Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA.

FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously.

An integrated strategy to study muscle development and myofilament structure in Caenorhabditis elegans.

A crucial step in the development of muscle cells in all metazoan animals is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. We propose that additional sarcomeric proteins exist for which there is a less severe, or entirely different, mutant phenotype produced in their absence. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. We generated two replicate long SAGE libraries for sorted embryonic muscle cells, identifying 7,974 protein-coding genes. A refined list of 3,577 genes expressed in muscle cells was compiled from the overlap between our SAGE data and available microarray data. Using the genes in our refined list, we have performed two separate RNA interference (RNAi) screens to identify novel genes that play a role in sarcomere assembly and/or maintenance in either embryonic or adult muscle. To identify muscle defects in embryos, we screened specifically for the Pat embryonic arrest phenotype. To visualize muscle defects in adult animals, we fed dsRNA to worms producing a GFP-tagged myosin protein, thus allowing us to analyze their myofilament organization under gene knockdown conditions using fluorescence microscopy. By eliminating or severely reducing the expression of 3,300 genes using RNAi, we identified 122 genes necessary for proper myofilament organization, 108 of which are genes without a previously characterized role in muscle. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known.

A gene expression fingerprint of C. elegans embryonic motor neurons.

BACKGROUND: Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo. RESULTS: Fluorescence Activated Cell Sorting (FACS) was used to isolate unc-4::GFP neurons from primary cultures of C. elegans embryonic cells. Microarray experiments detected 6,217 unique transcripts of which approximately 1,000 are enriched in unc-4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data include transcripts for receptors to a remarkably wide variety of signaling molecules. The added presence of a robust array of G-protein pathway components is indicative of complex and highly integrated mechanisms for modulating motor neuron activity. Over half of the enriched genes (537) have human homologs, a finding that could reflect substantial overlap with the gene expression repertoire of mammalian motor neurons. CONCLUSION: We have described a microarray-based method, MAPCeL, for profiling gene expression in specific C. elegans motor neurons and provide evidence that this approach can reveal candidate genes for key roles in the differentiation and function of these cells. These methods can now be applied to generate a gene expression map of the C. elegans nervous system.

Transcriptional regulation of secretory capacity by bZip transcription factors.

Cells of specialized secretory organs expand their secretory pathways to accommodate the increased protein load necessary for their function. The endoplasmic reticulum (ER), the Golgi apparatus and the secretory vesicles, expand not only the membrane components but also the protein machinery required for increased protein production and transport. Increased protein load causes an ER stress response akin to the Unfolded Protein Response (UPR). Recent work has implicated several bZip transcription factors in the regulation of protein components of the early secretory pathway necessary to alleviate this stress. Here, we highlight eight bZip transcription factors in regulating secretory pathway component genes. These include components of the three canonical branches of the UPR-ATF4, XBP1, and ATF6, as well as the five members of the Creb3 family of transcription factors. We review findings from both invertebrate and vertebrate model systems suggesting that all of these proteins increase secretory capacity in response to increased protein load. Finally, we propose that the Creb3 family of factors may have a dual role in secretory cell differentiation by also regulating the pathways necessary for cell cycle exit during terminal differentiation.

Changes in organelle position and epithelial architecture associated with loss of CrebA.

Drosophila CrebA facilitates high-level secretion by transcriptional upregulation of the protein components of the core secretory machinery. In CrebA mutant embryos, both salivary gland (SG) morphology and epidermal cuticle secretion are abnormal, phenotypes similar to those observed with mutations in core secretory pathway component genes. Here, we examine the cellular defects associated with CrebA loss in the SG epithelium. Apically localized secretory vesicles are smaller and less abundant, consistent with overall reductions in secretion. Unexpectedly, global mislocalization of cellular organelles and excess membrane accumulation in the septate junctions (SJs) are also observed. Whereas mutations in core secretory pathway genes lead to organelle localization defects similar to those of CrebA mutants, they have no effect on SJ-associated membrane. Mutations in tetraspanin genes, which are normally repressed by CrebA, have mild defects in SJ morphology that are rescued by simultaneous CrebA loss. Correspondingly, removal of several tetraspanins gives partial rescue of the CrebA SJ phenotype, supporting a role for tetraspanins in SJ organization.

The CrebA/Creb3-like transcription factors are major and direct regulators of secretory capacity.

Secretion occurs in all cells, with relatively low levels in most cells and extremely high levels in specialized secretory cells, such as those of the pancreas, salivary, and mammary glands. How secretory capacity is selectively up-regulated in specialized secretory cells is unknown. Here, we find that the CrebA/Creb3-like family of bZip transcription factors functions to up-regulate expression of both the general protein machinery required in all cells for secretion and of cell type-specific secreted proteins. Drosophila CrebA directly binds the enhancers of secretory pathway genes and is both necessary and sufficient to activate expression of every secretory pathway component gene examined thus far. Microarray profiling reveals that CrebA also up-regulates expression of genes encoding cell type-specific secreted components. Finally, we found that the human CrebA orthologues, Creb3L1 and Creb3L2, have the ability to up-regulate the secretory pathway in nonsecretory cell types.

The embryonic muscle transcriptome of Caenorhabditis elegans.

