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PAG-3, a Zn-finger transcription factor, determines neuroblast fate in C. elegans

Scott Cameron^1,2,*,, Scott G. Clark⁴, Joan B. McDermott⁵, Eric Aamodt⁵ and H. Robert Horvitz¹

¹ Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
² Division of Pediatric Hematology/Oncology, Children’s Hospital/Dana-Farber Cancer Institute, Boston, MA 02115, USA
⁴ Skirball Institute, New York University School of Medicine, New York, NY 10016, USA
⁵ Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA
^* Present address: Departments of Pediatrics and Molecular Biology, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Blvd, Dallas, TX 75390-9148, USA

View larger version (21K): [in a new window]   Fig. 1. Postembryonic development of the ventral nerve cord in C. elegans. (A) X: the positions of the ten cells that undergo programmed cell death. Filled circles indicate the six cells in the midbody that survive and differentiate to become VC motoneurons. Anterior is leftwards and ventral is downwards. (B) The postembryonic cell lineages are shown schematically, with vertical lines representing time and horizontal lines representing a cell division along the anteroposterior body axis. Each P cell divides to produce an anterior daughter, Pn.a, which is a neuroblast, and a posterior daughter, Pn.p, which is hypodermal. W, a neuroblast in the retrovesicular ganglion, follows a division pattern identical to that of Pn.a cells. (C) The nomenclature used to identify cells in the P cell lineages. Cell lineages are from Sulston and Horvitz (Sulston and Horvitz, 1977).

View larger version (24K): [in a new window]   Fig. 2. Numbers of cell corpses in pag-3 mutants. The numbers of cell deaths in pag-3 mutants was assayed by counting cell corpses in the ventral cords of 60-65 early L3 ced-1; pag-3 hermaphrodites using Nomarski microscopy. The difference between the observed number of corpses in ced-1 mutants and the number of deaths that occur in the corresponding lineages of developing animals can be accounted for by an incomplete block in engulfment of cell corpses in the ced-1 mutants and by a role for engulfment in cell killing (Ellis et al., 1991; Reddien et al., 2001). In pag-3 mutants, no cell corpses were observed in regions of the ventral cords that lack cell deaths in wild-type animals. For the analysis of pag-3 alleles, we constructed strains heterozygous for the indicated pag-3 alleles and for a small chromosomal deficiency (Df) which deletes the pag-3 locus (see Materials and Methods). Using an unpaired Student’s t-test, we found the differences in mean corpse number±s.e.m. between pag-3(ls20) and pag-3(ls20)/Df to be significant (P<0.02 for both anterior and posterior ventral cord data), while those between pag-3(n3098) and pag-3(n3098)/Df were not (P<0.40 and P<0.77, anterior and posterior, respectively). Anterior ventral cord, cell corpses generated by W, P1 and P2; posterior ventral cord, corpses generated by P9-P12.

View larger version (34K): [in a new window]   Fig. 3. P cell lineages in pag-3 mutants. Abnormalities in pag-3 lineages are indicated by bold lines; surviving cells that die in wild-type lineages are indicated by circles. Cells that die in wild-type animals are indicated by an x; additional cell deaths observed in pag-3 mutants are indicated by an underlined x. pag-3 mutants initiated P cell divisions at the same time during development as wild-type animals. The time scale in B applies to all lineages. (A) Lineages of wild-type animals from Sulston and Horvitz (Sulston and Horvitz, 1977). (B) Lineages of pag-3(n3098) mutants. (C) P9-P12 cell lineages of three pag-3(ls20) mutants. The variability we observed in the division patterns and cell fates in pag-3 mutants has been observed generally in mutants with cell lineage defects (Sternberg and Horvitz, 1984). In reiterated divisions, the time between mitoses became progressively longer and the morphology during mitosis more abnormal with each round of reiteration, often with what appeared to be aborted attempts at cell division. In those lineages in which the posterior daughter would normally have undergone programmed cell death, cell death often occurred later than observed in wild-type lineages and sometimes did not occur. (D) The cell fates in P cell lineages of wild-type and pag-3 mutant animals. A,B,C,D,E are distinct cell fates, representing cells that may divide, survive and differentiate, or undergo programmed cell death. VA, VB and VC are motoneuron classes (White et al., 1976). ‘Pn.aa’ is a neuroblast that divides to generate daughter cells like those normally generated by Pn.aa. (E) Experimentally determined cell fates in the Pn.a lineages of wild-type (Sulston and Horvitz, 1977; White et al., 1976; White et al., 1986) and pag-3 mutant animals. Cell fates were assessed as described in the text. ±VA is an abnormal VA cell (see text). The fate of the presumptive DAS cell in pag-3 mutants was not tested experimentally (DAS).

