Reprogramming of early embryonic blastomeres into endodermal progenitors by a Caenorhabditis elegans GATA factor

The END-1 GATA factor has been implicated in specifying endoderm in Caenorhabditis elegans and is the earliest known zygotic protein expressed in the lineage of E, the clonal endoderm progenitor. We report that ubiquitous end-1 expression during a critical period in embryogenesis causes all non-endodermal lineages to produce endoderm instead of ectoderm and/or mesoderm. END-1 expression bypasses the requirement for maternal SKN-1 and the maternal Wnt signaling pathway in endoderm formation. This suggests that a primary function of these maternal factors is to regulate zygotic end-1 expression, which is then sufficient to initiate the entire program for endoderm development.     

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.     

Anterior-posterior patterning within the Caenorhabditis elegans endoderm

The endoderm of higher organisms is extensively patterned along the anterior/posterior axis. Although the endoderm (gut or E lineage) of the nematode Caenorhabditis elegans appears to be a simple uniform tube, cells in the anterior gut show several molecular and anatomical differences from cells in the posterior gut. In particular, the gut esterase ges-1 gene, which is normally expressed in all cells of the endoderm, is expressed only in the anterior-most gut cells when certain sequences in the ges-1 promoter are deleted. Using such a deleted ges-1 transgene as a biochemical marker of differentiation, we have investigated the basis of anterior-posterior gut patterning in C. elegans. Although homeotic genes are involved in endoderm patterning in other organisms, we show that anterior gut markers are expressed normally in C. elegans embryos lacking genes of the homeotic cluster. Although signalling from the mesoderm is involved in endoderm patterning in other organisms, we show that ablation of all non-gut blastomeres from the C. elegans embryo does not affect anterior gut marker expression; furthermore, ectopic guts produced by genetic transformation express anterior gut markers generally in the expected location and in the expected number of cells. We conclude that anterior gut fate requires no specific cell-cell contact but rather is produced autonomously within the E lineage. Cytochalasin D blocking experiments fully support this conclusion. Finally, the HMG protein POP-1, a downstream component of the Wnt signalling pathway, has recently been shown to be important in many anterior/posterior fate decisions during C. elegans embryogenesis (Lin, R., Hill, R. J. and Priess, J. R. (1998) Cell 92, 229-239). When RNA-mediated interference is used to eliminate pop-1 function from the embryo, gut is still produced but anterior gut marker expression is abolished. We suggest that the C. elegans endoderm is patterned by elements of the Wnt/pop-1 signalling pathway acting autonomously within the E lineage.     

Analysis of a Caenorhabditis elegans Twist homolog identifies conserved and divergent aspects of mesodermal patterning

Mesodermal development is a multistep process in which cells become increasingly specialized to form specific tissue types. In Drosophila and mammals, proper segregation and patterning of the mesoderm involves the bHLH factor Twist. We investigated the activity of a Twist-related factor, CeTwist, during Caenorhabditis elegans mesoderm development. Embryonic mesoderm in C. elegans derives from a number of distinct founder cells that are specified during the early lineages; in contrast, a single blast cell (M) is responsible for all nongonadal mesoderm formation during postembryonic development. Using immunofluorescence and reporter fusions, we determined the activity pattern of the gene encoding CeTwist. No activity was observed during specification of mesodermal lineages in the early embryo; instead, the gene was active within the M lineage and in a number of mesodermal cells with nonstriated muscle fates. A role for CeTwist in postembryonic mesodermal cell fate specification was indicated by ectopic expression and genetic interference assays. These experiments showed that CeTwist was responsible for activating two target genes normally expressed in specific subsets of nonstriated muscles derived from the M lineage. In vitro and in vivo assays suggested that CeTwist cooperates with the C. elegans E/Daughterless homolog in directly activating these targets. The two target genes that we have studied, ceh-24 and egl-15, encode an NK-2 class homeodomain and an FGF receptor (FGFR) homolog, respectively. Twist activates FGFR and NK-homeodomain target genes during mesodermal patterning of Drosophila and similar target interactions have been proposed to modulate mesenchymal growth during closure of the vertebrate skull. These results suggest the possibility that a conserved pathway may be used for diverse functions in mesodermal specification.     

