The transcriptional regulator Id3 is expressed in cranial sensory placodes during early avian embryonic development.

The chick homologue of the helix-loop-helix gene Id3 was isolated, and its expression pattern was analyzed during early stages of chick development. Chick Id3 is dynamically expressed in the olfactory, lens, and otic placodes. It is also observed in the epiphysis, nephric primordium, stomodeum, dermomyotome, distal branchial arches, dorsolateral hindbrain, foregut endoderm, dorsal spinal cord, and somites.     

Temporally and spatially restricted expression of a gland cell gene during regeneration and in vitro transdifferentiation in the hydrozoan Podocoryne carnea

An antiserum to transdifferentiated striated muscle cells from the medusa of Podocoryne carnea was prepared and used to screen a gt11-expression library prepared from gonozoids of P. carnea. We isolated a cDNA clone termed Pod-EPPT with at least 63 tandem repeats of the tetrapeptide-motive glu-pro-pro-thr, named Pod-EPPT. Using Pod-EPPT as a molecular marker for head quality the morphological relationship between the two metagenic life stages of this hydroid, the polyp and the medusa, was studied. In situ hybridization demonstrated that expression of the gene corresponding is restricted to secretory cells in the endoderm of the oral hypostome region of polyps and medusae and, presumably, to progenitor cells of this type. Cells expressing Pod-EPPT could not be observed in the larval stage. During head regeneration in polyps, Pod-EPPT expression is upregulated soon after head removal in previously non-expressing cells and in newly differentiating secretory cells. This activation of a head-specific gene precedes the morphologically obvious events of head regeneration. Pod-EPPT is one of the genes that are activated during manubrium (mouth) regeneration from experimentally combined subumbrellar plate endoderm and striated muscle of the medusa.     

Temporally restricted expression of transcription factor betaFTZ-F1: significance for embryogenesis, molting and metamorphosis in Drosophila melanogaster

FTZ-F1, a member of the nuclear receptor superfamily, has been implicated in the activation of the segmentation gene fushi tarazu during early embryogenesis of Drosophila melanogaster. We found that an isoform of FTZ-F1, betaFTZ-F1, is expressed in the nuclei of almost all tissues slightly before the first and second larval ecdysis and before pupation. Severely affected ftz-f1 mutants display an embryonic lethal phenotype, but can be rescued by ectopic expression of betaFTZ-F1 during the period of endogenous betaFTZ-F1 expression in the wild type. The resulting larvae are not able to molt, but this activity is rescued again by forced expression of betaFTZ-F1, allowing progression to the next larval instar stage. On the other hand, premature expression of betaFTZ-F1 in wild-type larvae at mid-first instar or mid-second instar stages causes defects in the molting process. Sensitive periods were found to be around the time of peak ecdysteroid levels and slightly before the start of endogenous betaFTZ-F1 expression. A hypomorphic ftz-f1 mutant that arrests in the prepupal stage can also be rescued by ectopic, time-specific expression of betaFTZ-F1. Failure of salivary gland histolysis, one of the phenotypes of the ftz-f1 mutant, is rescued by forced expression of the ftz-f1 downstream gene BR-C during the late prepupal period. These results suggest that betaFTZ-F1 regulates genes associated with ecdysis and metamorphosis, and that the exact timing of its action in the ecdysone-induced gene cascade is important for proper development.     

The protein Id: a negative regulator of helix-loop-helix DNA binding proteins

We have isolated a cDNA clone encoding a novel helix-loop-helix (HLH) protein, Id. Id is missing the basic region adjacent to the HLH domain that is essential for specific DNA binding in another HLH protein, MyoD. An in vitro translation product of Id can associate specifically with at least three HLH proteins (MyoD, E12, and E47) and attenuate their ability to bind DNA as homodimeric or heterodimeric complexes. Id is expressed at varying levels in all cell lines tested. In three cell lines that can be induced to undergo terminal differentiation, Id RNA levels decrease upon induction. Transfection experiments indicate that over-expression of Id inhibits the trans-activation of the muscle creatine kinase enhancer by MyoD. Based on these findings, we propose that HLH proteins lacking a basic region may negatively regulate other HLH proteins through the formation of nonfunctional heterodimeric complexes.     

