Two enhancer regions in the mouse En-2 locus direct expression to the mid/hindbrain region and mandibular myoblasts.

An En-2/lacZ gene fusion containing 9.5 kb of En-2 genomic DNA was capable of directing lacZ expression in an En-2-specific manner both temporally and spatially during embryogenesis and in the adult. lacZ expression was confined in the embryo to cells within the mid/hindbrain and mandibular arch regions and in the adult to cells of the molecular and granular layers of the cerebellum, and within the pons and colliculi regions. Interestingly, in the adult, transgene expression patterns within the cerebellum in two lines appeared to mark distinct anterior-posterior compartments. Analysis of the expression pattern of this transgene, in fetal and adult mice lacking a functional En-2 protein, provided evidence that the En-2 gene in mouse is not autoregulated. Deletion analysis of the En-2 genomic region and the use of a heterologous promoter identified two enhancer-containing regions of 1.5 and 1.0 kb in length, 5' of the transcribed sequences, which independently directed expression in the embryo to either the mid/hindbrain region or mandibular myoblasts, respectively. The 1.5 kb fragment contains the most anterior neural enhancer and the 1.0 kb fragment, the earliest myogenic enhancer thus far characterized. These En-2-specific regulatory regions can now be used in a biochemical analysis to identify proteins important in anterior-posterior patterning of the vertebrate CNS and in the specification of muscle identity as well as in a mutational analysis to direct expression of other developmentally important genes to these regions.

[1]  M. Westerfield,et al.  Coordinate embryonic expression of three zebrafish engrailed genes. , 1992, Development.

[2]  J. Rossant,et al.  Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo. , 1992, Development.

[3]  M. Wassef,et al.  Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon. , 1992, Development.

[4]  D. Jacobs,et al.  Deformed autoregulatory element from Drosophila functions in a conserved manner in transgenic mice , 1992, Nature.

[5]  W. McGinnis,et al.  A human HOX4B regulatory element provides head-specific expression in Drosophila embryos , 1992, Nature.

[6]  Roel Nusse,et al.  Wnt genes , 1992, Cell.

[7]  Peter Gruss,et al.  Pax in development , 1992, Cell.

[8]  A. Joyner,et al.  The midbrain-hindbrain phenotype of Wnt-1− Wnt-1− mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum , 1992, Cell.

[9]  R. Krumlauf,et al.  Analysis of the murine Hox‐2.7 gene: conserved alternative transcripts with differential distributions in the nervous system and the potential for shared regulatory regions. , 1992, The EMBO journal.

[10]  C. A. Gardner,et al.  Expression patterns of engrailed‐like proteins in the chick embryo , 1992, Developmental dynamics : an official publication of the American Association of Anatomists.

[11]  D. Duboule,et al.  Comparison of mouse and human HOX‐4 complexes defines conserved sequences involved in the regulation of Hox‐4.4. , 1992, The EMBO journal.

[12]  William McGinnis,et al.  Homeobox genes and axial patterning , 1992, Cell.

[13]  H. Nakamura,et al.  Establishment of rostrocaudal polarity in tectal primordium: engrailed expression and subsequent tectal polarity. , 1991, Development.

[14]  R. Krumlauf,et al.  Multiple spatially specific enhancers are required to reconstruct the pattern of Hox-2.6 gene expression. , 1991, Genes & development.

[15]  C. A. Gardner,et al.  The cellular environment controls the expression of engrailed-like protein in the cranial neuroepithelium of quail-chick chimeric embryos. , 1991, Development.

[16]  P. O’Farrell,et al.  Multiple modes of engrailed regulation in the progression towards cell fate determination , 1991, Nature.

[17]  K. Schughart,et al.  A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs. , 1991, Development.

[18]  D. Noden.  Vertebrate craniofacial development: the relation between ontogenetic process and morphological outcome. , 1991, Brain, behavior and evolution.

[19]  P. O’Farrell,et al.  Active repression of transcription by the engrailed homeodomain protein. , 1991, The EMBO journal.

[20]  M. Wassef,et al.  Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en , 1991, Neuron.

[21]  P Gruss,et al.  Separate elements cause lineage restriction and specify boundaries of Hox-1.1 expression. , 1991, Development.

[22]  H. Arnold,et al.  Early expression of the myogenic regulatory gene, myf-5, in precursor cells of skeletal muscle in the mouse embryo. , 1991, Development.

[23]  A. Joyner,et al.  Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox. , 1991, Science.

[24]  C. Holt,et al.  Cephalic expression and molecular characterization of Xenopus En-2. , 1991, Development.

[25]  A. Joyner,et al.  Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum. , 1991, Development.

