The "Down syndrome critical region" of human chromosome 21 has been defined bas... more The "Down syndrome critical region" of human chromosome 21 has been defined based on the analysis of rare cases of partial trisomy 21. Evidence is accumulating that DYRK1A, one of the 20 genes located in this region, is an important candidate gene involved in the neurobiological alterations of Down syndrome. Both the structure of the DYRK1A gene and the sequence of the encoded protein kinase are highly conserved in evolution. The protein contains a unique assembly of structural motifs outside the catalytic domain, including a nuclear localization signal, a PEST region, and a repeat of 13 consecutive histidines. MNB/DYRK1A and related kinases are unique among serine/threonine-specific protein kinases in that their activity depends on tyrosine autophosphorylation in the catalytic domain. Also, evidence is accumulating that mRNA levels of MNB/DYRK1A are subject to tight regulation. A number of putative substrates of MNB/DYRK1A have emerged in the recent years, the majority of them being transcription factors. Although the function of MNB/DYRK1A in intracellular signalling and regulation of cell function is still poorly defined, current evidence suggests that the kinase may play a role in the regulation of gene expression.
pre-T cell receptor complex (Saint-Ruf et al., 1994). Targeted disruptions of the TCR and pT␣ ge... more pre-T cell receptor complex (Saint-Ruf et al., 1994). Targeted disruptions of the TCR and pT␣ genes have shown that the pre-TCR complex is important for the generation of ␣/ T cells (Mombaerts et al., 1992; Fehling et al., 1995). Cells that fail to express a functional pre
Transcriptional cascades responsible for initiating the formation of vertebrate embryonic structu... more Transcriptional cascades responsible for initiating the formation of vertebrate embryonic structures such as limbs are not well established. Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations along the anteroposterior axis of the embryo is not known. The T-box transcription factor TBX5 is important for normal heart and limb formation, but its role in early limb development is not well defined. We report that mouse embryos lacking Tbx5 do not form forelimb buds, although the patterning of the lateral plate mesoderm into the limb field is intact. Tbx5 is not essential for an early establishment of forelimb versus hindlimb identity. In the absence of Tbx5, the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression. Tbx5 directly activates the Fgf10 gene via a conserved binding site, providing a simple and direct mechanism for limb bud initiation. Lef1/Tcf1-dependent Wnt signaling is not essential for initiation of Tbx5 or Fgf10 transcription, but is required in concert with Tbx5 for maintenance of normal levels of Fgf10 expression. We conclude that Tbx5 is not essential for the early establishment of the limb field in the lateral plate mesoderm but is a primary and direct initiator of forelimb bud formation. These data suggest common pathways for the differentiation and growth of embryonic structures downstream of T-box genes.
Lymphoid enhancer factor (LEF1), a nuclear mediator of Wnt signaling, is required for the formati... more Lymphoid enhancer factor (LEF1), a nuclear mediator of Wnt signaling, is required for the formation of organs that depend on inductive interactions between epithelial and mesenchymal tissues. In previous tissue recombination experiments with normal and Lef1 −/− tooth germs, we found that the effect of LEF1 expression in the epithelium is tissue nonautonomous and transferred to the subjacent mesenchyme. Here we examine the molecular basis for LEF1 function and find that the epithelium of the developmentally arrested Lef1 −/− tooth rudiments fails to express Fgf4, Shh, and Bmp4, but not Wnt10a. We identify the Fgf4 gene as a direct transcriptional target for LEF1 and show that beads soaked with recombinant FGF4 protein can fully overcome the developmental arrest of Lef1 −/− tooth germs. In addition, we find that FGF4 beads induce rapidly the expression of Fgf3 in dental mesenchyme and that both epithelial and mesenchymal FGF proteins induce the delayed expression of Shh in the epithelium. Taken together, these data indicate that a single target of LEF1 can account for the function of LEF1 in tooth development and for a relay of a Wnt signal reception to a cascade of FGF signaling activities, allowing for a sequential and reciprocal communication between epithelium and mesenchyme.
Lef1 and other genes of the LEF1/TCF family of transcription factors are nuclear mediators of Wnt... more Lef1 and other genes of the LEF1/TCF family of transcription factors are nuclear mediators of Wnt signaling. Here we examine the expression pattern and functional importance of Lef1 in the developing forebrain of the mouse. Lef1 is expressed in the developing hippocampus, and LEF1-deficient embryos lack dentate gyrus granule cells but contain glial cells and interneurons in the region of the dentate gyrus. In mouse embryos homozygous for a Lef1-lacZ fusion gene, which encodes a protein that is not only deficient in DNA binding but also interferes with β-catenin-mediated transcriptional activation by other LEF1/TCF proteins, the entire hippocampus including the CA fields is missing. Thus, LEF1 regulates the generation of dentate gyrus granule cells, and together with other LEF1/TCF proteins, the development of the hippocampus.
