RESUMEN
Bop1 can promote cell proliferation and is a component of the Pes1-Bop1-WDR12 (PeBoW) complex that regulates ribosomal RNA processing and biogenesis. In embryos, however, bop1 mRNA is highly enriched in the neural plate, cranial neural crest and placodes, and potentially may interact with Six1, which also is expressed in these tissues. Recent work demonstrated that during development, Bop1 is required for establishing the size of the tadpole brain, retina and cranial cartilages, as well as controlling neural tissue gene expression levels. Herein, we extend this work by assessing the effects of Bop1 knockdown at neural plate and larval stages. Loss of Bop1 expanded neural plate gene expression domains (sox2, sox11, irx1) and reduced neural crest (foxd3, sox9), placode (six1, sox11, irx1, sox9) and epidermal (dlx5) expression domains. At larval stages, Bop1 knockdown reduced the expression of several otic vesicle genes (six1, pax2, irx1, sox9, dlx5, otx2, tbx1) and branchial arch genes that are required for chondrogenesis (sox9, tbx1, dlx5). The latter was not the result of impaired neural crest migration. Together these observations indicate that Bop1 is a multifunctional protein that in addition to its well-known role in ribosomal biogenesis functions during early development to establish the craniofacial precursor domains.
Asunto(s)
Cresta Neural , Factores de Transcripción , Cresta Neural/metabolismo , Factores de Transcripción/metabolismo , Cabeza , Cráneo/metabolismo , Ribosomas/metabolismo , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Branchio-oto-renal syndrome (BOR) is a disorder characterized by hearing loss, and craniofacial and/or renal defects. Variants in the transcription factor Six1 and its co-factor Eya1, both of which are required for otic development, are linked to BOR. We previously identified Sobp as a potential Six1 co-factor, and SOBP variants in mouse and humans cause otic phenotypes; therefore, we asked whether Sobp interacts with Six1 and thereby may contribute to BOR. Co-immunoprecipitation and immunofluorescence experiments demonstrate that Sobp binds to and colocalizes with Six1 in the cell nucleus. Luciferase assays show that Sobp interferes with the transcriptional activation of Six1+Eya1 target genes. Experiments in Xenopus embryos that either knock down or increase expression of Sobp show that it is required for formation of ectodermal domains at neural plate stages. In addition, altering Sobp levels disrupts otic vesicle development and causes craniofacial cartilage defects. Expression of Xenopus Sobp containing the human variant disrupts the pre-placodal ectoderm similar to full-length Sobp, but other changes are distinct. These results indicate that Sobp modifies Six1 function and is required for vertebrate craniofacial development, and identify Sobp as a potential candidate gene for BOR.
Asunto(s)
Desarrollo Óseo , Proteínas de Homeodominio/metabolismo , Metaloproteínas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Síndrome Branquio Oto Renal/embriología , Síndrome Branquio Oto Renal/genética , Núcleo Celular/metabolismo , Oído Interno/embriología , Oído Interno/metabolismo , Ectodermo/embriología , Ectodermo/metabolismo , Expresión Génica , Proteínas de Homeodominio/genética , Larva/crecimiento & desarrollo , Metaloproteínas/genética , Cresta Neural/embriología , Cresta Neural/metabolismo , Proteínas Nucleares/genética , Unión Proteica , Proteínas Tirosina Fosfatasas/metabolismo , Activación Transcripcional , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
BACKGROUND: Members of the sulfotransferase superfamily (SULT) influence the activity of a wide range of hormones, neurotransmitters, metabolites and xenobiotics. However, their roles in developmental processes are not well characterized even though they are expressed during embryogenesis. We previously found in a microarray screen that Six1 up-regulates LOC100037047, which encodes XB5850668.L, an uncharacterized sulfotransferase. RESULTS: Since Six1 is required for patterning the embryonic ectoderm into its neural plate, neural crest, preplacodal and epidermal domains, we used loss- and gain-of function assays to characterize the role of XB5850668.L during this process. Knockdown of endogenous XB5850668.L resulted in the reduction of epidermal, neural crest, cranial placode and otic vesicle gene expression domains, concomitant with neural plate expansion. Increased levels had minimal effects, but infrequently expanded neural plate and neural crest gene domains, and infrequently reduced cranial placode and otic vesicle gene domains. Mutation of two key amino acids in the sulfotransferase catalytic domain required for PAPS binding and enzymatic activity tended to reduce the effects of overexpressing the wild-type protein. CONCLUSIONS: Our analyses indicates that XB5850668.L is a member of the SULT2 family that plays important roles in patterning the embryonic ectoderm. Some aspects of its influence likely depend on sulfotransferase activity.