Temporally and spatially regulated expression of a candidate G-protein-coupled receptor during cerebral cortical development

Genes expressed in layer-specific patterns in the mammalian cerebral cortex may play a role in specifying the identity of different cortical layers. Using PCR-differential display, we identified a cDNA that encodes rCNL3, a gene cloned previously by sequence homology to G-protein-coupled receptors. rCNL3 is expressed predominantly in layers 2-4 of the young rat cortex and in the developing and adult striatum. Cortical expression of rCNL3 begins postnatally at P3 and continues at high levels until around P15, while striatal expression begins at E20 and continues through adulthood. rCNL3 expression is not detectable in the ventricular zone precursors that generate the neurons of layers 2-4. The expression pattern of rCNL3 in the developing cortex suggests that rCNL3 is not involved in the initial specification of laminar fate, but rather may be involved with later differentiation events within the superficial cortical layers.     

Spatially restricted patterns of expression of the homeobox-containing gene Hox 2.1. during mouse embryogenesis

The mouse Hox 2.1 gene contains a homeobox sequence and is therefore a candidate for a vertebrate gene involved in the control of embryonic patterning or positional specification. To investigate this possibility, we have used in situ hybridization to determine the pattern of Hox 2.1 expression during mouse embryogenesis. At 8.5 days post coitum, Hox 2.1 is expressed at a low level in the posterior neuroectoderm and mesoderm, and in the neuroectoderm of the presumptive hindbrain. At 12.5 days p.c., Hox 2.1 is expressed in an anteroposterior restricted domain extending from the hindbrain throughout the length of the spinal cord, predominantly in the dorsal region. Between 12.5 and 13.5 days p.c. the domain becomes localized to the occipital and cervical regions. We also detect Hox 2.1 RNA in the embryonic lung, stomach, mesonephros and metanephros, as well as in myenteric plexus, dorsal root ganglia and the nodose ganglion, and in mature granulocytes. The embryonic expression of Hox 2.1 in neural tissue is compared with that of Hox 3.1, which also shows anteroposterior restricted domains of gene expression. These patterns of expression are not clearly consistent with Hox 2.1 or Hox 3.1 having roles in segmental patterning. However, the data are consistent with these genes having regulatory roles in anteroposterior positional specification in the neuroectoderm and mesoderm, and suggest that Hox 2.1 may also have functions during organogenesis.     

Temporally and spatially regulated somatic mutagenesis in mice.

In mice transgenesis through oocyte injection or DNA recombination in embryonal stem (ES) cells allows mutations to be introduced into the germline. However, the earliest phenotype of the introduced mutation can eclipse later effects. We show in mice that site-specific genomic recombination can be induced in a selected cell type, B lymphocytes, at a chosen time. This precision of somatic mutagenesis was accomplished by limiting expression of a Cre recombinase-estrogen receptor fusion protein to B lymphocytes by use of tissue-specific elements in the promoter of the transgene employed. The expressed fusion protein remained inactive until derepressed by systemic administration of an exogenous ligand for the estrogen receptor, 4-OH-tamoxifen. Upon derepression the Cre recombinase enzyme deleted specific DNA segments, flanked by loxP sites, in B lymphocytes only. The efficiency of recombination in cells expressing the fusion protein could be varied from low levels to >80%, depending on the dose of ligand administered. Our work presents a paradigm applicable to other uses of site-specific recombination in somatic mutagenesis where both temporal and spatial regulation are desired.     

Regulation of Id1 and its association with basic helix-loop-helix proteins during nerve growth factor-induced differentiation of PC12 cells.

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of helix-loop-helix (HLH) proteins during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor. Gel mobility shift assays using PC12 cell nuclear extracts demonstrated that active basic HLH complexes exist throughout differentiation. Addition of exogeneous Id1 protein, a negative regulator of basic HLH proteins, disrupted specific complexes formed by PC12 cell nuclear extracts on a CANNTG consensus oligonucleotide. To identify possible novel basic HLH proteins in these complexes, a glutathione S-transferase-Id1 fusion protein was used to screen a PC12 cell cDNA expression library. A single clone representing the rat E2-2 gene was identified. Sequential immunoprecipitations with antibodies to each HLH protein revealed an association between Id1 and E2-2 that could be detected in both untreated and nerve growth factor-treated PC12 cell lysates. These experiments define a new HLH interaction between Id1 and E2-2 in neuronal cells and suggest that neuronal differentiation may be regulated by HLH proteins in a distinctive manner.