Major attention is being paid in recent years to the genes harbored within the so called Down syn... more Major attention is being paid in recent years to the genes harbored within the so called Down syndrome Critical Region of human chromosome 21. Among them, those genes with a possible brain function are becoming the focus of intense research due to the numerous neurobiological alterations and cognitive deficits that Down syndrome individuals have. MNB/DYRK1A is one of these genes. It encodes a protein kinase with unique genetic and biochemical properties, which have been evolutionarily conserved from insects to humans. MNB/DYRK1A is expressed in the developing brain where it seems to play a role in proliferation of neural progenitor cells, neurogenesis, and neuronal differentiation. Although at a lower level, MNB/DYRK1A is also expressed in the adult brain where, as judged by the phenotype of mutant and transgenic animals, it may be involved in learning and memory. Nevertheless, most of the molecular mechanisms underlying these functions remain to be unraveled. In this review we compile and discuss experimental evidences, which support the involvement of MNB/DYRK1A in several neuropathologies and cognitive deficits of Down syndrome.
Proceedings of the National Academy of Sciences of the United States of America, Jul 10, 2001
Members of the LEF-1͞TCF family of transcription factors have been implicated in mediating a nucl... more Members of the LEF-1͞TCF family of transcription factors have been implicated in mediating a nuclear response to Wnt signals by association with -catenin. Consistent with this view, mice carrying mutations in either the Wnt3a gene or in both transcription factor genes Lef1 and Tcf1 were previously found to show a similar defect in the formation of paraxial mesoderm in the gastrulating mouse embryo. In addition, mutations in the Brachyury gene, a direct transcriptional target of LEF-1, were shown to result in mesodermal defects. However, direct evidence for the role of LEF-1 and Brachyury in Wnt3a signaling has been limiting. In this study, we genetically examine the function of LEF-1 in the regulation of Brachyury expression and in signaling by Wnt3a. Analysis of the expression of Brachyury in Lef1 ؊/؊ Tcf1 ؊/؊ mice and studies of Brachyury:lacZ transgenes containing wild type or mutated LEF-1 binding sites indicate that Lef1 is dispensable for the initiation, but is required for the maintenance of Brachyury expression. We also show that the expression of an activated form of LEF-1, containing the -catenin activation domain fused to the amino terminus of LEF-1, can rescue a Wnt3a mutation. Together, these data provide genetic evidence that Lef1 mediates the Wnt3a signal and regulates the stable maintenance of Brachyury expression during gastrulation.
Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of t... more Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of the Notch pathway in vertebrate somitogenesis. Here, we examine the molecular basis for this presumed hierarchy and show that a targeted mutation of Lef1, which abrogates LEF1 function and impairs the activity of coexpressed TCF factors, affects the patterning of somites and the expression of components of the Notch pathway. LEF1 was found to bind multiple sites in the Dll1 promoter in vitro and in vivo. Moreover, mutations of LEF1-binding sites in the Dll1 promoter impair expression of a Dll1-LacZ transgene in the presomitic mesoderm. Finally, the induced expression of LEF1--catenin activates the expression of endogenous Dll1 in fibroblastic cells. Thus, Wnt signaling can affect the Notch pathway by a LEF1mediated regulation of Dll1.
Trabajo presentado al 17th Meeting of the Spanish Society for Developmental Biology (SEBD), celeb... more Trabajo presentado al 17th Meeting of the Spanish Society for Developmental Biology (SEBD), celebrado de forma virtual del 18 al 20 de noviembre de 2020.Peer reviewe
Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Ali... more Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019.También presentado al 16th Christmas Meeting del Instituto de Neurociencias, celebrado en Alicante del 19 al 20 de diciembre de 2019.The epithelial-mesenchymal transition (EMT) endows cells with migratory and invasive properties and it is crucial for the formation of many tissues and organs during embryonic development. This cellular program is triggered after the activation of transcription factors, referred to as EMT-TFs. In the adult, the reactivation of the EMT program contributes to the progression of diseases like fibrosis and cancer (Nieto et al. Cell, 2016). Prrx1, identified as a novel EMT-TF in our lab, produces three different splice isoforms, whose functions remain to be characterized. We have tested the ability of each of these isoforms to induce EMT in vitro, and only the longest isoform (Prrx1L) is able to do so. To test the role of the different isoforms in vivo, we have generated isoform-specific mutant mouse lines that we are characterizing using the phenotype of the Prrx1 null mice in the skeleton and the vasculature (J.F. Martin et al. Genes & Development, 1995, K. Ihida-Stansbury et al. Circulation research, 2004). Using the postnatal retina as a model to study vasculogenesis, we have observed that Prrx1 is specifically expressed in mural cells. Consistent with this expression pattern, we have seen that the Prrx1 isoforms are essential for the integrity of the vasculature in the P6 retina and adult lungs. We are using the in vivo matrigel plug assay to dissect the function of Prrx1 isoforms in mural cells and their interaction with endothelial cells.Peer reviewe
Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Ali... more Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019.También presentado al 16th Christmas Meeting del Instituto de Neurociencias, celebrado en Alicante del 19 al 20 de diciembre de 2019.Gene loss-of-function (LOF) studies are fundamental to address their relevance in a particular biological process. Abundant approaches exist to perform LOF depending on the type of experiment and organism being used. Morpholino oligonucleotidesmediated knockdown (MO) technologies have been widely used over 20 years to address the function of developmental genes in vertebrates. New gene editing technologies including the CRISPR/Cas9 system have now become the gold standard to test gene function. We have previously shown that a Prrx1a-dependent L/R asymmetric EMT induces differential L/R forces leading to the leftward displacement of the posterior pole of the heart, driving its normal left-handed position. This analysis was performed by MO-mediated knockdown and as previously published prrx1a mutant alleles in zebrafish do not present defects in heart laterality, we decided to check whether G0 CRISPR-generated prrx1a mutants were able to recapitulate the heart phenotypes of prrx1a morphants. We found that both MO-mediated knock down and CRISPR-mediated gene editing of prrx1a results in an efficient Prrx1a protein loss, while the previously published mutant alleles are likely not null. Consistent with this, prrx1a crispant embryos confirm the mesocardia phenotype previously found in the morphants as well as the observed reduction in atrial size. However, morphants show early defects in heart development including defects in spaw expression leading to heart jogging randomization, which is not observed in the crispants. These data indicate that the early phenotypes are prrx1a-independent, that heart laterality is instructed after jogging and confirm the role of Prrx1a in the process.Peer reviewe
Ectodermal dysplasia syndromes affect the development of several organs, including hair, teeth, a... more Ectodermal dysplasia syndromes affect the development of several organs, including hair, teeth, and glands. The recent cloning of two genes responsible for these syndromes has led to the identification of a novel TNF family ligand, ectodysplasin, and TNF receptor, edar. This has indicated a developmental regulatory role for TNFs for the first time. Our in situ hybridization analysis of the expression of ectodysplasin (encoded by the Tabby gene) and edar (encoded by the downless gene) during mouse tooth morphogenesis showed that they are expressed in complementary patterns exclusively in ectodermal tissue layer. Edar was expressed reiteratively in signaling centers regulating key steps in morphogenesis. The analysis of the effects of eight signaling molecules in the TGF, FGF, Hh, Wnt, and EGF families in tooth explant cultures revealed that the expression of edar was induced by activinA, whereas Wnt6 induced ectodysplasin expression. Moreover, ectodysplasin expression was downregulated in branchial arch epithelium and in tooth germs of Lef1 mutant mice, suggesting that signaling by ectodysplasin is regulated by LEF-1-mediated Wnt signals. The analysis of the signaling centers in tooth germs of Tabby mice (ectodysplasin null mutants) indicated that in the absence of ectodysplasin the signaling centers were small. However, no downstream targets of ectodysplasin signaling were identified among several genes expressed in the signaling centers. We conclude that ectodysplasin functions as a planar signal between ectodermal compartments and regulates the function, but not the induction, of epithelial signaling centers. This TNF signaling is tightly associated with epithelial-mesenchymal interactions and with other signaling pathways regulating organogenesis. We suggest that activin signaling from mesenchyme induces the expression of the TNF receptor edar in the epithelial signaling centers, thus making them responsive to Wnt-induced ectodysplasin from the nearby ectoderm. This is the first demonstration of integration of the Wnt, activin, and TNF signaling pathways.
The cholinergic enzyme acetylcholinesterase (AChE) and the catalytic component of the γ-secretase... more The cholinergic enzyme acetylcholinesterase (AChE) and the catalytic component of the γ-secretase complex, presenilin-1 (PS1), are known to interact. In this study, we investigate the consequences of AChE-PS1 interactions, particularly the influence of AChE in PS1 levels and γ-secretase activity. PS1 is able to co-immunoprecipitate all AChE variants (AChE-R and AChE-T) and molecular forms (tetramers and light subunits) present in the human brain. Over-expression of AChE-R or AChE-T, or their respective inactive mutants, all trigger an increase in PS1 protein levels. The AChE specie capable of triggering the biggest increase in PS1 levels is a complex of AChE with the membrane anchoring subunit proline-rich membrane anchor (PRiMA), which restricts the localization of the resulting AChE tetramer to the outer plasma membrane. Incubation of cultured cells with soluble AChE demonstrates that AChE is able to increase PS1 at both the protein and transcript levels. However, the increase of PS1 caused by soluble AChE is accompanied by a decrease in γ-secretase activity as shown by the reduction of the processing of the β-amyloid precursor protein. This inhibitory effect of AChE on γ-secretase activity was also demonstrated by directly assessing accumulation of CTF-APP in cell-free membrane preparations incubated with AChE. Our data suggest that AChE may function as an inhibitor of γ-secretase activity.