Asunto(s)
Ectodermo , Cresta Neural , Cresta Neural/metabolismo , Cráneo/metabolismo , Desarrollo Embrionario/genética , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Mcrs1 is a multifunctional protein that is critical for many cellular processes in a wide range of cell types. Previously, we showed that Mcrs1 binds to the Six1 transcription factor and reduces the ability of the Six1-Eya1 complex to upregulate transcription, and that Mcrs1 loss-of-function leads to the expansion of several neural plate genes, reduction of neural border and pre-placodal ectoderm (PPR) genes, and pleiotropic effects on various neural crest (NC) genes. Because the affected embryonic structures give rise to several of the cranial tissues affected in Branchio-otic/Branchio-oto-renal (BOR) syndrome, herein we tested whether these gene expression changes subsequently alter the development of the proximate precursors of BOR affected structures - the otic vesicles (OV) and branchial arches (BA). We found that Mcrs1 is required for the expression of several OV genes involved in inner ear formation, patterning and otic capsule cartilage formation. Mcrs1 knockdown also reduced the expression domains of many genes expressed in the larval BA, derived from either NC or PPR, except for emx2, which was expanded. Reduced Mcrs1 also diminished the length of the expression domain of tbx1 in BA1 and BA2 and interfered with cranial NC migration from the dorsal neural tube; this subsequently resulted in defects in the morphology of lower jaw cartilages derived from BA1 and BA2, including the infrarostral, Meckel's, and ceratohyal as well as the otic capsule. These results demonstrate that Mcrs1 plays an important role in processes that lead to the formation of craniofacial cartilages and its loss results in phenotypes consistent with reduced Six1 activity associated with BOR.
Asunto(s)
Región Branquial , Síndrome Branquio Oto Renal , Región Branquial/metabolismo , Síndrome Branquio Oto Renal/genética , Síndrome Branquio Oto Renal/metabolismo , Cartílago/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Cresta Neural , Placa Neural/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
The Six1 transcription factor plays a major role in craniofacial development. Mutations in SIX1 and its co-factor, EYA1, are causative for about 50% of Branchio-otic/Branchio-oto-renal syndrome (BOR) patients, who are characterized by variable craniofacial, otic and renal malformations. We previously screened for other proteins that might interact with Six1 to identify additional genes that may play a role in BOR, and herein characterize the developmental role of one of them, Microspherule protein 1 (Mcrs1). We found that in cultured cells, Mcrs1 bound to Six1 and in both cultured cells and embryonic ectoderm reduced Six1-Eya1 transcriptional activation. Knock-down of Mcrs1 in embryos caused an expansion of the domains of neural plate genes and two genes expressed in both the neural plate and neural crest (zic1, zic2). In contrast, two other genes expressed in pre-migratory neural crest (foxd3, sox9) were primarily reduced. Cranial placode genes showed a mixture of expanded and diminished expression domains. At larval stages, loss of Mcrs1 resulted in a significant reduction of otic vesicle gene expression concomitant with a smaller otic vesicle volume. Experimentally increasing Mcrs1 above endogenous levels favored the expansion of neural border and neural crest gene domains over cranial placode genes; it also reduced otic vesicle gene expression but not otic vesicle volume. Co-expression of Mcrs1 and Six1 as well as double knock-down and rescue experiments establish a functional interaction between Mcrs1 and Six1 in the embryo, and demonstrate that this interaction has an important role in the development of craniofacial tissues including the otic vesicle.
