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doi: 10.1242/10.1242/dev.00483

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The C. elegans Hand gene controls embryogenesis and early gonadogenesis

Laura D. Mathies1, Samuel T. Henderson2 and Judith Kimble1,*

1 Howard Hughes Medical Institute and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706-1544, USA
2 Institute for Behavioral Genetics, University of Colorado-Boulder, Boulder, CO 80309-0447, USA



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  		Fig. 1. Early gonadogenesis in C. elegans. Somatic gonadal precursors (SGPs: Z1 and Z4), dark gray; primordial germ cells (PGCs: Z2 and Z3), light gray. (A) SGPs are specified within the mesodermal layer (white circles) and then migrate to meet PGCs. (B) SGPs and PGCs coalesce into the gonadal primordium, which at this stage has a left-right orientation. (C) During embryo morphogenesis, the gonadal primordium shifts to an anterior-posterior orientation, and acquires left-right and dorsal ventral axes. The first SGP division is asymmetric and segregates the potential to make two regulatory cells: anchor cells (AC) and distal tip cells (DTC). Genes crucial for early SGP divisions are noted.

 


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  		Fig. 2. The hnd-1 gene encodes the C. elegans Hand transcription factor. (A) hnd-1 (formerly C44C10.8) genomic organization. White, untranslated regions; gray and black, coding region; black, basic helix-loop-helix (bHLH) domain. Brackets below mark end points of hnd-1 deletion. (B) Amino acid sequence alignment of bHLH domains of human (h), Xenopus (x), zebrafish (z), Drosophila (Dm) and C. elegans (Ce) HAND proteins. (C) hnd-1 reporters. Top, hnd-1(FL)::GFP inserts GFP after amino acid 163. Middle, hnd-1(N)::GFP fuses GFP to the N terminus of HND-1 and uses the unc-54 3'UTR (light gray). Bottom, hnd-1::GFPlacZ replaces most of the hnd-1 coding region with a GFPlacZ fusion and the unc-54 3'UTR (light gray). GFP and GFPlacZ are not to scale.

 


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  		Fig. 3. hnd-1 morphogenesis defect. (A,C,E) wild type, (B,D,F) hnd-1(q740). (A) Wild-type L1 with normal body morphology. The gonadal primordium is bracketed. Three cells are visible: Z1, black arrow; PGCs, white arrows; Z4 is in a different focal plane. (B) hnd-1 L1 with typical body shape defect (black arrow). Arrested larvae often have vacuoles in the head (arrowheads). (C) Wild-type pretzel-stage embryo is elongated and contains pharynx (ph) and gut. (D) hnd-1 pretzel-stage embryo has not elongated posteriorly (arrow), but has a fully developed pharynx and gut tissue, which can be disorganized. (E) Wild-type embryos stained with {alpha}-myosin antibodies. Two muscle quadrants are visible in this plane (arrowheads). (F) hnd-1 embryos generate four muscle quadrants, which can be disorganized posteriorly (arrow); three quadrants are visible in this plane (arrowheads). Scale bar: 10 µm.

 


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  		Fig. 4. hnd-1 gonadal defects. (A-D) L4 hnd-1(RNAi) hermaphrodites, DAPI stained to highlight nuclei. Dashed line delineates the extent of the gonad. Arrowhead, distal end; carat, center of gonad (vulva). (A,B) Anterior half of animal is on the left, posterior on the right. (A) Two-armed gonad. (B) One-armed gonad. (C) No apparent gonad. (D) Abnormal gonad.

 


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  		Fig. 5. Gonadal primordia in hnd-1 L1 larvae. All images are hnd-1(RNAi). Black arrow, SGP with name of cell; asterisk, PGC. (A-C) pes-1::GFP marks SGPs, GFP overlays DIC image. (A,B) Primordium with two SGPs. Right focal plane shows Z1 (A). Left focal plane shows Z4 (B). (C) Primordium with one SGP, left focal plane. (D) Primordium with no SGPs, two PGCs are present, but are separated. (E-J) lag-2::GFP marks SGPs. (E-G) Two SGPs in normal positions. Z1 is at anterior pole on right (E), Z4 is at posterior pole on left (F). Z1 and Z4 extend cytoplasmic processes to meet mid-ventrally (G, white arrow). (H-J) Primordium with one misplaced SGP. Z1 is displaced dorsally (H), Z4 is located at the posterior pole (I). Z1 and Z4 meet mid-dorsally via a thin cytoplasmic process (J, white arrow). Scale bar: 5 µm.

 


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  		Fig. 6. hnd-1::GFP expression. (A) Lineage diagram depicting cells that express hnd-1::GFP (green). Dashed lines indicate approximate stage of embryos in panels B-D. (B-H) Confocal images of embryos expressing hnd-1::GFP (green). (B-D) Projections of z-series; embryos were observed over time to identify cells. (B) hnd-1(N)::GFP expression is first detected in four granddaughters of MS, descendants of MS.ap and MS.pp, in four granddaughters of C, descendants of C.ap and C.pp (C.xp), and in two daughters of D. (C) hnd-1(FL)::GFP expression in granddaughters of MS.ap/MS.pp and daughters of C.ap/C.pp. (D) hnd-1(N)::GFP expression fades after the next division of most hnd-1-expressing cells but is retained in daughters of MS.appp and MS.pppp, and in daughters of C.ppp and C.app. (E-H) Embryos fixed and stained with {alpha}-HLH-1 or {alpha}-PGL-1 (red). (E) Unlike HLH-1 (red), which is detected in body muscle lineages throughout embryogenesis (Krause et al., 1990Go), hnd-1(N)::GFP is absent from body muscles by the comma-stage of embryogenesis; at this time, expression is seen in Z1/Z4. (F) hnd-1(N)::GFP is detected in Z1 and Z4 as they meet the PGCs (Z2, Z3), marked by PGL-1 (red). (G) Shortly after, expression is absent from Z1 and Z4. (H) hnd-1 embryos express hnd-1(N)::GFP in Z1/Z4.

 


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  		Fig. 7. Cell death in hnd-1 mutants. Gonadal primordia: black arrow, SGP with name of cell; asterisk, PGC. (A,B) ced-2 mutants have no corpses near gonad. Z1 is in the right plane (A) and Z4 is in the left plane (B). (C,D) ced-2; hnd-1 double mutant. Z1 is missing, but a cell corpse occurs in its place at the anterior pole of the gonadal primordium (C, open arrow). Z4 is present (D). Scale bar: 5 µm.

 

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© The Company of Biologists Ltd 2003