Developing axons must control their growth rate to follow the appropriate pathways and establish ... more Developing axons must control their growth rate to follow the appropriate pathways and establish specific connections. However, the regulatory mechanisms involved remain elusive. By combining live imaging with transplantation studies in mice, we found that spontaneous calcium activity in the thalamocortical system and the growth rate of thalamocortical axons were developmentally and intrinsically regulated. Indeed, the spontaneous activity of thalamic neurons governed axon growth and extension through the cortex in vivo. This activity-dependent modulation of growth was mediated by transcriptional regulation of Robo1 through an NF-B binding site. Disruption of either the Robo1 or Slit1 genes accelerated the progression of thalamocortical axons in vivo, and interfering with Robo1 signaling restored normal axon growth in electrically silent neurons. Thus, modifications to spontaneous calcium activity encode a switch in the axon outgrowth program that allows the establishment of specific neuronal connections through the transcriptional regulation of Slit1 and Robo1 signaling.
Targeted inactivation of the murine gene encoding the transcription factor LEF-1 abrogates the fo... more Targeted inactivation of the murine gene encoding the transcription factor LEF-1 abrogates the formation of organs that depend on epithelial-mesenchymal tissue interactions. In this study we have recombined epithelial and mesenchymal tissues from normal and LEF-1-deficient embryos at different stages of development to define the LEF-1-dependent steps in tooth and whisker organogenesis. At the initiation of organ development, formation of the epithelial primordium of the whisker but not tooth is dependent on mesenchymal Left gene expression. Subsequent formation of a whisker and tooth mesenchymal papilla and completion of organogenesis require transient expression of Lefl in the epithelium. These experiments indicate that the effect of Lefl expression is transmitted from one tissue to the other. In addition, the finding that the expression of Lefl can be activated by bone morphogenetic protein 4 (BMP-4) suggests a regulatory role of this transcription factor in BMP-mediated inductive tissue interactions.
Classical studies of cholinesterase activity during liver dysfunction have focused on butyrylchol... more Classical studies of cholinesterase activity during liver dysfunction have focused on butyrylcholinesterase (BuChE), whereas acetylcholinesterase (AChE) has not received much attention. In the current study, liver and plasma AChE levels were investigated in rats with cirrhosis induced after 3 weeks of bile duct ligation (BDL). BDL rats showed a pronounced decrease in liver AChE levels (ϳ50%) compared with sham-operated (non-ligated, NL) controls; whereas liver BuChE appeared unaffected. A selective loss of tetrameric (G 4) AChE was detected in BDL rats, an effect also observed in rats with carbon tetrachloride-induced cirrhosis. In accordance, SDS-PAGE analysis showed that the major 55-kd immunoreactive AChE band was decreased in BDL as compared with NL. A 65-kd band, attributed in part to inactive AChE, was increased as became the most abundant AChE subunit in BDL liver. The overall decrease in AChE activity in BDL liver was not accompanied by a reduction of AChE transcripts. The loss of G 4 was also reflected by changes observed in AChE glycosylation pattern attributable to different liver AChE forms being differentially glycosylated. BDL affects AChE levels in both hepatocytes and Kupffer cells; however, altered AChE expression was mainly reflected in an alteration in hepatocyte AChE pattern. Plasma from BDL rats had approximately 45% lower AChE activity than controls, displaying decreased G 4 levels and altered lectin-binding patterns. In conclusion, the liver is an important source of serum AChE; altered AChE levels may be a useful biomarker for liver cirrhosis.
Trabajo presentado al 17th Meeting of the Spanish Society for Developmental Biology (SEBD), celeb... more Trabajo presentado al 17th Meeting of the Spanish Society for Developmental Biology (SEBD), celebrado de forma virtual del 18 al 20 de noviembre de 2020.Peer reviewe
Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Ali... more Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019.También presentado al 16th Christmas Meeting del Instituto de Neurociencias, celebrado en Alicante del 19 al 20 de diciembre de 2019.The epithelial-mesenchymal transition (EMT) endows cells with migratory and invasive properties and it is crucial for the formation of many tissues and organs during embryonic development. This cellular program is triggered after the activation of transcription factors, referred to as EMT-TFs. In the adult, the reactivation of the EMT program contributes to the progression of diseases like fibrosis and cancer (Nieto et al. Cell, 2016). Prrx1, identified as a novel EMT-TF in our lab, produces three different splice isoforms, whose functions remain to be characterized. We have tested the ability of each of these isoforms to induce EMT in vitro, and only the longest isoform (Prrx1L) is able to do so. To test the role of the different isoforms in vivo, we have generated isoform-specific mutant mouse lines that we are characterizing using the phenotype of the Prrx1 null mice in the skeleton and the vasculature (J.F. Martin et al. Genes & Development, 1995, K. Ihida-Stansbury et al. Circulation research, 2004). Using the postnatal retina as a model to study vasculogenesis, we have observed that Prrx1 is specifically expressed in mural cells. Consistent with this expression pattern, we have seen that the Prrx1 isoforms are essential for the integrity of the vasculature in the P6 retina and adult lungs. We are using the in vivo matrigel plug assay to dissect the function of Prrx1 isoforms in mural cells and their interaction with endothelial cells.Peer reviewe
The "Down syndrome critical region" of human chromosome 21 has been defined bas... more The "Down syndrome critical region" of human chromosome 21 has been defined based on the analysis of rare cases of partial trisomy 21. Evidence is accumulating that DYRK1A, one of the 20 genes located in this region, is an important candidate gene involved in the neurobiological alterations of Down syndrome. Both the structure of the DYRK1A gene and the sequence of the encoded protein kinase are highly conserved in evolution. The protein contains a unique assembly of structural motifs outside the catalytic domain, including a nuclear localization signal, a PEST region, and a repeat of 13 consecutive histidines. MNB/DYRK1A and related kinases are unique among serine/threonine-specific protein kinases in that their activity depends on tyrosine autophosphorylation in the catalytic domain. Also, evidence is accumulating that mRNA levels of MNB/DYRK1A are subject to tight regulation. A number of putative substrates of MNB/DYRK1A have emerged in the recent years, the majority of them being transcription factors. Although the function of MNB/DYRK1A in intracellular signalling and regulation of cell function is still poorly defined, current evidence suggests that the kinase may play a role in the regulation of gene expression.
