Antebi A et al. (1996) East Coast Worm Meeting "daf-12 Coordinates Multiple Developmental Events"

Hedgecock EM, Antebi A

[East Coast Worm Meeting, 1996]

In the nematode, developmental events within or between tissues are precisely coordinated. The DAF-12 nuclear hormone receptor plays multiple roles in synchronizing development. As a heterochronic regulator, it instructs stage specific programs in both gonad and soma. In the dauer pathway, it selects dauer or continuous development in response to growth conditions. Gonadal, somatic and dauer functions are genetically separable, suggesting a complex locus, and alleles can be grouped into six idealized classes. In collaboration with Don Riddle, Pam Larsen and Wen Hui Yeh, we have begun a molecular analysis of alleles to correlate structure with function. So far, we have found a sharp clustering of alleles which selectively disrupt dauer formation within the zinc fingers of the receptor. A unifying hypothesis is that a hormone, acting through DAF-12 and related receptors, coordinates events in gonad and some, as well as dauer development. We suggest a simple model in which the commitments or starts to each developmental stage are synchronized by a hormonal pulse available to all tissues. Moreover, we propose that the starts to each developmental stage lie between the molts, as judged by new cuticle synthesis in the hypodermic and transitions in the migratory behavior of gonadal leader cells. These "parastages" may more accurately reflect the actual functional boundaries of developmental stages.

Antebi A et al. (1998) European Worm Meeting "The daf-12 nuclear receptor regulates developmental age and diapause"

Antebi A, Yeh W-H, Hedgecock EM, Snow MI, Riddle DL, Larsen PL

[European Worm Meeting, 1998]

Heterochronic genes record developmental age in the various tissues and select stage-appropriate programs for nematode life stages L1 through adult. Dauer pathway genes mediate commitment to reproductive growth or the L3 dauer diapause in response to environmental stimuli. daf-12 acts at the convergence of these pathways, regulating both developmental age and dauer diapause at larval stage L3. Mutants repeat L2 programs at L3 and later stages, and inappropriately choose between reproductive growth and dauer arrest irrespective of environmental conditions. In the adult, daf-12 mutants also enhance lifespan extension of daf-2 mutants from 2 fold to 4 fold (Larsen et al, 1995). daf-12 encodes a nuclear receptor most closely related to the drosophila DHR96 and vertebrate Vitamin D receptors. daf-12 , two related nematode receptors, and DHR96 define an ancient branch of the nuclear receptor family, sharing a conserved DNA recognition helix. daf-12 alleles fall into 6 classes based on dauer and heterochronic phenotypes. Molecular lesions within a class strikingly cluster in modular domains of the protein, with several implications for receptor function. daf-12 gives rise to 3 isoforms expressed throughout development. We suggest that daf-12 functional complexity can be explained by at least two activities, a and b required for commitment to reproductive growth and the dauer diapause respectively. The molecular identity of daf-12 as a nuclear receptor suggests a hormonal mechanism in regulatory circuits that record developmental age and control dauer diapause. Interactions with daf-2 suggest that adult longevity might also be influenced by heterochronic factors.

Antebi A et al. (1995) International C. elegans Meeting "daf-12, A HETEROCHRONIC GENE THAT CONTROLS CONTINUOUS AND DAUER DEVELOPMENT"

Antebi A, Hedgecock EM

[International C. elegans Meeting, 1995]

Heterochronic genes control the temporal program of larval development. Mutations can advance or delay the maturation of individual tissues. We have identified two heterochronic loci, daf-12 and mig-8, that control gonadal development. These mutations prevent or delay the cell migrations that determine the reflexed shape of the mature gonad (Mig phenotype). daf-12 also regulates hypodermal development. In mutant animals, the seam cells repeat the L1 molt division pattern at the L2 molt. During the L4 intermolt, seam cells often fail to make the larval/adult fate switch, and repeat their stem-cell like division pattern, resulting in gaps in the lateral alae (Lin phenotype). daf-12 also controls the choice between continuous versus dauer development. Depending upon allele, these mutants are defective in dauer formation (Daf-d phenotype), or form dauers constitutively under non-inducing conditions (Daf-c). daf-12 lies at the convergence of dauer and heterochronic pathways. In the dauer pathway, daf-12 integrates inputs from neurosensory and BMP signaling pathways to implement dauer or continuous development (Riddle et al. 1981; Estevez et al 1993; Thomas et al., 1993). In the heterochronic pathway (Ambros, 1989), daf-12 appears to act downstream of lin-14 (and its negative regulator lin-4), but upstream of lin-28 and lin-29. Like lin-14 and lin-28, daf-12 phenotypes are suppressed by dauer development. The various daf-12 phenotypes (Mig, Lin, Daf) are expressed by L3. Experiments with a heat-sensitive allele are consistent with a requirement for daf-12 by L2. daf-12 is a complex locus with six distinct classes of alleles which express either one, two or all three phenotypes. daf-12(+) encodes a nuclear hormone receptor (D. Riddle, P. Larsen, W.Yeh p.c.), suggesting that the temporal program of larval development is coordinated hormonally in nematodes.