[26]  C. Kimmel,et al.  Specification of jaw muscle identity in zebrafish: correlation with engrailed-homeoprotein expression. , 1990, Science.

[27]  F. Ruddle,et al.  Evidence for positive and negative regulation of the Hox-3.1 gene. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[28]  C Kress,et al.  Hox-2.3 upstream sequences mediate lacZ expression in intermediate mesoderm derivatives of transgenic mice. , 1990, Development.

[29]  M. Ross,et al.  Meander tail reveals a discrete developmental unit in the mouse cerebellum. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[30]  S. Martinez,et al.  Expression of the homeobox Chick-en gene in chick/quail chimeras with inverted mes-metencephalic grafts. , 1990, Developmental biology.

[31]  M. Horikoshi,et al.  Binding site-dependent direct activation and repression of in vitro transcription by Drosophila homeodomain proteins , 1990, Cell.

[32]  S. Martinez,et al.  Pluripotentiality of the 2-day-old avian germinative neuroepithelium. , 1990, Developmental biology.

[33]  R. Balling,et al.  Position-specific activity of the Hox1.1 promoter in transgenic mice. , 1990, Development.

[34]  C. Peschle,et al.  Region-specific enhancers near two mammalian homeo box genes define adjacent rostrocaudal domains in the central nervous system. , 1990, Genes & development.

[35]  S. Martinez,et al.  Rostral Cerebellum Originates from the Caudal Portion of the So‐Called ‘Mesencephalic’ Vesicle: A Study Using Chick/Quail Chimeras , 1989, The European journal of neuroscience.

[36]  K. G. Coleman,et al.  Expression of engrailed proteins in arthropods, annelids, and chordates , 1989, Cell.

[37]  A. Joyner,et al.  Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. , 1989, Science.

[38]  J. Rossant,et al.  Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice. , 1989, Development.

[39]  H. Willard,et al.  Chromosomal localization of the human homeo box-containing genes, EN1 and EN2. , 1989, Genomics.

[40]  P. O’Farrell,et al.  Activation and repression of transcription by homoeodomain-containing proteins that bind a common site , 1988, Nature.

[41]  A. Joyner,et al.  Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development. , 1988, Genes & development.

[42]  M. Levine,et al.  DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context , 1988, Molecular and cellular biology.

[43]  C. Tuggle,et al.  Spatial regulation of homeobox gene fusions in the embryonic central nervous system of transgenic mice , 1988, Neuron.

[44]  C. A. Gardner,et al.  Expression of an engrailed‐like gene during development of the early embryonic chick nervous system , 1988, Journal of neuroscience research.

[45]  P. O’Farrell,et al.  The sequence specificity of homeodomain-DNA interaction , 1988, Cell.

[46]  A. Joyner,et al.  Expression of the homeo box-containing gene En-2 delineates a specific region of the developing mouse brain. , 1988, Genes & development.

[47]  A. Joyner,et al.  En-1 and En-2, two mouse genes with sequence homology to the Drosophila engrailed gene: expression during embryogenesis. , 1987, Genes & development.

[48]  P. O’Farrell,et al.  The Drosophila developmental gene, engrailed, encodes a sequence-specific DNA binding activity , 1985, Nature.

[49]  K. G. Coleman,et al.  Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila engrailed gene , 1985, Cell.

[50]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[51]  M. Kozak.  Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. , 1983, Microbiological reviews.

[52]  T. Kornberg.  Engrailed: a gene controlling compartment and segment formation in Drosophila. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[53]  W. Hollander,et al.  Meander tail: a recessive mutant located in chromosome 4 of the mouse. , 1977, The Journal of heredity.

[54]  P. Lawrence,et al.  Control of compartment development by the engrailed gene in Drosophila , 1975, Nature.

[55]  A. Joyner,et al.  Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions. , 1992, Developmental genetics.

[56]  G. Lyons,et al.  Myogenesis in the mouse. , 1992, Ciba Foundation symposium.

[57]  J. Zákány,et al.  The use of lacZ gene fusions in the studies of mammalian development: developmental regulation of mammalian homeobox genes in the CNS. , 1990, Journal de physiologie.

[58]  M E Hallonet,et al.  A new approach to the development of the cerebellum provided by the quail-chick marker system. , 1990, Development.

[59]  K. G. Coleman,et al.  Expression of engrailed proteins in arthropods, annelids, and chordates. , 1989, Cell.

[60]  M. Ashburner.  A Laboratory manual , 1989 .

[61]  B. Hogan,et al.  Manipulating the mouse embryo: A laboratory manual , 1986 .