pre-T cell receptor complex (Saint-Ruf et al., 1994). Targeted disruptions of the TCR and pT␣ ge... more pre-T cell receptor complex (Saint-Ruf et al., 1994). Targeted disruptions of the TCR and pT␣ genes have shown that the pre-TCR complex is important for the generation of ␣/ T cells (Mombaerts et al., 1992; Fehling et al., 1995). Cells that fail to express a functional pre
Transcriptional cascades responsible for initiating the formation of vertebrate embryonic structu... more Transcriptional cascades responsible for initiating the formation of vertebrate embryonic structures such as limbs are not well established. Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations along the anteroposterior axis of the embryo is not known. The T-box transcription factor TBX5 is important for normal heart and limb formation, but its role in early limb development is not well defined. We report that mouse embryos lacking Tbx5 do not form forelimb buds, although the patterning of the lateral plate mesoderm into the limb field is intact. Tbx5 is not essential for an early establishment of forelimb versus hindlimb identity. In the absence of Tbx5, the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression. Tbx5 directly activates the Fgf10 gene via a conserved binding site, providing a simple and direct mechanism for limb bud initiation. Lef1/Tcf1-dependent Wnt signaling is not essential for initiation of Tbx5 or Fgf10 transcription, but is required in concert with Tbx5 for maintenance of normal levels of Fgf10 expression. We conclude that Tbx5 is not essential for the early establishment of the limb field in the lateral plate mesoderm but is a primary and direct initiator of forelimb bud formation. These data suggest common pathways for the differentiation and growth of embryonic structures downstream of T-box genes.
Lymphoid enhancer factor (LEF1), a nuclear mediator of Wnt signaling, is required for the formati... more Lymphoid enhancer factor (LEF1), a nuclear mediator of Wnt signaling, is required for the formation of organs that depend on inductive interactions between epithelial and mesenchymal tissues. In previous tissue recombination experiments with normal and Lef1 −/− tooth germs, we found that the effect of LEF1 expression in the epithelium is tissue nonautonomous and transferred to the subjacent mesenchyme. Here we examine the molecular basis for LEF1 function and find that the epithelium of the developmentally arrested Lef1 −/− tooth rudiments fails to express Fgf4, Shh, and Bmp4, but not Wnt10a. We identify the Fgf4 gene as a direct transcriptional target for LEF1 and show that beads soaked with recombinant FGF4 protein can fully overcome the developmental arrest of Lef1 −/− tooth germs. In addition, we find that FGF4 beads induce rapidly the expression of Fgf3 in dental mesenchyme and that both epithelial and mesenchymal FGF proteins induce the delayed expression of Shh in the epithelium. Taken together, these data indicate that a single target of LEF1 can account for the function of LEF1 in tooth development and for a relay of a Wnt signal reception to a cascade of FGF signaling activities, allowing for a sequential and reciprocal communication between epithelium and mesenchyme.
Lef1 and other genes of the LEF1/TCF family of transcription factors are nuclear mediators of Wnt... more Lef1 and other genes of the LEF1/TCF family of transcription factors are nuclear mediators of Wnt signaling. Here we examine the expression pattern and functional importance of Lef1 in the developing forebrain of the mouse. Lef1 is expressed in the developing hippocampus, and LEF1-deficient embryos lack dentate gyrus granule cells but contain glial cells and interneurons in the region of the dentate gyrus. In mouse embryos homozygous for a Lef1-lacZ fusion gene, which encodes a protein that is not only deficient in DNA binding but also interferes with β-catenin-mediated transcriptional activation by other LEF1/TCF proteins, the entire hippocampus including the CA fields is missing. Thus, LEF1 regulates the generation of dentate gyrus granule cells, and together with other LEF1/TCF proteins, the development of the hippocampus.