Antebi A et al. (1993) International C. elegans Meeting "Mutations in unc-39 perturb cell migration and cell fate."

Hedgecock EM, Antebi A

[International C. elegans Meeting, 1993]

Antebi A et al. (2000) European Worm Meeting "The DAF-12 nuclear receptor is temporally regulated and expressed in target tissues"

Antebi A, Kober-Eisermann C, Tait D

[European Worm Meeting, 2000]

In C. elegans, daf-12 regulates L3 larval stage options of reproductive growth and dauer diapause, and also influences adult longevity. daf-12 encodes a conserved member of the nuclear receptor family with a unique DNA recognition helix, the ESCKA helix. It has two paralogs in C. elegans (NHR-8, NHR48), an ortholog in Drosophila(DHR96) and related homologs (SXR and Vitamin D receptors) in vertebrates. The gene encodes two isoforms (A1 and A3) containing both the DNA and ligand binding domain and one (B1) containing the ligand binding domain only. To better understand daf-12 regulation we made a daf-12::GFP gene fusion tagging the first exon of the A isoforms in cosmid F11A1, a construct which presumably contains the entire daf-12 regulatory region. An integrated transgene rescues the Daf-d phenotype of null mutants, suggesting that the fusion protein is functional. The subcellular localization is nuclear as expected for a transcription factor. DAF-12::GFP is expressed in most cells, including phenotypically affected target tissues. It is expressed from embryo to adult, but with stage and tissue specific dynamics. In particular, it is upregulated in late L1 and L2, the time when the decision between dauer diapause and reproductive growth is made. It is down-regulated in L4, but expression persists in a handful of tissues in the adult, including nervous system, somatic gonad, and intestine. These results are consistent with daf-12s role as being part of regulatory circuits controlling dauer formation, developmental age and aging. We propose that organism-wide commitments to diapause versus reproductive growth entail a hormonal signal that acts through daf-12 in the target tissues. Within target cells themselves, daf-12 is temporally regulated by the heterochronic circuit.

Adam Antebi (2010) East Asia Worm Meeting "Nuclear receptorcontrol of C. elegans reproduction and survival"

Adam Antebi

[East Asia Worm Meeting, 2010]

In many species, endocrine systems coordinate the balance between growth, reproduction and survival. During C. elegans development, unfavorable conditions provoke arrest at the dauer diapause (a long-lived stage geared towards somatic endurance), while favorable conditions promote maturation to reproductive maturity.Dauer formation is governed by an endocrine network consisting of cGMP, TGF-beta, and Insulin/IGF signaling, whichultimately converge on the nuclear hormone receptor DAF-12. Steroidalligands of DAF-12, calledthe dafachronic acids, work as a switch inthe dauer decision: the unligandedreceptor specifies the dauer stage, while the liganded receptor permits reproductive development. Adult animals, too, have apparent reproductive modes and survival modes. Notably, when germline stem cells are removed, animals live 50-60% longer and are stress resistant. Extended survival in this pathway depends on DAF-12 signaling as well asthe DAF-16/FOXO transcription factor. Thus endocrine networksregulate organism-wide checkpoints at several times during the life cycle, mediating the balance between reproduction and survival.

Antebi A et al. (1997) International C. elegans Meeting "The daf-12 nuclear receptor advances life stages and specifies the dauer diapause."

