James D. McGhee

The GATA family (vertebrates and invertebrates)

Over the past year, vertebrate GATA factors have been found to participate directly in several signal-transduction pathways. Smad3, phosphorylated by TGF-beta signalling, interacts with GATA3 to induce differentiation of T helper cells. Hypertrophic stimuli act through RhoA GTPase and ROCK kinase to activate GATA4 in cardiac myocytes. In the liver, GATA4 is elevated by BMP and FGF signalling, and is able to bind to chromatin targets. Invertebrate GATA factors play a central role in specifying the mesendoderm.

The GATA-factor elt-2 is essential for formation of the Caenorhabditis elegans intestine

The Caenorhabditis elegans elt-2 gene encodes a single-finger GATA factor, previously cloned by virtue of its binding to a tandem pair of GATA sites that control the gut-specific ges-1 esterase gene. In the present paper, we show that elt-2 expression is completely gut specific, beginning when the embryonic gut has only two cells (one cell cycle prior to ges-1 expression) and continuing in every cell of the gut throughout the life of the worm. When elt-2 is expressed ectopically using a transgenic heat-shock construct, the endogenous ges-1 gene is now expressed in most if not all cells of the embryo; several other gut markers (including a transgenic elt-2-promoter::lacZ reporter construct designed to test for elt-2 autoregulation) are also expressed ectopically in the same experiment. These effects are specific in that two other C. elegans GATA factors (elt-1 and elt-3) do not cause ectopic gut gene expression. An imprecise transposon excision was identified that removes the entire elt-2 coding region. Homozygous elt-2 null mutants die at the L1 larval stage with an apparent malformation or degeneration of gut cells. Although the loss of elt-2 function has major consequences for later gut morphogenesis and function, mutant embryos still express ges-1. We suggest that elt-2 is part of a redundant network of genes that controls embryonic gut development; other factors may be able to compensate for elt-2 loss in the earlier stages of gut development but not in later stages. We discuss whether elements of this regulatory network may be conserved in all metazoa.

DNA synthesis and the control of embryonic gene expression in C. elegans

DNA synthesis in each cell lineage of the early C. elegans embryo was measured using microspectrofluorimetry. Aphidicolin was shown to inhibit DNA synthesis almost instantly and completely. Aphidicolin was then used to investigate how DNA synthesis controls expression of two biochemical markers that appear at different times during gut development: gut granules and a carboxylesterase. We show that marker expression is controlled neither by reaching the normal DNA: cytoplasm ratio, by counting the normal number of rounds of DNA synthesis, nor by a simple lengthening of the cell cycle. Instead, expression of both gut markers requires a short period of DNA synthesis in the first cell cycle after the gut has been clonally established.

ELT-2 is the predominant transcription factor controlling differentiation and function of the C. elegans intestine, from embryo to adult

Starting with SAGE-libraries prepared from C. elegans FAC-sorted embryonic intestine cells (8E-16E cell stage), from total embryos and from purified oocytes, and taking advantage of the NextDB in situ hybridization data base, we define sets of genes highly expressed from the zygotic genome, and expressed either exclusively or preferentially in the embryonic intestine or in the intestine of newly hatched larvae; we had previously defined a similarly expressed set of genes from the adult intestine. We show that an extended TGATAA-like sequence is essentially the only candidate for a cis-acting regulatory motif common to intestine genes expressed at all stages. This sequence is a strong ELT-2 binding site and matches the sequence of GATA-like sites found to be important for the expression of every intestinal gene so far analyzed experimentally. We show that the majority of these three sets of highly expressed intestinal-specific/intestinal-enriched genes respond strongly to ectopic expression of ELT-2 within the embryo. By flow-sorting elt-2(null) larvae from elt-2(+) larvae and then preparing Solexa/Illumina-SAGE libraries, we show that the majority of these genes also respond strongly to loss-of-function of ELT-2. To test the consequences of loss of other transcription factors identified in the embryonic intestine, we develop a strain of worms that is RNAi-sensitive only in the intestine; however, we are unable (with one possible exception) to identify any other transcription factor whose intestinal loss-of-function causes a phenotype of comparable severity to the phenotype caused by loss of ELT-2. Overall, our results support a model in which ELT-2 is the predominant transcription factor in the post-specification C. elegans intestine and participates directly in the transcriptional regulation of the majority (>80%) of intestinal genes. We present evidence that ELT-2 plays a central role in most aspects of C. elegans intestinal physiology: establishing the structure of the enterocyte, regulating enzymes and transporters involved in digestion and nutrition, responding to environmental toxins and pathogenic infections, and regulating the downstream intestinal components of the daf-2/daf-16 pathway influencing aging and longevity.

