Cristina Pujades

Cell segregation in the vertebrate hindbrain relies on actomyosin cables located at the interhombomeric boundaries

Segregating cells into compartments during embryonic development is essential for growth and pattern formation. Physical mechanisms shaping compartment boundaries were recently explored in Drosophila, where actomyosin‐based barriers were revealed to be important for keeping cells apart. In vertebrates, interhombomeric boundaries are straight interfaces, which often serve as signaling centers that pattern the surrounding tissue. Here, we demonstrate that in the hindbrain of zebrafish embryos cell sorting sharpens the molecular boundaries and, once borders are straight, actomyosin barriers are key to keeping rhombomeric cells segregated. Actomyosin cytoskeletal components are enriched at interhombomeric boundaries, forming cable‐like structures in the apical side of the neuroepithelial cells by the time morphological boundaries are visible. When myosin II function is inhibited, cable structures do not form, leading to rhombomeric cell mixing. Downregulation of EphA4a compromises actomyosin cables and cells with different rhombomeric identity intermingle, and the phenotype is rescued enhancing myosin II activity. Moreover, enrichment of actomyosin structures is obtained when EphA4 is ectopically expressed in even‐numbered rhombomeres. These findings suggest that mechanical barriers act downstream of EphA/ephrin signaling to segregate cells from different rhombomeres.

Patterning and cell fate in ear development

The inner ear is a complex structure responsible for the senses of audition and balance in vertebrates. The ear is organised into different sense organs that are specialised to detect specific stimuli such as sound and linear or angular accelerations. The elementary sensory unit of the ear consists of hair cells, supporting cells, neurons and Schwann cells. Hair cells are the mechano-electrical transducing elements, and otic neurons convey information coded in electrical impulses to the brain. With the exception of the Schwann cells, all cellular elements of the inner ear derive from the otic placode. This is an ectodermal thickening that is specified in the head ectoderm adjacent to the caudal hindbrain. The complex organisation of the ear requires precise coupling of regional specification and cell fate decisions during development, i.e. specificity in defining particular spatial domains containing particular cell types. Those decisions are taken early in development and are the subject of this article. We review here recent work on: i) early patterning of the otic placode, ii) the role of neural tube signals in the patterning of the otic vesicle, and iii) the genes underlying cell fate determination of neurons and sensory hair cells.

vHnf1 Regulates Specification of Caudal Rhombomere Identity in the Chick Hindbrain

The homeobox‐containing gene variant hepatocyte nuclear factor‐1 (vHnf1) has recently been shown to be involved in zebrafish caudal hindbrain specification, notably in the activation of MafB and Krox20 expression. We have explored this regulatory network in the chick by in ovo electroporation in the neural tube. We show that misexpression of vHnf1 confers caudal identity to more anterior regions of the hindbrain. Ectopic expression of mvHnf1 leads to ectopic activation of MafB and Krox20, and downregulation of Hoxb1 in rhombomere 4. Unexpectedly, mvhnf1 strongly upregulates Fgf3 expression throughout the hindbrain, in both a cell‐autonomous and a non‐cell‐autonomous manner. Blockade of FGF signaling correlates with a selective loss of MafB and Krox20 expression, without affecting the expression of vHnf1, Fgf3, or Hoxb1. Based on these observations, we propose that in chick, as in zebrafish, vHnf1 acts with FGF to promote caudal hindbrain identity by activating MafB and Krox20 expression. However, our data suggest differences in the vHnf1 downstream cascade in different vertebrates. Developmental Dynamics 234:567–576, 2005.

Analysis of mouse kreisler mutants reveals new roles of hindbrain-derived signals in the establishment of the otic neurogenic domain

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB--a gene transiently expressed in rhombomeres 5 and 6 of the developing hindbrain--in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalisation, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 mutant embryos, characterized by a downregulation or absence of Fgf3 in r5 and r6, display ectopic neuroblasts in the otic primordium. However, Fgf3-/-Fgf10-/- double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight the importance of hindbrain-derived signals in the regulation of otic neurogenesis.

