RESUMEN
A segment of 1,022 base pairs (bp) of the 5'-flanking region of the human albumin gene, fused to a reporter gene, directs hepatoma-specific transcription. Three functionally distinct regions have been defined by deletion analysis: (i) a negative element located between bp -673 and -486, (ii) an enhancer essential for efficient albumin transcription located between bp -486 and -221, and (iii) a promoter spanning a region highly conserved throughout evolution. Protein-binding studies have demonstrated that a liver trans-acting factor which interacts with the enhancer region is the well-characterized transcription factor LF-B1, which binds to promoters of several liver-specific genes. A synthetic oligodeoxynucleotide containing the LF-B1-binding site is sufficient to act as a tissue-specific transcriptional enhancer when placed in front of the albumin promoter. The fact that the same binding site functions in both an enhancer and a promoter suggests that these two elements influence the initiation of transcription through similar mechanisms.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Hígado/fisiología , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Albúmina Sérica/genética , Factores de Transcripción/fisiología , Secuencia de Bases , Línea Celular , Genes , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
In search of genes possibly involved in the regulation of hindbrain segmentation, we have isolated mouse cDNA clones corresponding to putative protein kinase genes by polymerase chain reaction amplification of cDNA from 9.5-day-old embryo hindbrains. In situ hybridization analysis revealed that one of these genes, Sek, was expressed in an alternating segment-restricted pattern in the developing hindbrain. Isolation and analysis of Sek cDNAs covering the entire coding sequence indicated that Sek encoded a putative receptor protein tyrosine kinase, belonging to the Eph family. These data are consistent with a role of the Sek gene product in a signal transduction process involved in pattern formation in the hindbrain.
Asunto(s)
Proteínas Fetales/genética , Regulación Enzimológica de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas Tirosina Quinasas/genética , Receptores de Superficie Celular/genética , Rombencéfalo/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Embrión de Mamíferos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , ARN/genética , ARN/aislamiento & purificación , Receptor EphA4 , Receptor EphA8 , Rombencéfalo/embriología , Homología de Secuencia de AminoácidoRESUMEN
alpha-Fetoprotein and albumin, two members of a multigene family, reversibly bind fatty acids with high affinity. The origin of alpha-fetoprotein (AFP) and albumin present in fetal tissues other than the liver and yolk sac is a subject of controversy. In this work, we have searched for the presence of the albumin and AFP mRNA molecules in different fetal organs of the baboon (Papio cinocephalus), using a highly sensitive gel-blot hybridization assay with human albumin and AFP cDNA probes. Large amounts of albumin and AFP mRNA molecules were found in the fetal liver; significant quantities were also present in the gastrointestinal tract and in the kidney. No detectable levels were found in the other tissues examined (brain, skin, spleen, pancreas, muscle, heart, thymus, placenta, and amnion). After injection of radiolabeled AFP into pregnant baboons, all fetal tissues took up the protein. White adipose tissue, kidney, intestine, lung, liver, and cerebral cortex showed a great uptake of exogenous AFP. [14C]Docosahexaenoic acid (22:6, n-3), injected at the same time, was actively transferred from the maternal compartment across the placenta and incorporated into cellular lipids by all fetal tissues and particularly by liver (around 70% of total incorporation). The levels of [14C]docosahexaenoic acid per gram of tissue increased in the order: maternal blood less than placenta less than fetal liver, indicating a selective accumulation of this fatty acid by the fetus. These results indicate that intracellular AFP in non-hepatic tissues of the developing baboon is, for the most part, of plasma origin.
Asunto(s)
Albúminas/metabolismo , Feto/metabolismo , ARN Mensajero/metabolismo , alfa-Fetoproteínas/metabolismo , Albúminas/genética , Animales , Sondas de ADN , Ácidos Docosahexaenoicos/metabolismo , Femenino , Hígado/metabolismo , Papio , Embarazo , ARN Mensajero/genética , Distribución Tisular , alfa-Fetoproteínas/genéticaRESUMEN
Administration of glucocorticoid hormones to the newborn rat results in a rapid decrease in the synthesis of alpha-fetoprotein (AFP) by the liver. The molecular basis of this hormonal action was investigated by examining the steady-state levels of AFP mRNA and albumin mRNA sequences in polysomal and total RNA preparations isolated from dexamethasone-treated and control animals. Following dexamethasone treatment the number of polysomal and total mRNA sequences hybridizable to specific (32P) cDNA probes was drastically decreased for AFP while it was unchanged for albumin. These data indicate that glucocorticoids exert a selective action on AFP mRNA levels and suggest that dexamethasone operate at the transcriptional level.