mex-1 and the general partitioning of cell fate in the early C. elegans embryo

It is thought that at least some of the initial specification of the five somatic founder cells of the C. elegans embryo occurs cell-autonomously through the segregation of factors during cell divisions. It has been suggested that in embryos from mothers homozygous for mutations in the maternal-effect gene mex-1, four blastomeres of the 8-cell embryo adopt the fate of the MS blastomere. It was proposed that mex-1 functions to localise or regulate factors that determine the fate of this blastomere. Here, a detailed cell lineage analysis of 9 mex-1 mutants reveals that the fates of all somatic founder cells are affected by mutations in this gene. We propose that mex-1, like the par genes, is involved in establishing the initial polarity of the embryo.     

Cerebral blood flow and left ventricular output in spontaneously breathing, newborn preterm infants treated with caffeine or aminophylline

Recent research into the development of the nervous system of the nematode Caenorhabditis elegans indicates the importance of multiple cell interactions and combinatorial gene expression. As many of the genes needed for C. elegans neuronal development have counterparts with similar activities in Drosophila melanogaster, the mechanisms of cell specification may be broadly conserved.     

Genes necessary for C. elegans cell and growth cone migrations

The migrations of cells and growth cones contribute to form and pattern during metazoan development. To study the mechanisms that regulate cell motility, we have screened for C. elegans mutants defective in the posteriorly directed migrations of the canal-associated neurons (CANs). Here we describe 14 genes necessary for CAN cell migration. Our characterization of the mutants has led to three conclusions. First, the mutations define three gene classes: genes necessary for cell fate specification, genes necessary for multiple cell migrations and a single gene necessary for final positioning of migrating cells. Second, cell interactions between the CAN and HSN, a neuron that migrates anteriorly to a position adjacent to the CAN, control the final destination of the HSN cell body. Third, C. elegans larval development requires the CANs. In the absence of CAN function, larvae arrest development, with excess fluid accumulating in their pseudocoeloms. This phenotype may reflect a role of the CANs in osmoregulation.     

Two distinct populations of primary cytotoxic cells infiltrating into allografted tumor rejection sites: infiltration of macrophages cytotoxic against allografted tumor precedes that of multiple sets of cytotoxic T lymphocytes with distinct specificity to alloantigens

The majority of cases with familial Alzheimer's disease (FAD) are linked to mutations of the presenilin (PS) genes. These genes show considerable sequence similarity to the sel-12 gene of Caenorhabditis elegans, which has been postulated to function in the facilitated signalling by lin-12 and glp-1. In order to analyse the functional conservation of the presenilins, we introduced the human PS-1 cDNA, as well as clinical and deletion mutant proteins, into sel-12 mutant animals and tested their potential to rescue the egg-laying defect. Human PS-1 expressed from the sel-12 promoter fully rescued the sel-12 phenotype, whereas two missense mutations, C410Y and A246E, identified in pedigrees with FAD, exhibited a strongly decreased rescuing activity. The large hydrophilic loop and transmembrane domain 7 are required for the biological activity of PS-1. PS-1 protein was proteolytically cleaved in C. elegans as it is in human cells. A PS-1 splice variant (FAD mutation deltaexon9) that does not undergo proteolytic cleavage also substituted for sel-12. The conservation of function of human PS-1 and C. elegans sel-12 suggests that presenilin proteins are required, directly or indirectly, for the proper operation of the Notch signalling pathway. FAD-associated mutant proteins tested showed different rescuing activities, indicating that they might affect different functional or regulatory aspects of PS-1. Proteolytic processing is not a prerequisite for PS-1 function in C. elegans.     

