Biomedical subjects

Miguel L Allende

Publications and source records attributed to Miguel L Allende.

15 recordsLinked to original sources

PubMed2006

Cloning of hif-1alpha and hif-2alpha and mRNA expression pattern during development in zebrafish.

Hypoxia-inducible factors (HIFs) regulate gene expression in response to hypoxia and in vertebrates they are known to participate in several developmental processes, including angiogenesis, vasculogenesis, heart and central nervous system development. Over the last decade, major progress in unraveling the molecular mechanisms that mediate regulation of HIF proteins by oxygen tension has been reported, but our knowledge on their developmental regulation during embryogenesis in model organisms is limited. Expression of hif-1alpha and hif-2alpha genes has been characterized during normal mouse development and they were found to be expressed from stages E7.5, later in E9.5 and E15.5 in several different tissues such as the brain, heart and blood vessels. However, there is no detailed temporal information on their expression at other embryonic stages, even though orthologous genes have been described in several different vertebrate species. In this study, we describe the cloning and detailed expression pattern of zebrafish hif-1alpha and hif-2alpha genes. Sequence analysis revealed that zebrafish Hif proteins are highly homologous to other vertebrate orthologues. Zebrafish hif-1alpha and hif-2alpha are both expressed throughout development in discrete territories in a dynamic pattern. Interestingly, in the notochord the expression of hif-1alpha is switched off, while hif-2alpha transcription is turned on, signifying that the two genes might have partially overlapping, although non-redundant functions in development. This is the first time that a detailed comparison of the expression of hif-1alpha and hif-2alpha is directly assessed in a vertebrate model system throughout development.

Animals↗

PubMed2006

Proneural gene requirement for hair cell differentiation in the zebrafish lateral line.

The lateral line system comprises an array of mechanosensory organs, the neuromasts, distributed over the body surface. Each neuromast consists of a patch of mechanosensory hair cells surrounded by support cells. We show that, in the zebrafish, two proneural genes are essential for differentiation of the hair cells, neuroD (nrd) and atonal homolog 1 (ath1). Gene knockdown experiments demonstrate that loss of function of either gene, but not of the related proneural gene neurogenin1 (ngn1), abrogate the appearance of hair cell markers. This is in contrast to other sensory systems, such as the neurons of the lateral line ganglion, where nrd is regulated by ngn1 and not by ath1. Overexpression of ath1 can induce nrd, and the phenotype produced by loss of ath1 function can be partially rescued by injection of nrd mRNA. This supports the conclusion that the activation of nrd probably requires ath1 in the hair cell lineage, whereas in sensory neurons nrd activation requires ngn1. We propose that the emergence of two atonal homologs, ath1 and ngn1, allowed the cellular segregation of mechanoreception and signal transmission that were originally performed by a single cell type as found in insects.

Animals↗

PubMed2006

Control of cell migration in the zebrafish lateral line: implication of the gene "tumour-associated calcium signal transducer," tacstd.

The sensory organs of the zebrafish lateral-line system (neuromasts) originate from migrating primordia that move along precise pathways. The posterior primordium, which deposits the neuromasts on the body and tail of the embryo, migrates along the horizontal myoseptum from the otic region to the tip of the tail. This migration is controlled by the chemokine SDF1, which is expressed along the prospective pathway, and by its receptor CXCR4, which is expressed by the migrating cells. In this report, we describe another zebrafish gene that is heterogeneously expressed in the migrating cells, tacstd. This gene codes for a membrane protein that is homologous to the TACSTD1/2 mammalian proteins. Inactivation of the zebrafish tacstd gene results in a decrease in proneuromast deposition, suggesting that tacstd is required for the deposition process.

Amino Acid Sequence↗

PubMed2006

Cracking the genome's second code: enhancer detection by combined phylogenetic footprinting and transgenic fish and frog embryos.