Asunto(s)
Embrión no Mamífero/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Proteínas de Unión al ARN/biosíntesis , Cráneo/embriología , Proteínas de Xenopus/biosíntesis , Animales , Ectodermo/embriología , Cresta Neural/embriología , Xenopus laevisRESUMEN
The specialized sensory organs of the vertebrate head are derived from thickened patches of cells in the ectoderm called cranial sensory placodes. The developmental program that generates these placodes and the genes that are expressed during the process have been studied extensively in a number of animals, yet very little is known about how these genes regulate one another. We previously found via a microarray screen that Six1, a known transcriptional regulator of cranial placode fate, up-regulates Irx1 in ectodermal explants. In this study, we investigated the transcriptional relationship between Six1 and Irx1 and found that they reciprocally regulate each other throughout cranial placode and otic vesicle formation. Although Irx1 expression precedes that of Six1 in the neural border zone, its continued and appropriately patterned expression in the pre-placodal region (PPR) and otic vesicle requires Six1. At early PPR stages, Six1 expands the Irx1 domain, but this activity subsides over time and changes to a predominantly repressive effect. Likewise, Irx1 initially expands Six1 expression in the PPR, but later represses it. We also found that Irx1 and Sox11, a known direct target of Six1, reciprocally affect each other. This work demonstrates that the interactions between Six1 and Irx1 are continuous during PPR and placode development and their transcriptional effects on one another change over developmental time.
Asunto(s)
Oído Interno/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Placa Neural/metabolismo , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Animales , Oído Interno/citología , Oído Interno/embriología , Ectodermo/citología , Ectodermo/embriología , Ectodermo/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Cabeza/embriología , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Proteínas del Tejido Nervioso/metabolismo , Placa Neural/citología , Placa Neural/embriología , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Mutations in SIX1 and in its co-factor, EYA1, underlie Branchiootorenal Spectrum disorder (BOS), which is characterized by variable branchial arch, otic and kidney malformations. However, mutations in these two genes are identified in only half of patients. We screened for other potential co-factors, and herein characterize one of them, Pa2G4 (aka Ebp1/Plfap). In human embryonic kidney cells, Pa2G4 binds to Six1 and interferes with the Six1-Eya1 complex. In Xenopus embryos, knock-down of Pa2G4 leads to down-regulation of neural border zone, neural crest and cranial placode genes, and concomitant expansion of neural plate genes. Gain-of-function leads to a broader neural border zone, expanded neural crest and altered cranial placode domains. In loss-of-function assays, the later developing otocyst is reduced in size, which impacts gene expression. In contrast, the size of the otocyst in gain-of-function assays is not changed but the expression domains of several otocyst genes are reduced. Together these findings establish an interaction between Pa2G4 and Six1, and demonstrate that it has an important role in the development of tissues affected in BOS. Thereby, we suggest that pa2g4 is a potential candidate gene for BOS.
Asunto(s)
Oído Interno/embriología , Oído Interno/metabolismo , Proteínas de Homeodominio/metabolismo , Cresta Neural/embriología , Cresta Neural/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Secuencia de Aminoácidos , Animales , Muerte Celular , Proliferación Celular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Cara/embriología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Placa Neural/embriología , Placa Neural/metabolismo , Unión Proteica , Dominios Proteicos , Cráneo/embriología , Cráneo/metabolismo , Transcripción Genética , Xenopus laevis/genéticaRESUMEN
In many animals, maternally synthesized mRNAs are critical for primary germ layer formation. In Xenopus, several maternal mRNAs are enriched in the animal blastomere progenitors of the embryonic ectoderm. We previously identified one of these, WW-domain binding protein 2 N-terminal like (wbp2nl), that others previously characterized as a sperm protein (PAWP) that promotes meiotic resumption. Herein we demonstrate that it has an additional developmental role in regionalizing the embryonic ectoderm. Knock-down of Wbp2nl in the dorsal ectoderm reduced cranial placode and neural crest gene expression domains and expanded neural plate domains; knock-down in ventral ectoderm reduced epidermal gene expression. Conversely, increasing levels of Wbp2nl in the neural plate induced ectopic epidermal and neural crest gene expression and repressed many neural plate and cranial placode genes. The effects in the neural plate appear to be mediated, at least in part, by down-regulating chd, a BMP antagonist. Because the cellular function of Wbp2nl is not known, we mutated several predicted motifs. Expressing mutated proteins in embryos showed that a putative phosphorylation site at Thr45 and an α-helix in the PH-G domain are required to ectopically induce epidermal and neural crest genes in the neural plate. An intact YAP-binding motif also is required for ectopic epidermal gene expression as well as for down-regulating chd. This work reveals novel developmental roles for a cytoplasmic protein that promotes epidermal and neural crest formation at the expense of neural ectoderm.