Congenital motor nystagmus linked to Xq26-q27

Congenital motor nystagmus (CMN) is a hereditary disorder characterized by bilateral ocular oscillations that begin in the first 6 mo of life. It must be distinguished from those genetic disorders-such as ocular albinism (OA), congenital stationary night blindness (CSNB), and blue-cone monochromatism (BCM)-in which nystagmus accompanies a clinically apparent defect in the visual sensory system. Although CMN is presumed to arise from a neurological abnormality of fixation, it is not known whether the molecular defect is located in the eye or in the brain. It may be inherited in an autosomal dominant, autosomal recessive, or X-linked pattern. Three families with CMN inherited in an X-linked, irregularly dominant pattern were investigated with linkage and candidate gene analysis. The penetrance among obligate female carriers was 54%. Evaluation of markers in the region of the genes for X-linked OA, CSNB, and BCM revealed no evidence of linkage, supporting the hypothesis that CMN represents a distinct entity. The gene was mapped to chromosome Xq26-q27 with the following markers: GATA172D05 (LOD score 3.164; recombination fraction [theta] = 0.156), DXS1047 (LOD score 10.296; theta = 0), DXS1192 (LOD score 8.174; theta = 0.027), DXS1232 (LOD score 6.015; theta = 0.036), DXS984 (LOD score 6.695; theta = 0), and GATA31E08 (LOD score 4.940; theta = 0.083). Assessment of haplotypes and multipoint linkage analysis, which gave a maximum LOD score of 10.790 with the 1-LOD-unit support interval spanning approximately 7 cM, place the gene in a region between GATA172D05 and DXS1192. Evaluation of candidate genes CDR1 and SOX3 did not reveal mutations in affected male subjects.     

Multiple isoforms of eukaryotic protein synthesis initiation factor 4E in Caenorhabditis elegans can distinguish between mono- and trimethylated mRNA cap structures

The rate-limiting step for cap-dependent translation initiation in eukaryotes is recruitment of mRNA to the ribosome. An early event in this process is recognition of the m7GTP-containing cap structure at the 5'-end of the mRNA by initiation factor eIF4E. In the nematode Caenorhabditis elegans, mRNAs from 70% of the genes contain a different cap structure, m32,2,7GTP. This cap structure is poorly recognized by mammalian elF4E, suggesting that C. elegans may possess a specialized form of elF4E that can recognize m32,2,7GTP. Analysis of the C. elegans genomic sequence data base revealed the presence of three elF4E-like genes, here named ife-1, ife-2, and ife-3. cDNAs for these three eIF4E isoforms were cloned and sequenced. Isoform-specific antibodies were prepared from synthetic peptides based on nonhomologous regions of the three proteins. All three eIF4E isoforms were detected in extracts of C. elegans and were retained on m7GTP-Sepharose. One eIF4E isoform, IFE-1, was also retained on m32,2,7GTP-Sepharose. Furthermore, binding of IFE-1 and IFE-2 to m7GTP-Sepharose was inhibited by m32,2,7GTP. These results suggest that IFE-1 and IFE-2 bind both m7GTP- and m32,2, 7GTP-containing mRNA cap structures, although with different affinities. In conjunction with IFE-3, these eIF4E isoforms would permit cap-dependent recruitment of all C. elegans mRNAs to the ribosome.     

A C. elegans Hox gene switches on, off, on and off again to regulate proliferation, differentiation and morphogenesis

Hox genes establish body pattern throughout the animal kingdom, but the role these genes play at the cellular level to modify and shape parts of the body remains a mystery. We find that the C. elegans Antennapedia homolog, mab-5, sequentially programs many independent events within individual cell lineages. In one body region, mab-5 first switches ON in a lineage to stimulate proliferation, then OFF to specify epidermal structures, then ON in just one branch of the lineage to promote neuroblast formation, and finally OFF to permit proper sense organ morphology. In a neighboring lineage, continuous mab-5 expression leads to a different pattern of development. Thus, this Hox gene achieves much of its power to diversify the anteroposterior axis through fine spatiotemporal differences in expression coupled with a changing pattern of cellular response.     

Adenovirus E1A expression controlled by the red pigment promoter in transgenic mice: a model for developmental abnormalities of the eye

Four transgenic founder mice were produced that carry the adenovirus E1A gene under control of the human red pigment promoter. Two of the founder animals passed the transgene to progeny, and one line was bred to homozygosity. The line of mice homozygous for the transgene expressed E1A-specific RNA at a low level in their eyes; no expression could be detected in other tissues that were tested. Either the founder animals or their progeny exhibited developmental abnormalities of their eyes, including a small eye phenotype, retinal dysplasia, anterior cleavage syndrome, and retinal pigment epithelium dysplasia. Individuals from the same line of mice exhibited subsets of the abnormalities, e.g., many animals had one normal size eye and one small eye, even though both eyes contained E1A-specific RNA. No tumors resulted from E1A expression in animals monitored for 22 months.