Genes involved in vertebrate development are unusually enriched for highly conserved non-coding sequence elements. These regions are readily detected in silico, by genome-wide sequence comparisons between different vertebrates, from mammals to fish (phylogenetic footprinting). It follows that sequence conservation must be the result of positive selection for an essential physiological role. An obvious possibility is that these conserved sequences possess regulatory or structural functions important for gene expression and, thus, an in vivo assay becomes necessary. We have developed a rapid testing system using zebrafish and Xenopus laevis embryos that allows us to assign transcriptional regulatory functions to conserved non-coding sequence elements. The sequences are cloned into a vector containing a minimal promoter and the GFP reporter, and are assayed for their putative cis-regulatory activity in zebrafish or Xenopus transgenic experiments. Vectors used include plasmid DNA and the Tol2 transposon system in fish and X. laevis. We have followed this logic to detect and analyze conserved elements in an intergenic region present in the Iroquois (Irx) gene clusters of zebrafish, Xenopus tropicalis, Fugu rubripes and mouse. We have assayed approximately 50 of these conserved elements and shown that the majority behave as modular positive regulatory elements (enhancers) that contribute to specific temporal and spatial domains that are part of the endogenous gene expression pattern. Moreover, comparison of the activity of cognate Irx enhancers from different organisms demonstrates that conservation of sequence is accompanied by in vivo functional conservation across species. Finally, for some of the most conserved elements, we have been able to identify a critical core sequence, essential for correct enhancer function.

Animals↗

PubMed2006

Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.

The combination of accessible embryology and forward genetic techniques has made zebrafish a powerful model system for the study of vertebrate development. One limitation of genetic analysis is that the study of gene function is usually limited to the first developmental event affected by a gene. In vivo electroporation has recently matured as a method for studying gene function at different developmental time points and in specific regions of the organism. The focal application of current allows macromolecules to be efficiently introduced into a targeted region at any time in the life cycle. Here we describe a rapid protocol by which DNA, RNA and morpholinos can all be precisely electroporated into zebrafish in a temporally and spatially controlled manner. This versatile technique allows gene function to be determined by both gain and loss of function analyses in specific regions at specific times. This is the first report that describes the electroporation of three different molecules into embryonic and larval zebrafish cells.

Animals↗

PubMed2005

Sub-lethal concentrations of waterborne copper are toxic to lateral line neuromasts in zebrafish (Danio rerio).

In teleosts, the lateral line system is composed of neuromasts containing hair cells that are analogous to those present in the inner ear of all vertebrates. In the zebrafish embryo and early larva, this system is composed of the anterior lateral line (ALL), which covers the head, and the posterior lateral line (PLL), present in the trunk and tail. The mechanosensory hair cells found in neuromasts can be labeled in vivo using fluorescent dyes such as 4-di-2-Asp (DiAsp) or FM1-43. We have studied the effects of water-borne copper exposure on the function of the lateral line system in zebrafish larvae. Our results show that transient incubation of post-hatching larvae for 2h with non-lethal concentrations of copper (1-50 microM CuSO4) induces cellular damage localized to neuromasts, apoptosis, and loss of hair cell markers. This effect is specific to copper, as other metals did not show these effects. Since hair cells in fish can regenerate, we followed the reappearance of viable hair cells in neuromasts after copper removal. In the PLL, we determined that there is a threshold concentration of copper above which regeneration does not occur, whereas, at lower concentrations, the length of time it takes for viable hair cells to reappear is dependent on the amount of copper used during the treatment. The ALL behaves differently though, as regeneration can occur even after treatments with concentrations of copper an order of magnitude higher than the one that irreversibly affects the PLL. Regeneration of hair cells is dependent on cell division within the neuromasts as damage that precludes proliferation prevents reappearance of this cell type.

Animals↗

PubMed2005

A functional survey of the enhancer activity of conserved non-coding sequences from vertebrate Iroquois cluster gene deserts.