Asunto(s)
Regulación de la Expresión Génica , Glucocorticoides/fisiología , Transcripción Genética , alfa-Fetoproteínas/genética , Animales , Animales Recién Nacidos/metabolismo , Dexametasona/farmacología , Polirribosomas , ARN Mensajero/análisis , Ratas , Ratas EndogámicasRESUMEN
A segment of the human alpha 1-antitrypsin (alpha 1AT) 5'-flanking region comprising nucleotides -137 to -37 from the start of transcription is sufficient to drive liver-specific transcription from the homologous alpha 1AT promoter and from the heterologous SV40 promoter. In this paper we characterize two proteins, LF-A1 and LF-B1, whose ability to bind wild-type and mutant alpha 1AT promoter segments correlates with the ability of these segments to activate transcription in vivo. DNase I protection and methylation interference analysis reveals that LF-A1 recognizes sequences present in the regulatory region of the human alpha 1-antitrypsin, apolipoprotein A1 and haptoglobin-related genes. These sequences share a common 5' TGG/A A/C CC 3' motif. LF-B1 binds to the palindrome 5' TGGTTAAT/ATTCACCA 3' which is present in the human alpha 1-antitrypsin gene between positions -78 and -62 from the start of transcription. LF-B1 also recognizes a related sequence present in the human albumin gene between -66 and -50. These results suggest that LF-A1 and LF-B1 are common positive trans-acting factors which are required for the expression of several genes in the hepatocyte.
Asunto(s)
Regulación de la Expresión Génica , Hígado/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , alfa 1-Antitripsina/genética , Albúminas/genética , Animales , Apolipoproteína A-I , Apolipoproteínas A/genética , Secuencia de Bases , Sitios de Unión , Inducción Enzimática , Genes Virales , Haptoglobinas/genética , Humanos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Homología de Secuencia de Ácido Nucleico , Virus 40 de los Simios/genética , alfa 1-Antitripsina/biosíntesisRESUMEN
Alpha-fetoprotein (AFP) is mainly synthesized by the fetal liver, the yolk sac and, to a much lower extent, by a few non-hepatic fetal tissues (i.e. kidney, pancreas, lung). This property is considered to be lost in mature quiescent cells of the adult. In the present we have studied the expression of AFP mRNA sequences in phytohemagglutinin (PHA)-stimulated normal human T-lymphocytes and in several human lymphoma cell lines. The amount of mRNA transcripts detected in quiescent T-lymphocytes by dot and Northern blot analysis was very low. It increased rapidly after PHA-activation, reached a maximum at 72 hours (six fold the level observed for quiescent T-lymphocytes) and decreased thereafter. The lymphoma cell lines Daudi, Raji, Rh6 et CEM, all expressed elevated levels of AFP mRNA. The transcripts had the size expected for human AFP, suggesting that they were functional and probably translated into protein. The possible role of AFP synthesis in lymphocyte blastogenesis and in lymphoma growth is discussed in relation with the strong binding affinity of this protein for polyunsaturated fatty acids.