Asunto(s)
Proteínas Portadoras/metabolismo , Ectodermo/embriología , Ectodermo/metabolismo , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Proteínas de Plasma Seminal/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Unión al ADN , Epidermis/embriología , Epidermis/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Mesodermo/metabolismo , Mutación/genética , Cresta Neural/embriología , Cresta Neural/metabolismo , Placa Neural/embriología , Placa Neural/metabolismo , Fenotipo , Dominios Proteicos , Transporte de Proteínas , Proteínas de Plasma Seminal/química , Proteínas de Plasma Seminal/genética , Alineación de Secuencia , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn, and Sox11 each induced explants made from ventral, epidermis-producing blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activities are required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing blastomeres significantly increased the expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage blastomeres to a neural fate. We demonstrate that mouse and human homologs of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and blastomere explants. genesis 54:334-349, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Diferenciación Celular/genética , Ectodermo/crecimiento & desarrollo , Factores de Transcripción Forkhead/genética , Placa Neural/crecimiento & desarrollo , Animales , Blastómeros/metabolismo , Blástula/crecimiento & desarrollo , Ectodermo/metabolismo , Factores de Transcripción Forkhead/biosíntesis , Gástrula/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Placa Neural/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Proteínas de Xenopus/biosíntesis , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrollo , Cigoto/crecimiento & desarrolloRESUMEN
BACKGROUND: Six1 plays an important role in the development of several vertebrate organs, including cranial sensory placodes, somites, and kidney. Although Six1 mutations cause one form of branchio-otic syndrome (BOS), the responsible gene in many patients has not been identified; genes that act downstream of Six1 are potential BOS candidates. RESULTS: We sought to identify novel genes expressed during placode, somite and kidney development by comparing gene expression between control and Six1-expressing ectodermal explants. The expression patterns of 19 of the significantly up-regulated and 11 of the significantly down-regulated genes were assayed from cleavage to larval stages. A total of 28/30 genes are expressed in the otocyst, a structure that is functionally disrupted in BOS, and 26/30 genes are expressed in the nephric mesoderm, a structure that is functionally disrupted in the related branchio-otic-renal (BOR) syndrome. We also identified the chick homologues of five genes and show that they have conserved expression patterns. CONCLUSIONS: Of the 30 genes selected for expression analyses, all are expressed at many of the developmental times and appropriate tissues to be regulated by Six1. Many have the potential to play a role in the disruption of hearing and kidney function seen in BOS/BOR patients.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Riñón/embriología , Cráneo/embriología , Somitos/embriología , Proteínas de Xenopus/metabolismo , Animales , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Riñón/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Cráneo/citología , Somitos/citología , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
FoxD4/5, a forkhead transcription factor, plays a critical role in establishing and maintaining the embryonic neural ectoderm. It both up-regulates genes that maintain a proliferative, immature neural ectoderm and down-regulates genes that promote the transition to a differentiating neural plate. We constructed deletion and mutant versions of FoxD4/5 to determine which domains are functionally responsible for these opposite activities, which regulate the critical developmental transition of neural precursors to neural progenitors to differentiating neural plate cells. Our results show that up-regulation of genes that maintain immature neural precursors (gem, zic2) requires the Acidic blob (AB) region in the N-terminal portion of the protein, indicating that the AB is the transactivating domain. Additionally, down-regulation of those genes that promote the transition to neural progenitors (sox) and those that lead to neural differentiation (zic, irx) involves: 1) an interaction with the Groucho co-repressor at the Eh-1 motif in the C-terminus; and 2) sequence downstream of this motif. Finally, the ability of FoxD4/5 to induce the ectopic expression of neural precursor genes in the ventral ectoderm also involves both the AB region and the Eh-1 motif; FoxD4/5 accomplishes ectopic neural induction by both activating neural precursor genes and repressing BMP signaling and epidermal genes. This study identifies the specific, conserved domains of the FoxD4/5 protein that allow this single transcription factor to regulate a network of genes that controls the transition of a proliferative neural ectodermal population to a committed neural plate population poised to begin differentiation.