Recent studies of the genome architecture of vertebrates have uncovered two unforeseen aspects of its organization. First, large regions of the genome, called gene deserts, are devoid of protein-coding sequences and have no obvious biological role. Second, comparative genomics has highlighted the existence of an array of highly conserved non-coding regions (HCNRs) in all vertebrates. Most surprisingly, these structural features are strongly associated with genes that have essential functions during development. Among these, the vertebrate Iroquois (Irx) genes stand out on both fronts. Mammalian Irx genes are organized in two clusters (IrxA and IrxB) that span >1 Mb each with no other genes interspersed. Additionally, a large number of HCNRs exist within Irx clusters. We have systematically examined the enhancer activity of HCNRs from the IrxB cluster using transgenic Xenopus and zebrafish embryos. Most of these HCNRs are active in subdomains of endogenous Irx expression, and some are candidates to contain shared enhancers of neighboring genes, which could explain the evolutionary conservation of Irx clusters. Furthermore, HCNRs present in tetrapod IrxB but not in fish may be responsible for novel Irx expression domains that appeared after their divergence. Finally, we have performed a more detailed analysis on two IrxB ultraconserved non-coding regions (UCRs) duplicated in IrxA clusters in similar relative positions. These four regions share a core region highly conserved among all of them and drive expression in similar domains. However, inter-species conserved sequences surrounding the core, specific for each of these UCRs, are able to modulate their expression.

Animals↗

PubMed2005

Early stages of zebrafish eye formation require the coordinated activity of Wnt11, Fz5, and the Wnt/beta-catenin pathway.

During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. We show that two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/beta-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/beta-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results allow us to postulate a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells.

Animals↗

PubMed2004

Zebrafish cellular nucleic acid-binding protein: gene structure and developmental behaviour.

Here we analyse the structural organisation and expression of the zebrafish cellular nucleic acid-binding protein (zCNBP) gene and protein. The gene is organised in five exons and four introns. A noteworthy feature of the gene is the absence of a predicted promoter region. The coding region encodes a 163-amino acid polypeptide with the highly conserved general structural organisation of seven CCHC Zn knuckle domains and an RGG box between the first and the second Zn knuckles. Although theoretical alternative splicing is possible, only one form of zCNBP is actually detected. This form is able to bind to single-stranded DNA and RNA probes in vitro. The analysis of zCNBP developmental expression shows a high amount of CNBP-mRNA in ovary and during the first developmental stages. CNBP-mRNA levels decrease while early development progresses until the midblastula transition (MBT) stage and increases again thereafter. The protein is localised in the cytoplasm of blastomeres whereas it is mainly nuclear in developmental stages after the MBT. These findings suggest that CNBP is a strikingly conserved single-stranded nucleic acid-binding protein which might interact with maternal mRNA during its storage in the embryo cell cytoplasm. It becomes nuclear once MBT takes place possibly in order to modulate zygotic transcription and/or to associate with newly synthesised transcripts.

Alternative Splicing↗

PubMed2004

Role of BMP signaling and the homeoprotein Iroquois in the specification of the cranial placodal field.

Different types of placodes originate at the anterior border of the neural plate but it is still an unresolved question whether individual placodes arise as distinct ectodermal specializations in situ or whether all or a subset of the placodes originate from a common preplacodal field. We have analyzed the expression and function of the homeoprotein Iro1 in Xenopus and zebrafish embryos, and we have compared its expression with several preplacodal and placodal markers. Our results indicate that the iro1 genes are expressed in the preplacodal region, being one of the earliest markers for this area. We show that an interaction between the neural plate and the epidermis is able to induce the expression of several preplacodal markers, including Xiro1, by a similar mechanism to that previously shown for neural crest induction. In addition, we analyzed the role of BMP in the specification of the preplacodal field by studying the expression of the preplacodal markers Six1, Xiro1, and several specific placodal markers. We experimentally modified the level of BMP activity by three different methods. First, we implanted beads soaked with noggin in early neurula stage Xenopus embryos; second, we injected the mRNA that encodes a dominant negative of the BMP receptor into Xenopus and zebrafish embryos; and third, we grafted cells expressing chordin into zebrafish embryos. The results obtained using all three methods show that a reduction in the level of BMP activity leads to an expansion of the preplacodal and placodal region similar to what has been described for neural crest regions. By using conditional constructs of Xiro1, we performed gain and loss of function experiments. We show that Xiro1 play an important role in the specification of both the preplacodal field as well as individual placodes. We have also used inducible dominant negative and activator constructs of Notch signaling components to analyze the role of these factors on placodal development. Our results indicate that the a precise level of BMP activity is required to induce the neural plate border, including placodes and neural crest cells, that in this border the iro1 gene is activated, and that this activation is required for the specification of the placodes.