Parental DNA strands segregate randomly during embryonic development of Caenorhabditis elegans

The fate of gamete DNA was followed in the next generation embryos of the nematode C. elegans. Either male worms or spermless hermaphrodites were grown on bromodeoxyuridine-containing E. coli in order to label germ-line DNA. Matings then produced embryos in which only the DNA strands provided by the gametes contained label. This original gamete DNA could be detected during embryonic development by using a fluorescently labeled monoclonal antibody specific to bromodeoxyuridine. Both the number and position of fluorescent spots in the embryo indicate that gamete DNA strands segregate randomly during development. Random segregation of parental DNA strands rules out models of development that invoke chromosome imprinting or immortal DNA strands.

The C. elegans pvf-1 gene encodes a PDGF/VEGF-like factor able to bind mammalian VEGF receptors and to induce angiogenesis

Members of the platelet‐derived growth factor/vascular endothelial growth factor (PDGF/ VEGF) family have been implicated in a variety of functions in vertebrates, especially angiogenesis. Here we identify and characterize a PDGF/VEGF‐like factor (named PVF‐1) from the nematode C. elegans. We show that PVF‐1 has biochemical properties similar to vertebrate PDGF/VEGF growth factors. More important, PVF‐1 binds to the human receptors VEGFR‐1 (Flt‐1) and VEGFR‐2 (KDR) and is able to induce angiogenesis in two model systems derived from vertebrates. Our results highlight the widespread evolutionary conservation of this important class of growth factors and raise the possibility that C. elegans can provide a simple experimental system in which to investigate how these factors function.—Tarsitano, M., De Falco, S., Colonna, V., McGhee, J. D., Persico, M. G. The C. elegans pvf‐1 gene encodes a PDGF/VEGF‐like factor able to bind mammalian VEGF receptors and to induce angiogenesis. FASEB J. 20, 227–233 (2006)

The Caenorhabditis elegans intestine

The transcriptional regulatory hierarchy that controls development of the Caenorhabditis elegans endoderm begins with the maternally provided SKN‐1 transcription factor, which determines the fate of the EMS blastomere of the four‐cell embryo. EMS divides to produce the posterior E blastomere (the clonal progenitor of the intestine) and the anterior MS blastomere, a major contributor to mesoderm. This segregation of lineage fates is controlled by an intercellular signal from the neighboring P2 blastomere and centers on the HMG protein POP‐1. POP‐1 would normally repress the endoderm program in both E and MS but two consequences of the P2‐to‐EMS signal are that POP‐1 is exported from the E‐cell nucleus and the remaining POP‐1 is converted to an endoderm activator by complexing with SYS‐1, a highly diverged β‐catenin. In the single E cell, a pair of genes encoding small redundant GATA‐type transcription factors, END‐1 and END‐3, are transcribed under the combined control of SKN‐1, the POP‐1/SYS‐1 complex, as well as the redundant pair of MED‐1/2 GATA factors, themselves direct zygotic targets of SKN‐1 in the EMS cell. With the expression of END‐1/END‐3, the endoderm is specified. END‐1 and END‐3 then activate transcription of a further set of GATA‐type transcription factors that drive intestine differentiation and function. One of these factors, ELT‐2, appears predominant; a second factor, ELT‐7, is partially redundant with ELT‐2. The mature intestine expresses several thousand genes, apparently all controlled, at least in part, by cis‐acting GATA‐type motifs. WIREs Dev Biol 2013, 2:347–367. doi: 10.1002/wdev.93

An acid phosphatase as a biochemical marker for intestinal development in the nematode Caenorhabditis elegans.