Antebi A, Hedgecock EM

[International C. elegans Meeting, 1997]

C. elegans has six recognized stages, embryo, larval stages L1-L4, and adult, with options for the dauer diapause at the L3 stage. A handful of heterochronic loci specify developmental age in the various tissues. The daf-12 nuclear receptor (see Snow et al.,) is the first identified heterochronic gene that coordinates stage-specific programs throughout gonad and soma. In addition, it plays an instructive role in the dauer pathway, a neuroendocrine signaling pathway controlling the dauer diapause and adult lifespan (Larsen et al., 1995). Mutants generally fail to express programs of L3 and later larval stages on schedule, often repeating cell lineage, pathfinding, and molting programs of the previous stage. Mutants also inappropriately choose between continuous and dauer development, as revealed by dauer defective and dauer constitutive alleles. Alleles fall into six classes based on somatic, gonadal and dauer phenotypes. Correlatively, alleles within a class molecularly cluster within DNA or ligand binding domains of the predicted protein. Genetic interactions suggest that daf-12 acts downstream of lin-14 but upstream of lin-28. daf-12 couples the heterochronic and dauer-signaling pathways, revealing how environmental factors can regulate life history, including perhaps senescence itself. Interestingly, this gene belongs to an ancient branch of the nuclear receptor family, suggesting a conserved role in insects and perhaps other phyla.

Antebi A (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Steroid control of C. elegans life plan and life span"

Antebi A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The steroid hormone receptor DAF-12 lies at the nexus of pathways regulating dauer diapause, developmental timing, and life span. I will discuss our current view of how this signaling pathway integrates environmental cues to coordinate various aspects of animal life history.

A Antebi et al. (1999) International C. elegans Meeting "Molecular genetic analysis of daf-12 and related receptors"

A Antebi, C Kober-Eisermann

[International C. elegans Meeting, 1999]

daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age. In general, nuclear receptors have a modular structure that includes a variable N-terminus with transactivation function, a pair of C2 Zn fingers comprising the DNA binding domain (DBD), a hinge region of variable length, and a C-terminal ligand binding domain (LBD) that contains dimerization and transactivation helixes. At least three daf-12 isoforms are identified by RT-PCR, including one isoform which contains the LBD but lacks the DBD. Most daf-12 alleles are lesions in DBD or LBD, but mutants can be grouped into 6 phenotypic classes based on their dauer defective phenotypes and their heterochronic phenotypes in gonadal and extragonadal tissues. Candidate molecular null alleles, which disrupt DBD and LBD, are dauer defective and have impenetrant heterochronic phenotypes. By contrast, recessive gain-of-function alleles, which retain the DBD but truncate the LBD, are dauer defective and have penetrant heterochronic phenotypes. We propose that products from gain-of-function alleles interfere with the activity of some other locus. Interestingly, two other nematode receptors related to daf-12 , F33D4.1 and ZK662.3 , are found in the C. elegans genome, and are potential candidates for the proposed redundant activity. All three nematode receptors are homologous to the Drosophila DHR96 , revealing that this branch of the nuclear receptor family is phyletically conserved. Like daf-12 , F33D4.1 and ZK662.3 give rise to various alternately spliced products. To investigate the function of these genes, we are generating mutations by reverse genetics and constructing GFP fusion proteins.

Snow MI et al. (1997) International C. elegans Meeting "ANALYSIS OF THE daf-12 GENE TRANSCRIPTS"

Yeh W-H, Larsen PL, Hedgecock EM, Snow MI, Riddle DL, Antebi A

[International C. elegans Meeting, 1997]

Mutations in daf-12 render the animals unable to form dauer larvae; thus, wild type DAF-12 function is required to promote dauer development. The daf-12 gene functions at a late step in the pathway for dauer development and it may be part of a negative regulatory loop with the daf-2 gene. The daf-12 gene was cloned by Tc1 transposon tagging and the cDNA sequence indicates that it encodes a member of the steroid/thyroid hormone receptor superfamily. A mixed stage Northern blot of the daf-12 gene shows three mRNAs. RT-PCR isolation of cDNAs corresponding to the mRNAs yielded three isoforms, two of which lack the zinc finger domain. The daf-12 mutants have been classified into 6 categories based on detailed analysis of the dauer-defective, dauer-constitutive, abnormal cell migration, and abnormal cell lineage phenotypes and the mutations sequenced (see Antebi and Hedgecock abstract). Analysis of the isoform structures and the site of mutations suggests that the nonsense mutations in the 5 prime region may not be nulls, if there is regulatory action by the shorter isoforms. We are in the process of determining when the daf-12 gene is expressed by developmental Northern analysis of RNA from larvae under different growth conditions.