Animals↗

PubMed2004

The Irx gene family in zebrafish: genomic structure, evolution and initial characterization of irx5b.

Genes of the iroquois ( Iro/Irx) family are highly conserved from Drosophila to mammals and they have been implicated in a number of developmental processes. In flies, the Iro genes participate in patterning events in the early larva and in imaginal disk specification. In vertebrates, the Irx genes regulate developmental events during gastrulation, nervous system regionalization, activation of proneural genes and organ patterning. The Iro genes in Drosophila and the Irx genes of mammals show a clustered organization in the genome. Flies have a single cluster comprising three genes while mammals have two clusters also having three genes each. Moreover, experimental evidence in flies shows that transcriptional regulatory elements are shared among genes within the Iro cluster, suggesting that the same may be true in vertebrates. To date, the genomic organization of the Irx genes in non-mammalian species has not been studied. In this work, we have isolated the irx5b gene from zebrafish, Danio rerio, and have characterized its expression pattern. Furthermore, we have identified the complete set of Irx genes in two fish species, the zebrafish and pufferfish, Takifugu rubripes, and have determined the genomic organization of these genes. Our analysis indicates that early in fish evolutionary history, the Irx gene clusters have been duplicated and that subsequent events have maintained the clustered organization for some of the genes, while others have been lost. In total there are 11 existing Irx genes in zebrafish and 10 in pufferfish. We propose a new nomenclature for the zebrafish Irx genes based on the analysis of their sequences and their genomic relationships.

Animals↗

PubMed2004

Cloning, expression pattern and essentiality of the high-affinity copper transporter 1 (ctr1) gene in zebrafish.

The high-affinity copper transporter 1 (Ctr1) is a highly conserved transmembrane protein that mediates the internalization of copper ions from the extracellular medium. In this study, we have isolated the zebrafish ctr1 gene. The zebrafish ctr1 cDNA encodes a protein with 69% identity to the human orthologue and shows conservation of specific amino acid residues involved in copper transport. We find only a single ctr1 gene in the zebrafish genome which maps to linkage group 5. The genomic structure of the zebrafish gene shows that it consists of five exons and that exon-intron boundaries are absolutely conserved with the mammalian ctr1 genes. Expression in embryos was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Zebrafish ctr1 is maternally loaded, and transcripts can be detected throughout development and in adult fish. Distribution of ctr1 message appears ubiquitous during early stages becoming restricted to the brain and ventral tissues by 24 h post fertilization (hpf). Beginning at 3 days post fertilization (dpf), expression is found mainly in the developing intestine. Specific knockdown of ctr1 by antisense morpholino oligonucleotides (MOs) causes early larval lethality. Defects include cell death in tissues where ctr1 is most heavily expressed, a finding similar to that described for a mouse knockout of mCtr1. Despite the existence of at least one other copper transport mechanism in the fish, our studies show that zebrafish ctr1 is an essential gene for development.

Amino Acid Sequence↗

PubMed2004

Transcription factor Ap-2alpha is necessary for development of embryonic melanophores, autonomic neurons and pharyngeal skeleton in zebrafish.