Asunto(s)
Ectodermo/embriología , Factores de Transcripción Forkhead/química , Regulación del Desarrollo de la Expresión Génica , Placa Neural/embriología , Activación Transcripcional , Proteínas de Xenopus/química , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Ectodermo/citología , Ectodermo/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Placa Neural/citología , Placa Neural/metabolismo , Estructura Terciaria de Proteína , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
Introduction: The Six1 transcription factor plays important roles in the development of cranial sensory organs, and point mutations underlie craniofacial birth defects. Because Six1's transcriptional activity can be modulated by interacting proteins, we previously screened for candidate interactors and identified zinc-finger MYM-containing protein 4 (Zmym4) by its inclusion of a few domains with a bona fide cofactor, Sine oculis binding protein (Sobp). Although Zmym4 has been implicated in regulating early brain development and certain cancers, its role in craniofacial development has not previously been described. Methods: We used co-immunoprecipitation and luciferase-reporter assays in cultured cells to test interactions between Zmym4 and Six1. We used knock-down and overexpression of Zmym4 in embryos to test for its effects on early ectodermal gene expression, neural crest migration and craniofacial cartilage formation. Results: We found no evidence that Zmym4 physically or transcriptionally interacts with Six1 in cultured cells. Nonetheless, knockdown of endogenous Zmym4 in embryos resulted in altered early cranial gene expression, including those expressed in the neural border, neural plate, neural crest and preplacodal ectoderm. Experimentally increasing Zmym4 levels had minor effects on neural border or neural plate genes, but altered the expression of neural crest and preplacodal genes. At larval stages, genes expressed in the otic vesicle and branchial arches showed reduced expression in Zmym4 morphants. Although we did not detect defects in neural crest migration into the branchial arches, loss of Zmym4 resulted in aberrant morphology of several craniofacial cartilages. Discussion: Although Zmym4 does not appear to function as a Six1 transcriptional cofactor, it plays an important role in regulating the expression of embryonic cranial genes in tissues critical for normal craniofacial development.
RESUMEN
FoxD4L1/D5 is a forkhead transcription factor that functions as both a transcriptional activator and repressor. FoxD4L1/D5 acts upstream of several other neural transcription factors to maintain neural fate, regulate neural plate patterning, and delay the expression of neural differentiation factors. To identify a more complete list of downstream genes that participate in these earliest steps of neural ectodermal development, we carried out a microarray analysis comparing gene expression in control animal cap ectodermal explants (ACs), which will form epidermis, to that in FoxD4L1/D5-expressing ACs. Forty-four genes were tested for validation by RT-PCR of ACs and/or in situ hybridization assays in embryos; 86% of those genes up-regulated and 100% of those genes down-regulated in the microarray were altered accordingly in one of these independent assays. Eleven of these 44 genes are of unknown function, and we provide herein their developmental expression patterns to begin to reveal their roles in ectodermal development.
