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.     

The C. elegans lethal gut-obstructed gob-1 gene is trehalose-6-phosphate phosphatase

We identified the gob-1 (gut-obstructed) gene in a forward genetic screen for intestinal defects in the nematode Caenorhabditis elegans. gob-1 loss of function results in early larval lethality, at least in part because of a blocked intestinal lumen and consequent starvation. The gob-1 gene is first expressed in the 8E cell stage of the embryonic intestine, and the GATA factor ELT-2 is sufficient but not necessary for this early phase of gob-1 expression; gob-1 expression later becomes widespread in embryos, larvae, and adults. GOB-1 is a member of the HAD-like hydrolase superfamily and shows a robust and specific phosphatase activity for the substrate trehalose-6-phosphate. Trehalose is a glucose disaccharide found in bacteria, fungi, plants, insects, and nematodes but not in mammals. Trehalose plays a number of critical roles such as providing flexible energy reserves and contributing to thermal and osmotic stress resistance. In budding yeast and in plants, the intermediate in trehalose synthesis, trehalose-6-phosphate, has additional critical but less well-defined roles in controlling glycolysis and carbohydrate metabolism. Strong loss-of-function mutants in the C. elegans tps-1 and tps-2 genes (which encode the two trehalose phosphate synthases responsible for trehalose-6-phosphate synthesis) completely suppress the lethality associated with gob-1 loss of function. The suppression of gob-1 lethality by ablation of TPS-1 and TPS-2, the upstream enzymes in the trehalose synthesis pathway, suggests that gob-1 lethality results from a toxic build-up of the intermediate trehalose-6-phosphate, not from an absence of trehalose. GOB-1 is the first trehalose-6-phosphate phosphatase to be identified in nematodes and, because of its associated lethality and distinctive sequence properties, provides a new and attractive target for anti-parasitic drugs.     

A gain-of-function mutation in oma-1, a C. elegans gene required for oocyte maturation,results in delayed degradation of maternal proteins and embryonic lethality

In vertebrates, oocytes undergo maturation, arrest in metaphase II, and can then be fertilized by sperm. Fertilization initiates molecular events that lead to the activation of early embryonic development. In Caenorhabditis elegans, where no delay between oocyte maturation and fertilization is apparent, oocyte maturation and fertilization must be tightly coordinated. It is not clear what coordinates the transition from an oocyte to an embryo in C. elegans, but regulated turnover of oocyte-specific proteins contributes to the process. We describe here a gain-of-function mutation (zu405) in a gene that is essential for oocyte maturation, oma-1. In wild type animals, OMA-1 protein is expressed at a high level exclusively in oocytes and newly fertilized embryos and is degraded rapidly after the first mitotic division. The zu405 mutation results in improper degradation of the OMA-1 protein in embryos. In oma-1(zu405) embryos, the C blastomere is transformed to the EMS blastomere fate, resulting in embryonic lethality. We show that degradation of several maternally supplied cell fate determinants, including SKN-1, PIE-1, MEX-3, and MEX-5, is delayed in oma-1(zu405) mutant embryos. In wild type embryos, SKN-1 functions in EMS for EMS blastomere fate specification. A decreased level of maternal SKN-1 protein in the C blastomere relative to EMS is believed to be responsible for this cell expressing the C, instead of the EMS, fate. Delayed degradation of maternal SKN-1 protein in oma-1(zu405) embryos and resultant elevated levels in C blastomere is likely responsible for the observed C-to-EMS blastomere fate transformation. These observations suggest that oma-1, in addition to its role in oocyte maturation, contributes to early embryonic development by regulating the temporal degradation of maternal proteins in early C. elegans embryos.     