Cell Lineage Analysis Reveals Three Different Progenitor Pools for Neurosensory Elements in the Otic Vesicle

In the inner ear, sensory versus neuronal specification is achieved through few well-defined bHLH transcription factors. However, the molecular mechanisms regulating the generation of the appropriate cell type in the correct place and at the correct time are not completely understood yet. Various studies have shown that hair cell- and neuron-specifying genes partially overlap in the otic territory, suggesting that mutual interactions among these bHLH factors could direct the generation of the two cell types from a common neurosensory progenitor. Although there is little evidence for a clonal relationship between macular hair cells and sensory neurons, the existence of a single progenitor able to give both sensory and neuronal cell types remains an open question. Here, we identified a population of common neurosensory progenitors in the zebrafish inner ear and studied the proneural requirement for cell fate decision within this population. Expression analysis reveals that proneural genes for hair cells and neurons overlap within the posteromedial otic epithelium. Combined results from single-cell lineage and functional studies on neurog1 and neuroD1 further demonstrate the following: (1) in the anterior region of the ear, neuronal and sensory lineages have already segregated at the onset of proneural gene expression and are committed to a given fate very early; (2) in contrast, the posteromedial part of the ear harbors a population of common progenitors giving both neurons and hair cells until late stages; and finally (3) neuroD1 is required within this pool of bipotent progenitors to generate the hair cell fate.

Clonal analysis in mice underlines the importance of rhombomeric boundaries in cell movement restriction during hindbrain segmentation.

Background Boundaries that prevent cell movement allow groups of cells to maintain their identity and follow independent developmental trajectories without the need for ongoing instructive signals from surrounding tissues. This is the case of vertebrate rhombomeric boundaries. Analysis in the developing chick hindbrain provided the first evidence that rhombomeres are units of cell lineage. The appearance of morphologically visible rhombomeres requires the segment restricted expression of a series of transcription factors, which position the boundaries and prefigure where morphological boundaries will be established. When the boundaries are established, when the cells are committed to a particular rhombomere and how they are organized within the hindbrain are important questions to our understanding of developmental regionalization. Methodology/Principal Findings Sophisticated experimental tools with high-resolution analysis have allowed us to explore cell lineage restriction within the hindbrain in mouse embryos. This novel strategy is based on knock-in alleles of ubiquitous expression and allows unrestricted clonal analysis of cell lineage from the two-cell stage to the adult mouse. Combining this analysis with statistical and mathematical tools we show that there is lineage compartmentalization along the anteroposterior axis from very early stages of mouse embryonic development. Conclusions Our results show that the compartment border coincides with the morphological boundary in the mouse hindbrain. The restriction of the cells to cross rhombomeric boundaries seen in chick is also observed in mouse. We show that the rhombomeric boundaries themselves are involved in cell movement restriction, although an underlying pre-pattern during early embryonic development might influence the way that cell populations organize.

Hedgehog Signaling Governs the Development of Otic Sensory Epithelium and Its Associated Innervation in Zebrafish

The inner ear is responsible for the perception of motion and sound in vertebrates. Its functional unit, the sensory patch, contains mechanosensory hair cells innervated by sensory neurons from the statoacoustic ganglion (SAG) that project to the corresponding nuclei in the brainstem. How hair cells develop at specific positions, and how otic neurons are sorted to specifically innervate each endorgan and to convey the extracted information to the hindbrain is not completely understood. In this work, we study the generation of macular sensory patches and investigate the role of Hedgehog (Hh) signaling in the production of their neurosensory elements. Using zebrafish transgenic lines to visualize the dynamics of hair cell and neuron production, we show that the development of the anterior and posterior maculae is asynchronic, suggesting they are independently regulated. Tracing experiments demonstrate the SAG is topologically organized in two different neuronal subpopulations, which are spatially segregated and innervate specifically each macula. Functional experiments identify the Hh pathway as crucial in coordinating the production of hair cells in the posterior macula, and the formation of its specific innervation. Finally, gene expression analyses suggest that Hh influences the balance between different SAG neuronal subpopulations. These results lead to a model in which Hh orients functionally the development of inner ear towards an auditory fate in all vertebrate species.