Asunto(s)
Ectodermo/embriología , Redes Reguladoras de Genes , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Hibridación in Situ , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Xenopus/genéticaRESUMEN
Six family transcription factors play important roles in craniofacial development. Their transcriptional activity can be modified by cofactor proteins. Two Six genes and one cofactor gene (Eya1) are involved in the human Branchio-otic (BO) and Branchio-otic-renal (BOR) syndromes. However, mutations in Six and Eya genes only account for approximately half of these patients. To discover potential new causative genes, we searched the Xenopus genome for orthologues of Drosophila cofactor proteins that interact with the fly Six-related factor, SO. We identified 33 Xenopus genes with high sequence identity to 20 of the 25 fly SO-interacting proteins. We provide the developmental expression patterns of the Xenopus orthologues for 11 of the fly genes, and demonstrate that all are expressed in developing craniofacial tissues with at least partial overlap with Six1/Six2. We speculate that these genes may function as Six-interacting partners with important roles in vertebrate craniofacial development and perhaps congenital syndromes.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Biología Computacional , Embrión no Mamífero/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/embriología , Factores de Transcripción/genética , Proteínas de Xenopus/genéticaRESUMEN
foxD5 is expressed in the nascent neural ectoderm concomitant with several other neural-fate specifying transcription factors. We used loss-of-function and gain-of-function approaches to analyze the functional position of foxD5 amongst these other factors. Loss of FoxD5 reduces the expression of sox2, sox11, soxD, zic1, zic3 and Xiro1-3 at the onset of gastrulation, and of geminin, sox3 and zic2, which are maternally expressed, by late gastrulation. At neural plate stages most of these genes remain reduced, but the domains of zic1 and zic3 are expanded. Increased FoxD5 induces geminin and zic2, weakly represses sox11 at early gastrula but later (st12) induces it; weakly represses sox2 and sox3 transiently and strongly represses soxD, zic1, zic3 and Xiro1-3. The foxD5 effects on zic1, zic3 and Xiro1-3 involve transcriptional repression, whereas those on geminin and zic2 involve transcriptional activation. foxD5's effects on geminin, sox11 and zic2 occur at the onset of gastrulation, whereas the other genes require earlier foxD5 activity. geminin, sox11 and zic2, each of which is up-regulated directly by foxD5, are all required to account for foxD5 phenotypes, indicating that this triad constitutes a transcriptional network rather than linear path that coordinately up-regulates genes that promote an immature neural fate and inhibits genes that promote the onset of neural differentiation. We also show that foxD5 promotes an ectopic neural fate in the epidermis by reducing BMP signaling. Several of the genes that are repressed by foxD5 in turn reduce foxD5 expression, contributing to the medial-lateral patterning of the neural plate.
Asunto(s)
Ectodermo/metabolismo , Factores de Transcripción Forkhead/metabolismo , Sistema Nervioso/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriología , Xenopus/metabolismo , Animales , Diferenciación Celular/genética , Ectodermo/embriología , Embrión no Mamífero , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Modelos Neurológicos , Sistema Nervioso/embriología , Proteínas de Xenopus/genéticaRESUMEN
Single-nucleotide mutations in human SIX1 result in amino acid substitutions in either the protein-protein interaction domain or the homeodomain, and cause â¼4% of branchio-otic (BOS) and branchio-oto-renal (BOR) cases. The phenotypic variation between patients with the same mutation, even within affected members of the same family, make it difficult to functionally distinguish between the different SIX1 mutations. We made four of the BOS/BOR substitutions in the Xenopus Six1 protein (V17E, R110W, W122R, Y129C), which is 100% identical to human in both the protein-protein interaction domain and the homeodomain, and expressed them in embryos to determine whether they cause differential changes in early craniofacial gene expression, otic gene expression or otic morphology. We confirmed that, similar to the human mutants, all four mutant Xenopus Six1 proteins access the nucleus but are transcriptionally deficient. Analysis of craniofacial gene expression showed that each mutant causes specific, often different and highly variable disruptions in the size of the domains of neural border zone, neural crest and pre-placodal ectoderm genes. Each mutant also had differential effects on genes that pattern the otic vesicle. Assessment of the tadpole inner ear demonstrated that while the auditory and vestibular structures formed, the volume of the otic cartilaginous capsule, otoliths, lumen and a subset of the hair cell-containing sensory patches were reduced. This detailed description of the effects of BOS/BOR-associated SIX1 mutations in the embryo indicates that each causes subtle changes in gene expression in the embryonic ectoderm and otocyst, leading to inner ear morphological anomalies.