Asunto(s)
Regulación de la Expresión Génica , Activación de Linfocitos , Linfocitos/metabolismo , Fitohemaglutininas/farmacología , ARN Mensajero/genética , Linfocitos T/metabolismo , alfa-Fetoproteínas/genética , Sondas de ADN , Humanos , Cinética , Linfoma , Hibridación de Ácido Nucleico , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The body mass of male emperor penguins is approximately 38 kg at the beginning of the 4-mo winter fast connected with breeding, and it is an estimated approximately 18 kg in leanest birds at time of spontaneous refeeding. For a 38- to 18-kg range, we investigated the changes in the rate of body mass loss, body composition, and plasma concentrations of uric acid and urea. After the first few days (phase I) a steady state (phase II) was reached in the proportions of the energy derived from proteins and lipids with proteins accounting for a constant 4%, and the remaining 96% being from lipids. The same proportions were maintained until body mass had decreased to 24 kg. Below this value the proportion of energy derived from proteins increased progressively (phase III), being 14 times higher at 18 kg than during phase II. Rate of body mass loss and plasma uric acid and urea concentrations closely reflected the changes in protein utilization: being at a low and steady value throughout phase II and increasing during phase III. Emperor penguins also fast during the spring, but for periods of only 2-3 wk. We found a 2.5 times higher value for rate of body mass loss, uric acid, and urea during spring phase II, suggesting lower effectiveness in protein sparing at that time. It may be attributed to the lower initial lipid reserves of spring birds. Would these findings be generalized to the wide variety of birds and mammals that spontaneously fast under natural conditions?(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aves/fisiología , Ayuno , Metabolismo de los Lípidos , Proteínas/metabolismo , Tejido Adiposo , Animales , Composición Corporal , Metabolismo Energético , Masculino , Valores de Referencia , Factores de Tiempo , Urea/sangre , Ácido Úrico/sangreRESUMEN
The nuclear protein LF-B1 (also referred to as HNF-1) is a transcription activator required for the expression of several liver-specific genes. LF-B1 has been purified to homogeneity from rat liver nuclear extracts. The sequence of the protein has been partially determined and, subsequently, overlapping cDNA clones containing the entire open reading frame of LF-B1 were isolated. The full-length cDNA encodes a 628 amino acid protein and directs the synthesis in vitro of a protein capable of binding DNA with the same specificity as LF-B1. The cDNA was recombined into a vaccinia virus vector and active LF-B1 was obtained from infected HeLa cells. Addition of the vaccinia recombinant protein to rat spleen extracts results in activation of transcription of an LF-B1-dependent promoter. The DNA binding domain of LF-B1 is located in the amino-terminal part of the protein and displays distant structural similarity to the homeobox domain. The distribution of LF-B1 mRNA is restricted to liver, which correlates with the tissue-specific expression of its target genes.
Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Genes Homeobox , Hígado/análisis , Proteínas Nucleares/aislamiento & purificación , Factores de Transcripción/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía de Afinidad , Clonación Molecular , ADN/aislamiento & purificación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidad de Órganos , Ratas , Ratas Endogámicas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres (r) with distinct identities. Recent studies have uncovered regulatory links between transcription factors governing this process, but little is known of how these relate to molecules mediating cell-cell signalling. The Eph receptor tyrosine kinase gene EphA4 (Sek-1) is expressed in r3 and r5, and function-blocking experiments suggest that it is involved in restricting intermingling of cells between odd- and even-numbered rhombomeres. We have analysed the cis-acting regulatory sequences of the EphA4 gene in transgenic mice and identified a 470 bp enhancer element that drives specific expression in r3 and r5. Within this element, we have identified eight binding sites for the Krox-20 transcription factor that is also expressed in r3 and r5. Mutation of these binding sites abolishes r3/r5 enhancer activity and ectopic expression of Krox-20 leads to ectopic activation of the enhancer. These data indicate that Krox-20 is a direct transcriptional activator of EphA4. Together with evidence that Krox-20 regulates Hox gene expression, our findings reveal a mechanism by which the identity and movement of cells are coupled such that sharply restricted segmental domains are generated.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fetales/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Tirosina Quinasas Receptoras/genética , Rombencéfalo/embriología , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Animales , Secuencia de Bases , Sitios de Unión , Tipificación del Cuerpo/genética , Huella de ADN , Proteína 2 de la Respuesta de Crecimiento Precoz , Elementos de Facilitación Genéticos/genética , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Notocorda/química , Mutación Puntual , ARN Mensajero/análisis , Receptor EphA4 , Rombencéfalo/química , SomitosRESUMEN
Embryonic Purkinje cells (PCs) from cerebellar primordia grafted in adult pcd mutant cerebellum replace missing PCs of the host, and become synaptically integrated into the defective cerebellar circuit. This process of neuronal replacement starts with the invasion of grafted PCs into the host cerebellum, and their radial migration through its molecular layer. The present study is aimed at determining whether the glial axes for this migration are embryonic radial glial cells that comigrate with the grafted PCs, or adult Bergmann fibers of the host, transiently reexpressing the molecular cues needed for their guidance of the migration. Transplants from a transgenic mouse line (Krox-20/lacZ14) in which Bergmann fibers could be identified by lacZ expression reveal that, despite the presence of X-gal-stained Bergmann fibers in the graft remnants and of grafted PCs in the host molecular layer, all Bergmann fibers in the host cerebellum lack of beta-galactosidase activity. Thus, these migratory axes belong to the host, not to the donor. Transplants from normal isogenic mouse embryos show that during the radial migration of grafted PCs (7 d after grafting) the involved host Bergmann fibers reexpress nestin (identified with monoclonal antibody Rat-401 immunostaining), normally expressed only by immature Bergmann fibers. Five days later, when grafted PCs have arrested their migration, host Bergmann fibers again become Rat-401 negative. These results indicate that embryonic PCs can trigger in adult cerebellum the molecular changes necessary for their own migration and ultimate synaptic integration in the host cortical circuitry.