Major attention is being paid in recent years to the genes harbored within the so called Down syn... more Major attention is being paid in recent years to the genes harbored within the so called Down syndrome Critical Region of human chromosome 21. Among them, those genes with a possible brain function are becoming the focus of intense research due to the numerous neurobiological alterations and cognitive deficits that Down syndrome individuals have. MNB/DYRK1A is one of these genes. It encodes a protein kinase with unique genetic and biochemical properties, which have been evolutionarily conserved from insects to humans. MNB/DYRK1A is expressed in the developing brain where it seems to play a role in proliferation of neural progenitor cells, neurogenesis, and neuronal differentiation. Although at a lower level, MNB/DYRK1A is also expressed in the adult brain where, as judged by the phenotype of mutant and transgenic animals, it may be involved in learning and memory. Nevertheless, most of the molecular mechanisms underlying these functions remain to be unraveled. In this review we compile and discuss experimental evidences, which support the involvement of MNB/DYRK1A in several neuropathologies and cognitive deficits of Down syndrome.
Proceedings of the National Academy of Sciences of the United States of America, Jul 10, 2001
Members of the LEF-1͞TCF family of transcription factors have been implicated in mediating a nucl... more Members of the LEF-1͞TCF family of transcription factors have been implicated in mediating a nuclear response to Wnt signals by association with -catenin. Consistent with this view, mice carrying mutations in either the Wnt3a gene or in both transcription factor genes Lef1 and Tcf1 were previously found to show a similar defect in the formation of paraxial mesoderm in the gastrulating mouse embryo. In addition, mutations in the Brachyury gene, a direct transcriptional target of LEF-1, were shown to result in mesodermal defects. However, direct evidence for the role of LEF-1 and Brachyury in Wnt3a signaling has been limiting. In this study, we genetically examine the function of LEF-1 in the regulation of Brachyury expression and in signaling by Wnt3a. Analysis of the expression of Brachyury in Lef1 ؊/؊ Tcf1 ؊/؊ mice and studies of Brachyury:lacZ transgenes containing wild type or mutated LEF-1 binding sites indicate that Lef1 is dispensable for the initiation, but is required for the maintenance of Brachyury expression. We also show that the expression of an activated form of LEF-1, containing the -catenin activation domain fused to the amino terminus of LEF-1, can rescue a Wnt3a mutation. Together, these data provide genetic evidence that Lef1 mediates the Wnt3a signal and regulates the stable maintenance of Brachyury expression during gastrulation.
Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of t... more Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of the Notch pathway in vertebrate somitogenesis. Here, we examine the molecular basis for this presumed hierarchy and show that a targeted mutation of Lef1, which abrogates LEF1 function and impairs the activity of coexpressed TCF factors, affects the patterning of somites and the expression of components of the Notch pathway. LEF1 was found to bind multiple sites in the Dll1 promoter in vitro and in vivo. Moreover, mutations of LEF1-binding sites in the Dll1 promoter impair expression of a Dll1-LacZ transgene in the presomitic mesoderm. Finally, the induced expression of LEF1--catenin activates the expression of endogenous Dll1 in fibroblastic cells. Thus, Wnt signaling can affect the Notch pathway by a LEF1mediated regulation of Dll1.
Trabajo presentado al 17th Meeting of the Spanish Society for Developmental Biology (SEBD), celeb... more Trabajo presentado al 17th Meeting of the Spanish Society for Developmental Biology (SEBD), celebrado de forma virtual del 18 al 20 de noviembre de 2020.Peer reviewe
Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Ali... more Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019.También presentado al 16th Christmas Meeting del Instituto de Neurociencias, celebrado en Alicante del 19 al 20 de diciembre de 2019.The epithelial-mesenchymal transition (EMT) endows cells with migratory and invasive properties and it is crucial for the formation of many tissues and organs during embryonic development. This cellular program is triggered after the activation of transcription factors, referred to as EMT-TFs. In the adult, the reactivation of the EMT program contributes to the progression of diseases like fibrosis and cancer (Nieto et al. Cell, 2016). Prrx1, identified as a novel EMT-TF in our lab, produces three different splice isoforms, whose functions remain to be characterized. We have tested the ability of each of these isoforms to induce EMT in vitro, and only the longest isoform (Prrx1L) is able to do so. To test the role of the different isoforms in vivo, we have generated isoform-specific mutant mouse lines that we are characterizing using the phenotype of the Prrx1 null mice in the skeleton and the vasculature (J.F. Martin et al. Genes & Development, 1995, K. Ihida-Stansbury et al. Circulation research, 2004). Using the postnatal retina as a model to study vasculogenesis, we have observed that Prrx1 is specifically expressed in mural cells. Consistent with this expression pattern, we have seen that the Prrx1 isoforms are essential for the integrity of the vasculature in the P6 retina and adult lungs. We are using the in vivo matrigel plug assay to dissect the function of Prrx1 isoforms in mural cells and their interaction with endothelial cells.Peer reviewe
Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Ali... more Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019.También presentado al 16th Christmas Meeting del Instituto de Neurociencias, celebrado en Alicante del 19 al 20 de diciembre de 2019.Gene loss-of-function (LOF) studies are fundamental to address their relevance in a particular biological process. Abundant approaches exist to perform LOF depending on the type of experiment and organism being used. Morpholino oligonucleotidesmediated knockdown (MO) technologies have been widely used over 20 years to address the function of developmental genes in vertebrates. New gene editing technologies including the CRISPR/Cas9 system have now become the gold standard to test gene function. We have previously shown that a Prrx1a-dependent L/R asymmetric EMT induces differential L/R forces leading to the leftward displacement of the posterior pole of the heart, driving its normal left-handed position. This analysis was performed by MO-mediated knockdown and as previously published prrx1a mutant alleles in zebrafish do not present defects in heart laterality, we decided to check whether G0 CRISPR-generated prrx1a mutants were able to recapitulate the heart phenotypes of prrx1a morphants. We found that both MO-mediated knock down and CRISPR-mediated gene editing of prrx1a results in an efficient Prrx1a protein loss, while the previously published mutant alleles are likely not null. Consistent with this, prrx1a crispant embryos confirm the mesocardia phenotype previously found in the morphants as well as the observed reduction in atrial size. However, morphants show early defects in heart development including defects in spaw expression leading to heart jogging randomization, which is not observed in the crispants. These data indicate that the early phenotypes are prrx1a-independent, that heart laterality is instructed after jogging and confirm the role of Prrx1a in the process.Peer reviewe
Ectodermal dysplasia syndromes affect the development of several organs, including hair, teeth, a... more Ectodermal dysplasia syndromes affect the development of several organs, including hair, teeth, and glands. The recent cloning of two genes responsible for these syndromes has led to the identification of a novel TNF family ligand, ectodysplasin, and TNF receptor, edar. This has indicated a developmental regulatory role for TNFs for the first time. Our in situ hybridization analysis of the expression of ectodysplasin (encoded by the Tabby gene) and edar (encoded by the downless gene) during mouse tooth morphogenesis showed that they are expressed in complementary patterns exclusively in ectodermal tissue layer. Edar was expressed reiteratively in signaling centers regulating key steps in morphogenesis. The analysis of the effects of eight signaling molecules in the TGF, FGF, Hh, Wnt, and EGF families in tooth explant cultures revealed that the expression of edar was induced by activinA, whereas Wnt6 induced ectodysplasin expression. Moreover, ectodysplasin expression was downregulated in branchial arch epithelium and in tooth germs of Lef1 mutant mice, suggesting that signaling by ectodysplasin is regulated by LEF-1-mediated Wnt signals. The analysis of the signaling centers in tooth germs of Tabby mice (ectodysplasin null mutants) indicated that in the absence of ectodysplasin the signaling centers were small. However, no downstream targets of ectodysplasin signaling were identified among several genes expressed in the signaling centers. We conclude that ectodysplasin functions as a planar signal between ectodermal compartments and regulates the function, but not the induction, of epithelial signaling centers. This TNF signaling is tightly associated with epithelial-mesenchymal interactions and with other signaling pathways regulating organogenesis. We suggest that activin signaling from mesenchyme induces the expression of the TNF receptor edar in the epithelial signaling centers, thus making them responsive to Wnt-induced ectodysplasin from the nearby ectoderm. This is the first demonstration of integration of the Wnt, activin, and TNF signaling pathways.
The cholinergic enzyme acetylcholinesterase (AChE) and the catalytic component of the γ-secretase... more The cholinergic enzyme acetylcholinesterase (AChE) and the catalytic component of the γ-secretase complex, presenilin-1 (PS1), are known to interact. In this study, we investigate the consequences of AChE-PS1 interactions, particularly the influence of AChE in PS1 levels and γ-secretase activity. PS1 is able to co-immunoprecipitate all AChE variants (AChE-R and AChE-T) and molecular forms (tetramers and light subunits) present in the human brain. Over-expression of AChE-R or AChE-T, or their respective inactive mutants, all trigger an increase in PS1 protein levels. The AChE specie capable of triggering the biggest increase in PS1 levels is a complex of AChE with the membrane anchoring subunit proline-rich membrane anchor (PRiMA), which restricts the localization of the resulting AChE tetramer to the outer plasma membrane. Incubation of cultured cells with soluble AChE demonstrates that AChE is able to increase PS1 at both the protein and transcript levels. However, the increase of PS1 caused by soluble AChE is accompanied by a decrease in γ-secretase activity as shown by the reduction of the processing of the β-amyloid precursor protein. This inhibitory effect of AChE on γ-secretase activity was also demonstrated by directly assessing accumulation of CTF-APP in cell-free membrane preparations incubated with AChE. Our data suggest that AChE may function as an inhibitor of γ-secretase activity.