The genes that control development of embryonic melanocytes are poorly defined. Although transcription factor Ap-2alpha is expressed in neural crest (NC) cells, its role in development of embryonic melanocytes and other neural crest derivatives is unclear because mouse Ap-2alpha mutants die before melanogenesis. We show that zebrafish embryos injected with morpholino antisense oligonucleotides complementary to ap-2alpha (ap-2alpha MO) complete early morphogenesis normally and have neural crest cells. Expression of c-kit, which encodes the receptor for the Steel ligand, is reduced in these embryos, and, similar to zebrafish c-kit mutant embryos, embryonic melanophores are reduced in number and migration. The effects of ap-2alpha MO injected into heterozygous and homozygous c-kit mutants support the notion that Ap-2alpha works through C-kit and additional target genes to mediate melanophore cell number and migration. In contrast to c-kit mutant embryos, in ap-2alpha MO-injected embryos, melanophores are small and under-pigmented, and unexpectedly, analysis of mosaic embryos suggests Ap-2alpha regulates melanophore differentiation through cell non-autonomous targets. In addition to melanophore phenotypes, we document reduction of other neural crest derivatives in ap-2alpha MO-injected embryos, including jaw cartilage, enteric neurons, and sympathetic neurons. These results reveal that Ap-2alpha regulates multiple steps of melanophore development, and is required for development of other neuronal and non-neuronal neural crest derivatives.

Amino Acid Sequence↗

PubMed2002

Zebrafish yolk-specific not really started (nrs) gene is a vertebrate homolog of the Drosophila spinster gene and is essential for embryogenesis.

By using retroviral insertional mutagenesis in zebrafish, we have identified a recessive lethal mutation in the not really started (nrs) gene. The nrs mutation disrupts a gene located in linkage group 3 that is highly homologous to the spinster gene identified in Drosophila and to spinster orthologs identified in mammals. In flies, spinster encodes a membrane protein involved in lysosomal metabolism and programmed cell death in the central nervous system and in the ovary. In nrs mutant fish embryos, we detect an opaque substance in the posterior yolk cell extension at approximately 1 day after fertilization. This material progressively accumulates and by 48 hr after fertilization fills the entire yolk. By day 3 of embryogenesis, mutant embryos are severely reduced in size compared with their wild-type siblings and they die a few hours later. By in situ hybridization, we show that the nrs mRNA is expressed in the yolk cell at the time the mutant phenotype becomes apparent. In wild-type embryos, nrs message is present maternally and zygotically throughout embryogenesis and is also detected in adult animals. In nrs homozygous mutant embryos, nrs transcripts are undetectable at the time the phenotype becomes apparent, indicating that the retroviral insertion has most likely abolished expression of the nrs gene. Finally, the nrs phenotype can be partially rescued by microinjection of nrs encoding DNA. These results suggest that the nrs mutation affects an essential gene encoding a putative membrane-bound protein expressed specifically in the yolk cell during zebrafish embryogenesis.

Amino Acid Sequence↗

PubMed2002

Expression and splice variant analysis of the zebrafish tcf4 transcription factor.

Wnt signalling has been implicated in antero-posterior patterning of the vertebrate embryonic body axis and in a number of other developmental processes. One of the downstream effectors of Wnt signalling is the beta-catenin protein which complexes with members of the Lef/tcf transcription factor family. In the zebrafish, specification of the head has been shown to be dependent on the Tcf3 protein which acts as a repressor of the posteriorizing activity of Wnt (Nature 407 (2000) 913). Here, we report the cloning and expression pattern of the zebrafish tcf4 gene. In embryos, we find that the tcf4 gene is highly regulated at the level of RNA splicing such that the variant proteins that are produced contain or lack domains proposed to be essential in repression or activation of transcription. Expression of tcf4 mRNA is first detected in a graded fashion in the anterior brain and subsequently becomes restricted to the dorsal diencephalon and anterior midbrain. There is also transient expression in the anterior rhombomeres of the hindbrain and in the developing gut.

Alternative Splicing↗