Asunto(s)
Cerebelo/embriología , Trasplante de Tejido Fetal , Proteínas del Tejido Nervioso , Células de Purkinje/trasplante , Animales , Anticuerpos Monoclonales , Movimiento Celular/fisiología , Cerebelo/citología , Proteínas de Unión al ADN , Proteína 2 de la Respuesta de Crecimiento Precoz , Expresión Génica , Proteínas de Filamentos Intermediarios/metabolismo , Operón Lac , Ratones , Ratones Mutantes , Ratones Transgénicos , Fibras Nerviosas/metabolismo , Nestina , Células de Purkinje/fisiología , Factores de Transcripción , beta-Galactosidasa/metabolismoRESUMEN
Hybridization of alpha-fetoprotein clones cDNA with human DNAs digested by seven restriction endonucleases reveals one polymorphism. This polymorphism, detected after restriction by MspI, is at low frequency (f = 0.02) and is validated by family analysis. It corresponds to an intronic sequence, for a methylated site external to the probes utilized.
Asunto(s)
Polimorfismo Genético , alfa-Fetoproteínas/genética , Enzimas de Restricción del ADN , Humanos , Hibridación de Ácido NucleicoRESUMEN
Rot analysis of hybridization data using highly labeled alpha-fetoprotein (AFP) and albumin (32P)cDNA probes has been used to quantitate AFP and albumin mRNA sequences in RNA preparations from different subcellular fractions of developing rat liver and Morris hepatoma 7777. In addition, size analysis of these mRNA sequences has been carried out by electrophoretic fractionation on agarose gels containing methylmercury hydroxyde and hybridization to radioactive cloned albumin and AFP cDNA probes. In all the tissues examined (fetal, newborn and adult rat liver, and hepatoma 7777) most of the albumin and AFP mRNA sequences were found associated with the polysomes as mature mRNA molecules; less than 2% of these sequences were present in the nuclear or the non polysomal cytoplasmic compartments. The number of AFP mRNA molecules was found to decrease in parallel in all the cellular compartments during rat liver development. In Morris hepatoma 7777 the content of albumin mRNA was considerably decreased in all the cellular fractions as compared to normal liver. These results demonstrate that post-transcriptional control mechanisms leading to an accumulation of non-functional mRNA molecules are not implicated in the changes of expression of albumin and AFP genes during rat liver development and neoplasia.
Asunto(s)
Genes , Neoplasias Hepáticas Experimentales/metabolismo , Hígado/metabolismo , ARN Mensajero/genética , Albúmina Sérica/genética , Transcripción Genética , alfa-Fetoproteínas/genética , Animales , Animales Recién Nacidos , ADN/metabolismo , Cinética , Hibridación de Ácido Nucleico , Polirribosomas/metabolismo , ARN Ribosómico/genética , Ratas , Fracciones Subcelulares/metabolismoRESUMEN
We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.
Asunto(s)
Genes , Hígado/embriología , Albúmina Sérica/genética , Transcripción Genética , alfa-Fetoproteínas/genética , Animales , Femenino , Feto/metabolismo , Edad Gestacional , Hígado/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos C57BL , Hibridación de Ácido Nucleico , Poli A/genética , Polirribosomas/metabolismo , Embarazo , ARN/genética , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Saco Vitelino/metabolismoRESUMEN
Hybridization of albumin clones cDNA with human DNAs digested by several restriction endonucleases reveals two HaeIII polymorphisms. The first polymorphism, H1, is of low frequency (f1 = 0.05); the second, which is validated by family analysis, occurs frequently (f2 = 0.21) and is an intronic polymorphism, probably of substitution--base type.