Developing axons must control their growth rate to follow the appropriate pathways and establish ... more Developing axons must control their growth rate to follow the appropriate pathways and establish specific connections. However, the regulatory mechanisms involved remain elusive. By combining live imaging with transplantation studies in mice, we found that spontaneous calcium activity in the thalamocortical system and the growth rate of thalamocortical axons were developmentally and intrinsically regulated. Indeed, the spontaneous activity of thalamic neurons governed axon growth and extension through the cortex in vivo. This activity-dependent modulation of growth was mediated by transcriptional regulation of Robo1 through an NF-B binding site. Disruption of either the Robo1 or Slit1 genes accelerated the progression of thalamocortical axons in vivo, and interfering with Robo1 signaling restored normal axon growth in electrically silent neurons. Thus, modifications to spontaneous calcium activity encode a switch in the axon outgrowth program that allows the establishment of specific neuronal connections through the transcriptional regulation of Slit1 and Robo1 signaling.
Targeted inactivation of the murine gene encoding the transcription factor LEF-1 abrogates the fo... more Targeted inactivation of the murine gene encoding the transcription factor LEF-1 abrogates the formation of organs that depend on epithelial-mesenchymal tissue interactions. In this study we have recombined epithelial and mesenchymal tissues from normal and LEF-1-deficient embryos at different stages of development to define the LEF-1-dependent steps in tooth and whisker organogenesis. At the initiation of organ development, formation of the epithelial primordium of the whisker but not tooth is dependent on mesenchymal Left gene expression. Subsequent formation of a whisker and tooth mesenchymal papilla and completion of organogenesis require transient expression of Lefl in the epithelium. These experiments indicate that the effect of Lefl expression is transmitted from one tissue to the other. In addition, the finding that the expression of Lefl can be activated by bone morphogenetic protein 4 (BMP-4) suggests a regulatory role of this transcription factor in BMP-mediated inductive tissue interactions.
Classical studies of cholinesterase activity during liver dysfunction have focused on butyrylchol... more Classical studies of cholinesterase activity during liver dysfunction have focused on butyrylcholinesterase (BuChE), whereas acetylcholinesterase (AChE) has not received much attention. In the current study, liver and plasma AChE levels were investigated in rats with cirrhosis induced after 3 weeks of bile duct ligation (BDL). BDL rats showed a pronounced decrease in liver AChE levels (ϳ50%) compared with sham-operated (non-ligated, NL) controls; whereas liver BuChE appeared unaffected. A selective loss of tetrameric (G 4) AChE was detected in BDL rats, an effect also observed in rats with carbon tetrachloride-induced cirrhosis. In accordance, SDS-PAGE analysis showed that the major 55-kd immunoreactive AChE band was decreased in BDL as compared with NL. A 65-kd band, attributed in part to inactive AChE, was increased as became the most abundant AChE subunit in BDL liver. The overall decrease in AChE activity in BDL liver was not accompanied by a reduction of AChE transcripts. The loss of G 4 was also reflected by changes observed in AChE glycosylation pattern attributable to different liver AChE forms being differentially glycosylated. BDL affects AChE levels in both hepatocytes and Kupffer cells; however, altered AChE expression was mainly reflected in an alteration in hepatocyte AChE pattern. Plasma from BDL rats had approximately 45% lower AChE activity than controls, displaying decreased G 4 levels and altered lectin-binding patterns. In conclusion, the liver is an important source of serum AChE; altered AChE levels may be a useful biomarker for liver cirrhosis.
Trabajo presentado al 17th Meeting of the Spanish Society for Developmental Biology (SEBD), celeb... more Trabajo presentado al 17th Meeting of the Spanish Society for Developmental Biology (SEBD), celebrado de forma virtual del 18 al 20 de noviembre de 2020.Peer reviewe
Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Ali... more Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019.También presentado al 16th Christmas Meeting del Instituto de Neurociencias, celebrado en Alicante del 19 al 20 de diciembre de 2019.The epithelial-mesenchymal transition (EMT) endows cells with migratory and invasive properties and it is crucial for the formation of many tissues and organs during embryonic development. This cellular program is triggered after the activation of transcription factors, referred to as EMT-TFs. In the adult, the reactivation of the EMT program contributes to the progression of diseases like fibrosis and cancer (Nieto et al. Cell, 2016). Prrx1, identified as a novel EMT-TF in our lab, produces three different splice isoforms, whose functions remain to be characterized. We have tested the ability of each of these isoforms to induce EMT in vitro, and only the longest isoform (Prrx1L) is able to do so. To test the role of the different isoforms in vivo, we have generated isoform-specific mutant mouse lines that we are characterizing using the phenotype of the Prrx1 null mice in the skeleton and the vasculature (J.F. Martin et al. Genes & Development, 1995, K. Ihida-Stansbury et al. Circulation research, 2004). Using the postnatal retina as a model to study vasculogenesis, we have observed that Prrx1 is specifically expressed in mural cells. Consistent with this expression pattern, we have seen that the Prrx1 isoforms are essential for the integrity of the vasculature in the P6 retina and adult lungs. We are using the in vivo matrigel plug assay to dissect the function of Prrx1 isoforms in mural cells and their interaction with endothelial cells.Peer reviewe
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