View larger version (53K): [in a new window]   Fig. 4. VC and VC-like neurons in pag-3 mutants. (A-F) Anterior is leftwards, and ventral is downwards. (A) Wild-type hermaphrodite stained with antiserum against the neuropeptide FMRFamide, which is expressed by the VC motoneurons. The three VC motoneurons anterior to the vulva are indicated, with a detailed view of the inset containing VC3 shown in C. In C, FMRFamide staining is visible in the cytoplasm surrounding the VC3 nucleus; nuclei are stained with DAPI. (B) pag-3(n3098) hermaphrodite stained with antiserum against the neuropeptide FMRFamide. Seven FMRFamide-positive nuclei anterior to the vulva are shown, with a detailed view of the inset shown in (D). (E-G) A chromosomally integrated Plin-11gfp reporter, nIs106, was used to determine the number of VC and VC-like neurons in wild-type and pag-3 mutant animals. In wild-type animals, the Plin-11gfp reporter is expressed in vulval cells and some head neurons, as well as in the six VC motoneurons (Freyd, 1991). (E) Adult nIs106 hermaphrodite. VC motoneuron nuclei are indicated. Vulval fluorescence obscures one and often two of the VC nuclei that flank the vulva (VC4 and VC5) (White et al., 1976), as in this image of a wild-type animal in which the VC4 nucleus is not visible. (F) Adult nIs106 pag-3(n3098) hermaphrodite. (G) Number of fluorescent nuclei seen in the ventral cords of adult hermaphrodites. pag-3(ls64) is R115opal (Jia et al., 1997). No attempt was made to count nuclei obscured by vulval fluorescence.

View larger version (37K): [in a new window]   Fig. 5. Expression pattern of PAG-3 during postembryonic development of the ventral nerve cord. (A,B) PAG-3 antibody staining of wild-type L1/L2 larvae. In all panels, anterior is leftwards and ventral is downwards. (C,D) DAPI co-staining of animals in A,B. In the postembryonic ventral cord, anterior lineages generally divide earlier than posterior lineages (Sulston and Horvitz, 1977). (A,C) In this animal the P6.aa, P7.aa and P8.aa neuroblasts have divided, while P10.aa and P6.ap are in the process of mitosis. The P9.aa and P11.aa neuroblasts have not yet divided. P12.aa and P12.ap are out of the focal plane in the preanal ganglion. PAG-3 protein was detected in the Pn.aa, but not in the Pn.ap, neuroblasts. By examining the staining of many animals at various stages of development, we could trace PAG-3 protein continuously from the Pn.aa neuroblasts to all three neurons generated by them. In A,C, the Pn.aaa neuroblasts were larger and appeared brighter than their posterior sister cells, the Pn.aap cells, which terminally differentiate (P3-P8.aap) or undergo programmed cell death (P1-P2.aap, P9-P12.aap). (B,D) PAG-3 protein in each of the three terminally differentiating neurons generated by the Pn.aa neuroblasts. (E) Diagram of P cell lineage with cells containing PAG-3 protein indicated by green lines. Pn.aa neuroblasts did not contain detectable PAG-3 when they were generated, but became positive a short time later.

View larger version (57K): [in a new window]   Fig. 6. Identification of PAG-3-staining ventral cord neurons in adult animals. Otherwise wild-type adult C. elegans carrying an integrated Punc-4lacZ transgene were co-stained with PAG-3 antiserum and monoclonal antibody directed against ß-galactosidase. DAPI was used to identify nuclei. (A-D) The posterior ventral cord; anterior is leftwards and ventral is downwards. VA11 and VA12 are marked with open triangles. (E-H) The retrovesicular ganglion and anterior ventral cord; ventral view, anterior is leftwards. AVFR and AVFL are marked with filled triangles. (A) DAPI, (B) ß-galactosidase expression in VA11 and VA12 (Miller and Niemeyer, 1995). (C) PAG-3 staining. (D) Merged image. (E) DAPI staining of nuclei in the retrovesicular ganglion. (F) ß-galactosidase expression in five cells in the retrovesicular ganglion. Punc-4lacZ is expressed in the AVF interneurons, the VA motoneurons, and additional cells in the retrovesicular ganglion (Miller and Niemeyer, 1995). (G) PAG-3 staining. By several criteria (see text), the two anterior-most PAG-3 expressing cells in the retrovesicular ganglion are the right and left AVF interneurons. The two more posterior PAG-3-positive cells in the retrovesicular ganglion are the embryonically generated right and left RIG interneurons; they are marked with asterisks. (H) Merged image.