Asunto(s)
Síndrome Branquio Oto Renal/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Mutación/genética , Cráneo/embriología , Secuencia de Aminoácidos , Animales , Oído , Células HEK293 , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Humanos , Cresta Neural/metabolismo , Membrana Otolítica/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Transcripción Genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genéticaRESUMEN
Congenital hearing loss is an important clinical problem because, without early intervention, affected children do not properly acquire language and consequently have difficulties developing social skills. Although most newborns in the US are screened for hearing deficits, even earlier diagnosis can be made with prenatal genetic screening. Genetic screening that identifies the relevant mutated gene can also warn about potential congenital defects in organs not related to hearing. We will discuss efforts to identify new candidate genes that underlie the Branchiootorenal spectrum disorders in which affected children have hearing deficits and are also at risk for kidney defects. Mutations in two genes, SIX1 and EYA1, have been identified in about half of the patients tested. To uncover new candidate genes, we have used the aquatic animal model, Xenopus laevis, to identify genes that are part of the developmental genetic pathway of Six1 during otic and kidney development. We have already identified a large number of potential Six1 transcriptional targets and candidate co-factor proteins that are expressed at the right time and in the correct tissues to interact with Six1 during development. We discuss the advantages of using this system for gene discovery in a human congenital hearing loss syndrome.
Asunto(s)
Síndrome Branquio Oto Renal/genética , Xenopus/genética , Animales , Humanos , Riñón/embriología , Riñón/fisiología , Transcripción Genética/genéticaRESUMEN
FoxD4L1 is a forkhead transcription factor that expands the neural ectoderm by down-regulating genes that promote the onset of neural differentiation and up-regulating genes that maintain proliferative neural precursors in an immature state. We previously demonstrated that binding of Grg4 to an Eh-1 motif enhances the ability of FoxD4L1 to down-regulate target neural genes but does not account for all of its repressive activity. Herein we analyzed the protein sequence for additional interaction motifs and secondary structure. Eight conserved motifs were identified in the C-terminal region of fish and frog proteins. Extending the analysis to mammals identified a high scoring motif downstream of the Eh-1 domain that contains a tryptophan residue implicated in protein-protein interactions. In addition, secondary structure prediction programs predicted an α-helical structure overlapping with amphibian-specific Motif 6 in Xenopus, and similarly located α-helical structures in other vertebrate FoxD proteins. We tested functionality of this site by inducing a glutamine-to-proline substitution expected to break the predicted α-helical structure; this significantly reduced FoxD4L1's ability to repress zic3 and irx1. Because this mutation does not interfere with Grg4 binding, these results demonstrate that at least two regions, the Eh-1 motif and a more C-terminal predicted α-helical/Motif 6 site, additively contribute to repression. In the N-terminal region we previously identified a 14 amino acid motif that is required for the up-regulation of target genes. Secondary structure prediction programs predicted a short ß-strand separating two acidic domains. Mutant constructs show that the ß-strand itself is not required for transcriptional activation. Instead, activation depends upon a glycine residue that is predicted to provide sufficient flexibility to bring the two acidic domains into close proximity. These results identify conserved predicted motifs with secondary structures that enable FoxD4L1 to carry out its essential functions as both a transcriptional repressor and activator of neural genes.
Asunto(s)
Biología Computacional , Secuencia Conservada , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/metabolismo , Silenciador del Gen , Activación Transcripcional , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Concentración de Iones de Hidrógeno , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismoRESUMEN
We investigated the role of the Notch signaling pathway in regulating several transcription factors that stabilize a neural fate and expand the neural plate. Increased Notch signaling in a neural lineage via a constitutively activated form (NICD) up-regulated geminin and zic2 in a cell-autonomous manner, and expanded the neural plate domains of sox11, sox2, and sox3. Loss- and gain-of-function assays show that foxD5 acts upstream of notch1 gene expression. Decreasing Notch signaling with an anti-morphic form of a Notch ligand (X-Delta-1(STU)) showed that the foxD5-mediated expansion of the sox gene neural plate domains requires Notch signaling. However, geminin and zic2 appear to be dually regulated by foxD5 and Notch1 signaling. These studies demonstrate that: (1) Notch signaling acts downstream of foxD5 to promote the expression of a subset of neural ectodermal transcription factors; and (2) Notch signaling and the foxD5 transcriptional pathway together maintain the neural plate in an undifferentiated state. Developmental Dynamics 238:1358-1365, 2009. (c) 2009 Wiley-Liss, Inc.