FGF signaling controls caudal hindbrain specification through Ras-ERK1/2 pathway

BackgroundDuring early steps of embryonic development the hindbrain undergoes a regionalization process along the anterior-posterior (AP) axis that leads to a metameric organization in a series of rhombomeres (r). Refinement of the AP identities within the hindbrain requires the establishment of local signaling centers, which emit signals that pattern territories in their vicinity. Previous results demonstrated that the transcription factor vHnf1 confers caudal identity to the hindbrain inducing Krox20 in r5 and MafB/Kreisler in r5 and r6, through FGF signaling [1].ResultsWe show that in the chick hindbrain, Fgf3 is transcriptionally activated as early as 30 min after mvHnf1 electroporation, suggesting that it is a direct target of this transcription factor. We also analyzed the expression profiles of FGF activity readouts, such as MKP3 and Pea3, and showed that both are expressed within the hindbrain at early stages of embryonic development. In addition, MKP3 is induced upon overexpression of mFgf3 or mvHnf1 in the hindbrain, confirming vHnf1 is upstream FGF signaling. Finally, we addressed the question of which of the FGF-responding intracellular pathways were active and involved in the regulation of Krox20 and MafB in the hindbrain. While Ras-ERK1/2 activity is necessary for MKP3, Krox20 and MafB induction, PI3K-Akt is not involved in that process.ConclusionBased on these observations we propose that vHnf1 acts directly through FGF3, and promotes caudal hindbrain identity by activating MafB and Krox20 via the Ras-ERK1/2 intracellular pathway.

Increase in a 55-kDa keratin-like protein in the nuclear matrix of rat liver cells during proliferative activation.

We have identified a protein (p55) with a molecular weight of 55 kDa and a pI of 6.2, which was strongly increased in the nuclear matrix of rat liver cells during proliferative activation. This protein is highly insoluble since it could not be solubilized either by detergents or by alkaline extraction. We have obtained three partial amino acid sequences which revealed that p55 has a high homology with cytokeratins. Polyclonal antibodies raised against p55 were used to carry out Western blot and immunocytochemical studies which indicated that p55 was localized only in the nuclei, specifically in the nuclear matrix. Autoradiographic experiments revealed that not all the cells presenting an increase in p55 incorporated [3H]thymidine, indicating that this protein is not related to DNA replication. Immunocytochemical studies also revealed that during mitosis p55 is localized surrounding the chromosomes and associated with the mitotic apparatus, suggesting that p55 is involved in the separation of chromosomes during cell division.

Advances in the development of novel antioxidant therapies as an approach for fetal alcohol syndrome prevention

Ethanol is the most common human teratogen, and its consumption during pregnancy can produce a wide range of abnormalities in infants known as fetal alcohol spectrum disorder (FASD). The major characteristics of FASD can be divided into: (i) growth retardation, (ii) craniofacial abnormalities, and (iii) central nervous system (CNS) dysfunction. FASD is the most common cause of nongenetic mental retardation in Western countries. Although the underlying molecular mechanisms of ethanol neurotoxicity are not completely determined, the induction of oxidative stress is believed to be one central process linked to the development of the disease. Currently, there is no known effective strategy for prevention (other than alcohol avoidance) or treatment. In the present review we will provide the state of art in the evidence for the use of antioxidants as a potential therapeutic strategy for the treatment using whole-embryo and culture cells models of FASD. We conclude that the imbalance of the intracellular redox state contributes to the pathogenesis observed in FASD models, and we suggest that antioxidant therapy can be considered a new efficient strategy to mitigate the effects of prenatal ethanol exposure.