drosophila disrupts distal limb development and induces distal-to-proximal transformations. We suggest that restriction of Meis1/Hth to proximal regions of the vertebrate and insect limb is essential to specify cell fates and differentiation patterns along the P–D axis of the limb.

CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses

CCR6 expression in dendritic, T, and B cells suggests that this beta-chemokine receptor may regulate the migration and recruitment of antigen-presenting and immunocompetent cells during inflammatory and immunological responses. Here we demonstrate that CCR6-/- mice have underdeveloped Peyers patches, in which the myeloid CD11b+ CD11c+ dendritic-cell subset is not present in the subepithelial dome. CCR6-/- mice also have increased numbers in T-cell subpopulations within the intestinal mucosa. In 2,4-dinitrofluorobenzene-induced contact hypersensitivity (CHS) studies, CCR6-/- mice developed more severe and more persistent inflammation than wild-type (WT) animals. Conversely, in a delayed-type hypersensitivity (DTH) model induced with allogeneic splenocytes, CCR6-/- mice developed no inflammatory response. The altered responses seen in the CHS and DTH assays suggest the existence of a defect in the activation and/or migration of the CD4(+) T-cell subsets that downregulate or elicit the inflammation response, respectively. These findings underscore the role of CCR6 in cutaneous and intestinal immunity and the utility of CCR6-/- mice as a model to study pathologies in these tissues. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish

The zebrafish heart has the capacity to regenerate after ventricular resection. Although this regeneration model has proved useful for the elucidation of certain regeneration mechanisms, it is based on the removal of heart tissue rather than its damage. Here, we characterize the cellular response and regenerative capacity of the zebrafish heart after cryoinjury, an alternative procedure that more closely models the pathophysiological process undergone by the human heart after myocardial infarction (MI). Localized damage was induced in 25% of the ventricle by cryocauterization (CC). During the first 24 hours post-injury, CC leads to cardiomyocyte death within the injured area and the near coronary vasculature. Cell death is followed by a rapid proliferative response in endocardium, epicardium and myocardium. During the first 3 weeks post-injury cell debris was cleared and the injured area replaced by a massive scar. The fibrotic tissue was subsequently degraded and replaced by cardiac tissue. Although animals survived CC, their hearts showed nonhomogeneous ventricular contraction and had a thickened ventricular wall, suggesting that regeneration is associated with processes resembling mammalian ventricular remodeling after acute MI. Our results provide the first evidence that, like mammalian hearts, teleost hearts undergo massive fibrosis after cardiac damage. Unlike mammals, however, the fish heart can progressively eliminate the scar and regenerate the lost myocardium, indicating that scar formation is compatible with myocardial regeneration and the existence of endogenous mechanisms of scar regression. This finding suggests that CC-induced damage in zebrafish could provide a valuable model for the study of the mechanisms of scar removal post-MI.

SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation.

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non‐essential role in embryo development. Rescued SOCS3‐deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3‐deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho‐1) suppresses giant cell differentiation. Conversely, SOCS3‐deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3‐deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation.

Epithelial-to-Mesenchymal and Endothelial-to-Mesenchymal Transition From Cardiovascular Development to Disease

Cellular switching from an epithelial-to-mesenchymal phenotype, and conversely from a mesenchymal-to-epithelial phenotype, are important biological programs that are operative from conception to death in mammalian organisms. Indeed, the capacity of cells to switch between these states has been fundamental to the generation of complex body patterns throughout evolution. Phenotypic switching from an epithelial to mesenchymal cell, termed epithelial-to-mesenchymal transition (EMT), was a paradigm that evolved from numerous observations on early embryonic development, the foundations of which date back to the 1920s and the pioneering work of Johannes Holtfreter on embryo formation and differentiation.1,2 By the late 1960s, seminal chick embryo studies by Elizabeth Hay3 led to the first formal description that epithelial cells can undergo a dramatic phenotypic transformation and give rise to embryonic mesoderm.4 Subsequent studies have revealed that this process is reversible (mesenchymal-to-epithelial transition [MET]), and gradually the term ‘transition” has come to replace ‘transformation.”nnGiven that EMT/MET was initially identified and described by developmental biologists, it is perhaps not surprising that these processes are best understood during embryonic implantation and development. As explored in this review, it is now known that successive waves of cellular transition, from an epithelial to mesenchymal and then back to an epithelial state, are required for normal embryonic patterning and organ formation. In addition, numerous studies that span a broad spectrum of physiological and pathological conditions have expanded our knowledge of EMT/MET and now provide evidence for the important role played by these processes in various adult conditions including fibrosis, wound repair, inflammation, and malignancy. Indeed, our conceptual framework now also encompasses several variations and subcategories of cellular phenotypic switching, including endothelial-to-mesenchymal transition (EndMT).nnIn this review, epithelial, endothelial, and mesenchymal phenotypic cellular switching will be explored in the cardiovascular system, spanning cardiovascular development through to adult …