Multiple regulatory elements with spatially and temporally distinct activities control the expression of the epithelial differentiation gene lin-26 in C. elegans

Epithelial differentiation is a very early event during development of most species. The nematode Caenorhabditis elegans, with its well-defined and invariant lineage, offers the possibility to link cell lineage, cell fate specification and gene regulation during epithelial differentiation. Here, we focus on the regulation of the gene lin-26, which is required for proper differentiation of epithelial cells in the ectoderm and mesoderm (somatic gonad). lin-26 expression starts in early embryos and remains on throughout development, in many cell types originating from different sublineages. Using GFP reporters and mutant rescue assays, we performed a molecular dissection of the lin-26 promoter and could identify almost all elements required to establish its complex spatial and temporal expression. Most of these elements act redundantly, or synergistically once combined, to drive expression in cells related by function. We also show that lin-26 promoter elements mediate activation in the epidermis (hypodermis) by the GATA factor ELT-1, or repression in the foregut (pharynx) by the FoxA protein PHA-4. Taken together, our data indicate that lin-26 regulation is achieved to a large extent through tissue-specific cis-regulatory elements.     

Expression of 'segmentation' genes during larval and juvenile development in the polychaetes Capitella sp. I and H. elegans

Polychaete annelids and arthropods are both segmented protostome invertebrates. To investigate whether the segmented body plan of these two phyla share a common molecular ground pattern, we report the developmental expression of orthologues of the arthropod segment polarity genes engrailed (en), hedgehog (hh), and wingless (wg/Wnt1) in larval and juvenile stages of the polychaete annelid Capitella sp. I and en in a second polychaete, Hydroides elegans. Temporally, neither Wnt1 nor hh are detected in the segmented region of the larval body until after morphological segmentation is apparent. Expression of CapI-Wnt1 is limited to a ring of ectoderm marking the future anus during larval segmentation. CapI-hh is expressed in a ring of the hindgut internal to that of CapI-Wnt1, as well as in a subset of ventral nerve cord neurons, anterior gut tissue, and mesoderm. In both H. elegans and Capitella sp. I, en is expressed in a spatially and temporally dynamic manner in segmentally iterated structures as well as a population of cells that migrate internally from ectoderm to mesoderm, possibly representing a population of ecto-mesodermal precursors. Significantly, the expression patterns we report for wg, en, and hh orthologues in Capitella sp. I and for en in larval development of H. elegans are not comparable to the highly conserved ectodermal segment polarity pattern observed in arthropods at any life history stage, consistent with distinct origins of segmentation between annelids and arthropods.     

Vitamin A deficiency in the late gastrula stage rat embryo results in a one to two vertebral anteriorization that extends throughout the axial skeleton

Vitamin A and its metabolites are known to be involved in patterning the vertebrate embryo. Study of the effect of vitamin A on axial skeletal patterning has been hindered by the fact that deficient embryos do not survive past midgestation. In this study, pregnant vitamin A-deficient rats were maintained on a purified diet containing limiting amounts of all-trans retinoic acid (12 microg atRA/g diet) and given a daily oral bolus dose of retinol starting at embryonic day 0.5, 8.25, 8.5, 8.75, 9.25, 9.5, 9.75, or 10.5. Embryos were recovered at E21.5 for analysis of the skeleton and at earlier times for analysis of select mRNAs. Normal axial skeletal development and patterning were observed in embryos from pregnant animals receiving retinol starting on or before E8.75. Delay of retinol supplementation to E9.5 or later resulted in a marked increase in both occurrence and severity of skeletal malformations, extending from the craniocervical to sacral regions. Embryos from the groups receiving retinol starting at E9.5 and E9.75 had one-vertebral anterior transformations of the cervical, thoracic, lumbar, and sacral vertebrae. Few embryos survived in the E10.5 group, but these embryos yielded the most severe and extensive anteriorization events. The skeletal alterations seen in vitamin A deficiency are associated with posterior shifts in the mesodermal expression of Hoxa-4, Hoxb-3, Hoxd-3, Hoxd-4, and Hoxa-9 mRNAs, whereas the anterior domains of Hoxb-4 and Cdx2 expression are unaltered. This work defines a critical window of development in the late gastrula-stage embryo when vitamin A is essential for normal axial skeletal patterning and shows that vitamin A deficiency causes anterior homeotic transformations extending from the cervical to lumbosacral regions.