BACKGROUND: The force generating mechanism of muscle is evolutionarily ancient; the fundamental structural and functional components of the sarcomere are common to motile animals throughout phylogeny. Recent evidence suggests that the transcription factors that regulate muscle development are also conserved. Thus, a comprehensive description of muscle gene expression in a simple model organism should define a basic muscle transcriptome that is also found in animals with more complex body plans. To this end, we applied microarray profiling of Caenorhabtidis elegans cells (MAPCeL) to muscle cell populations extracted from developing C. elegans embryos. RESULTS: We used fluorescence-activated cell sorting to isolate myo-3::green fluorescent protein (GFP) positive muscle cells, and their cultured derivatives, from dissociated early C. elegans embryos. Microarray analysis identified 7,070 expressed genes, 1,312 of which are enriched in the myo-3::GFP positive cell population relative to the average embryonic cell. The muscle enriched gene set was validated by comparisons with known muscle markers, independently derived expression data, and GFP reporters in transgenic strains. These results confirm the utility of MAPCeL for cell type specific expression profiling and reveal that 60% of these transcripts have human homologs. CONCLUSION: This study provides a comprehensive description of gene expression in developing C. elegans embryonic muscle cells. The finding that more than half of these muscle enriched transcripts encode proteins with human homologs suggests that mutant analysis of these genes in C. elegans could reveal evolutionarily conserved models of muscle gene function, with ready application to human muscle pathologies.

UNC-4 represses CEH-12/HB9 to specify synaptic inputs to VA motor neurons in C. elegans.

In Caenorhabditis elegans, VA and VB motor neurons arise as lineal sisters but synapse with different interneurons to regulate locomotion. VA-specific inputs are defined by the UNC-4 homeoprotein and its transcriptional corepressor, UNC-37/Groucho, which function in the VAs to block the creation of chemical synapses and gap junctions with interneurons normally reserved for VBs. To reveal downstream genes that control this choice, we have employed a cell-specific microarray strategy that has now identified unc-4-regulated transcripts. One of these genes, ceh-12, a member of the HB9 family of homeoproteins, is normally restricted to VBs. We show that expression of CEH-12/HB9 in VA motor neurons in unc-4 mutants imposes VB-type inputs. Thus, this work reveals a developmental switch in which motor neuron input is defined by differential expression of transcription factors that select alternative presynaptic partners. The conservation of UNC-4, HB9, and Groucho expression in the vertebrate motor circuit argues that similar mechanisms may regulate synaptic specificity in the spinal cord.

Cell-specific microarray profiling experiments reveal a comprehensive picture of gene expression in the C. elegans nervous system.

BACKGROUND: With its fully sequenced genome and simple, well-defined nervous system, the nematode Caenorhabditis elegans offers a unique opportunity to correlate gene expression with neuronal differentiation. The lineal origin, cellular morphology and synaptic connectivity of each of the 302 neurons are known. In many instances, specific behaviors can be attributed to particular neurons or circuits. Here we describe microarray-based methods that monitor gene expression in C. elegans neurons and, thereby, link comprehensive profiles of neuronal transcription to key developmental and functional properties of the nervous system. RESULTS: We employed complementary microarray-based strategies to profile gene expression in the embryonic and larval nervous systems. In the MAPCeL (Microarray Profiling C. elegans cells) method, we used fluorescence activated cell sorting (FACS) to isolate GFP-tagged embryonic neurons for microarray analysis. To profile the larval nervous system, we used the mRNA-tagging technique in which an epitope-labeled mRNA binding protein (FLAG-PAB-1) was transgenically expressed in neurons for immunoprecipitation of cell-specific transcripts. These combined approaches identified approximately 2,500 mRNAs that are highly enriched in either the embryonic or larval C. elegans nervous system. These data are validated in part by the detection of gene classes (for example, transcription factors, ion channels, synaptic vesicle components) with established roles in neuronal development or function. Of particular interest are 19 conserved transcripts of unknown function that are also expressed in the mammalian brain. In addition to utilizing these profiling approaches to define stage-specific gene expression, we also applied the mRNA-tagging method to fingerprint a specific neuron type, the A-class group of cholinergic motor neurons, during early larval development. A comparison of these data to a MAPCeL profile of embryonic A-class motor neurons identified genes with common functions in both types of A-class motor neurons as well as transcripts with roles specific to each motor neuron type. CONCLUSION: We describe microarray-based strategies for generating expression profiles of embryonic and larval C. elegans neurons. These methods can be applied to particular neurons at specific developmental stages and, therefore, provide an unprecedented opportunity to obtain spatially and temporally defined snapshots of gene expression in a simple model nervous system.

acr-16 encodes an essential subunit of the levamisole-resistant nicotinic receptor at the Caenorhabditis elegans neuromuscular junction.

The Caenorhabditis elegans neuromuscular junction (NMJ) contains three pharmacologically distinct ionotropic receptors: gamma-aminobutyric acid receptors, levamisole-sensitive nicotinic receptors, and levamisole-insensitive nicotinic receptors. The subunit compositions of the gamma-aminobutyric acid- and levamisole-sensitive receptors have been elucidated, but the levamisole-insensitive acetylcholine receptor is uncharacterized. To determine which of the approximately 40 putative nicotinic receptor subunit genes in the C. elegans genome encodes the levamisole-resistant receptor, we utilized MAPCeL, a microarray profiling strategy. Of seven nicotinic receptor subunit transcripts found to be enriched in muscle, five encode the levamisole receptor subunits, leaving two candidates for the levamisole-insensitive receptor: acr-8 and acr-16. Electrophysiological analysis of the acr-16 deletion mutant showed that the levamisole-insensitive muscle acetylcholine current was eliminated, whereas deletion of acr-8 had no effect. These data suggest that ACR-16, like its closest vertebrate homolog, the nicotinic receptor alpha7-subunit, may form homomeric receptors in vivo. Genetic ablation of both the levamisole-sensitive receptor and acr-16 abolished all cholinergic synaptic currents at the NMJ and severely impaired C. elegans locomotion. Therefore, ACR-16-containing receptors account for all non-levamisole-sensitive nicotinic synaptic signaling at the C. elegans NMJ. The determination of subunit composition for all three C. elegans body wall muscle ionotropic receptors provides a critical foundation for future research at this tractable model synapse.