Antagonism between extradenticle function and Hedgehog signalling in the developing limb

The Drosophila homeobox gene extradenticle (exd) encodes a highly conserved cofactor of Hox proteins. exd activity is regulated post-translationally by a mechanism involving nuclear translocation,; only nuclear Exd protein is functional. The exd gene is required for patterning of the proximal region of the leg,, whereas patterning of the distal region requires signalling by the Wingless (Wg) and Decapentaplegic (Dpp), proteins, which are in turn activated by Hedgehog (Hh). Here we show that exd function and Dpp/Wg signalling are antagonistic and divide the leg into two mutually exclusive domains. In the proximal domain, exd activity prevents cells from responding to Dpp and Wg. Conversely, in the distal domain, exd function is suppressed by the Dpp/Wg response gene Distal-less (Dll), which prevents the nuclear transport of Exd. We also found that the product of a murine homologue of exd (Pbx1) is regulated at the subcellular level, and that its pattern of nuclear localization in the mouse limb resembles that of Exd in the Drosophila leg. These findings suggest that the division of the limb into two antagonistic domains, as defined by exd (Pbx1) function and Hh signalling, may be a general feature of limb development.

Myc-driven endogenous cell competition in the early mammalian embryo

The epiblast is the mammalian embryonic tissue that contains the pluripotent stem cells that generate the whole embryo. We have established a method for inducing functional genetic mosaics in the mouse. Using this system, here we show that induction of a mosaic imbalance of Myc expression in the epiblast provokes the expansion of cells with higher Myc levels through the apoptotic elimination of cells with lower levels, without disrupting development. In contrast, homogeneous shifts in Myc levels did not affect epiblast cell viability, indicating that the observed competition results from comparison of relative Myc levels between epiblast cells. During normal development we found that Myc levels are intrinsically heterogeneous among epiblast cells, and that endogenous cell competition refines the epiblast cell population through the elimination of cells with low relative Myc levels. These results show that natural cell competition in the early mammalian embryo contributes to the selection of the epiblast cell pool.

Identification of the vertebrate Iroquois homeobox gene family with overlapping expression during early development of the nervous system

In Drosophila the decision processes between the neural and epidermal fate for equipotent ectodermal cells depend on the activity of proneural genes. Members of the Drosophila Iroquois-Complex (Iro-C) positively regulate the activity of certain proneural AS-C genes during the formation of external sensory organs. We have identified and characterized three mouse Iroquois-related genes: Irx1, -2 and -3, which have a homeodomain very similar to that of the Drosophila Iro-C genes. The sequence similarity implies that these three genes represent a separate homeobox family. All three genes are expressed with distinct spatio/temporal patterns during early mouse embryogenesis. These patterns implicate them in a number of embryonic developmental processes: the A/P and D/V patterning of specific regions of the central nervous system (CNS), and regionalization of the otic vesicle, branchial epithelium and limbs.

Ezh1 Is Required for Hematopoietic Stem Cell Maintenance and Prevents Senescence-like Cell Cycle Arrest

Polycomb group (PcG) proteins are key epigenetic regulators of hematopietic stem cell (HSC) fate. The PcG members Ezh2 and Ezh1 are important determinants of embryonic stem cell identity, and the transcript levels of these histone methyltransferases are inversely correlated during development. However, the role of Ezh1 in somatic stem cells is largely unknown. Here we show that Ezh1 maintains repopulating HSCs in a slow-cycling, undifferentiated state, protecting them from senescence. Ezh1 ablation induces significant loss of adult HSCs, with concomitant impairment of their self-renewal capacity due to a potent senescence response. Epigenomic and gene expression changes induced by Ezh1 deletion in senesced HSCs demonstrated that Ezh1-mediated PRC2 activity catalyzes monomethylation and dimethylation of H3K27. Deletion of Cdkn2a on the Ezh1 null background rescued HSC proliferation and survival. Our results suggest that Ezh1 is an important histone methyltransferase for HSC maintenance.