Asunto(s)
Albúminas/genética , Mapeo Cromosómico , Enzimas de Restricción del ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Polimorfismo Genético , ADN/genética , Femenino , Genes , Humanos , Masculino , Hibridación de Ácido Nucleico , LinajeRESUMEN
The effect on ketonemia of alternate exposure to ambient temperatures (Ta) of 25 and 5 degrees C was investigated in fasting geese. Three experimental birds were compared to three controls continuously exposed to 25 degrees C Ta while fasting. During the first 9 days of fasting, when both groups were exposed to 25 degrees C, plasma concentration of beta-hydroxybutyrate (beta-OHB) increased similarly in both, from 0.10 +/- 0.02 to 6.62 +/- 0.71 mmol X L-1. It later plateaued at 8-9 mmol X L-1 in the control birds. When the experimental birds were exposed to 5 degrees C Ta between the 9th and 15th day of the fast, it increased further during the first 24 h but thereafter decreased of 57%, from 8.62 +/- 1.56 to 3.73 +/- 1.24 mmol X L-1. This decrease was reversed within the 6 days of return to 25 degrees C Ta. In both groups, plasma acetoacetate (AcAc) concentration remained very low during the fast: 51 +/- 1 mumol X L-1. This reversible cold-induced effect on ketonemia may be used for investigating the possible role of ketone bodies in protein sparing during fasting.
Asunto(s)
Frío , Ayuno , Cuerpos Cetónicos/sangre , Ácido 3-Hidroxibutírico , Acetoacetatos/sangre , Animales , Gansos , Hidroxibutiratos/sangre , Masculino , Proteínas/metabolismo , Factores de TiempoRESUMEN
In the hindbrain region of the developing CNS, anteroposterior patterning involves a transient segmentation process which leads to the formation of morphological bulges called rhombomeres. The rhombomeres constitute cell lineage restriction units and participate in the establishment of a metameric pattern which is responsible for the segmental organisation of motor and reticular neurons. Like Drosophila compartments, rhombomeres also constitute domains of specific gene expression. Genes expressed in a rhombomere-specific manner so far identified encode various types of putative regulatory molecules, including transcription factors, like Hox proteins, the zinc finger protein Krox-20 and the basic domain leucine-zipper protein kreisler, and receptor type molecules, like Sek-1, a member of the EPH family of tyrosine kinase receptors. Such genes are thought to play a role either in the definition of segmental territories or in the specification of the identity of the rhombomeres. Initial analysis of the function of some of these genes have indeed supported this hypothesis. This is the case for the Krox-20 gene. It is expressed within the developing hindbrain in two transverse domains which prefigure and then coincide with r3 and r5. We have inactivated Krox-20 by homologous recombination in ES cells and demonstrated that the mutation leads to the deletion of r3 and r5. The mutation introduced into the Krox-20 gene involved the in-frame insertion of the lacZ coding sequence. This allowed us to follow the late expression pattern of the gene and to identify two additional phenotypes, affecting myelination of the peripheral nervous system and endochondral ossification. The lacZ reporter also permitted a detailed analysis of the expression of Krox-20 in peripheral glial cells, revealing important steps in the control of their development. Recently we have performed a detailed analysis of specific neuronal populations affected by the mutation which shed new light on the role of Krox-20 in the segmentation and on the physiological consequences of its inactivation. We have also identified several new members of the Sek-1 family of receptor tyrosine kinases, which are also expressed in a rhombomere-specific manner. Finally, we have provided evidence that Krox-20 is as a key regulator of r3/r5-specific transcription, controlling the expression of at least five other regulator genes in these rhombomeres. In three cases, Hoxb-2, Hoxa-2 and Sek-1, we could demonstrate that Krox-20 was directly involved in the transcriptional activation of these genes.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes Homeobox/genética , Genes/genética , Rombencéfalo/embriología , Factores de Transcripción/genética , Animales , Proteínas de Unión al ADN/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz , Desarrollo Embrionario y Fetal , Regulación del Desarrollo de la Expresión Génica , Rombencéfalo/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The transcription factor genes Hoxa1 and Krox-20 have been shown to play important roles in vertebrate hindbrain segmentation. In this report, we present evidence for novel functions of these genes which co-operate in specifying cellular identity in rhombomere (r) 3. Although Hoxa1 has not been observed to be expressed rostrally to the prospective r3/r4 boundary, its inactivation results in (i) the appearance of patches of cells presenting an r2-like molecular identity within r3, (ii) early neuronal differentiation in r3, normally characteristic of even-numbered rhombomeres, and (iii) abnormal navigation of r3 motor axons, similar to that observed in even-numbered rhombomeres. These phenotypic manifestations become more severe in the context of the additional inactivation of one allele of the Krox-20 gene, demonstrating that Hoxa1 and Krox-20 synergize in a dosage-dependent manner to specify r3 identity and odd- versus even-numbered rhombomere characters. In addition, these data suggest that the control of the development of r3 may not be autonomous but dependent on interactions with Hoxa1-expressing cells.