We describe an acid phosphatase enzyme (EC 3.1.3.2) that is localized to the intestine of the nematode Caenorhabditis elegans and that should serve as a convenient biochemical marker for gut differentiation. In adult worms, acid phosphatase activity is located along the edge of the gut lumen in the vicinity of the intestinal brush border. All but the anterior six cells of the intestine stain for phosphatase activity; the nonstaining cells all descend from the Ea(l/r)(a/p)a cells. Acid phosphatase activity is low in oocytes and early embryos but increases substantially when embryos reach late morphogenesis stage; this increase corresponds to the appearance of a major band of acid phosphatase activity detectable on isoelectric focusing gels. We designate this band as the product of the pho-1 gene. The pattern of acid phosphatase expression in several embryonic mutants suggests that pho-1 expression in the developing intestine is lineage autonomous. We induced an isoelectric focusing variant in the pho-1 enzyme and used this to map the pho-1 locus about 1.5 map units to the left of center of chromosome II. We purified the pho-1 enzyme to homogeneity (6500-fold purification; 4% recovery of activity); the pho-1 acid phosphatase is a homodimeric glycoprotein with a subunit molecular weight of 55,000 Da. This paper establishes a new experimental system with which to investigate the molecular basis of lineage-specific gene expression during C. elegans development.

Bacterial residence time in the intestine of Caenorhabditis elegans

We fed adult Caenorhabditis elegans fluorescent microspheres mixed with their Escherichia coli food and then measured the total fluorescence of expelled faeces as a function of time after transfer to unlabelled bacteria. Intestinal clearance obeys a simple first-order decay or dilution curve: we estimate that 43 ± 10% of the maximum intestinal volume is expelled in each defecation and the average residence time of a bead (by inference, a bacterium) is less than 2 min. Our results raise questions how bacteria can be sufficiently digested in this brief period to provide energy and material to sustain the high rate of C. elegans oocyte production.

Interference Between the PHA-4 and PEB-1 Transcription Factors in Formation of the Caenorhabditis elegans Pharynx

PHA-4 is a forkhead/winged helix transcription factor that acts as an organ identity factor in the development of the Caenorhabditis elegans pharynx. PEB-1 is a novel DNA-binding protein also involved in pharyngeal morphogenesis. PHA-4 and PEB-1 bind at overlapping sites on the C183 sequence element that controls pharynx-specific expression of the C. elegans myo-2 gene. It has been suggested that PHA-4 and PEB-1 act cooperatively on the C183 sequence. In this study, we test this model and assess the C183-dependent transcriptional activity of PHA-4 and PEB-1, both individually and in combination. We show that PHA-4 and PEB-1 are both modest transcriptional activators in yeast but that co-expression of the two factors does not result in significantly increased expression of a C183-regulated reporter gene. Electrophoretic mobility-shift assays provide no evidence for the formation of a PHA-4/PEB-1 complex in vitro but rather show that PHA-4 and PEB-1 cannot bind C183 simultaneously. As we have reported previously, ectopic expression of PHA-4 in C. elegans causes ectopic expression of a C183-regulated reporter gene. We show that ectopic expression of PEB-1 cannot cause ectopic expression of the same reporter but rather ectopic PEB-1 inhibits reporter gene activation by PHA-4. Overall, our results do not support a model in which PHA-4 and PEB-1 synergize in vivo but rather support a model in which PEB-1 may negatively modulate PHA-4s ability to activate transcription through C183 during formation of the C. elegans pharynx.