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First published online December 17, 2003
doi: 10.1242/10.1242/dev.00903

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Hyzic, the Hydra homolog of the zic/odd-paired gene, is involved in the early specification of the sensory nematocytes

Molecular Cell Biology, Darmstadt University of Technology, Schnittspahnstraße 10, 64287 Darmstadt, Germany

View larger version (50K): [in a new window]   Fig. 1. Sequence analysis of Hyzic. (A) Schematic of the predicted HyZic protein showing the zinc finger domain located in the C-terminal part. Numbers represent amino acid positions. (B) Alignment of the zinc finger domain of HyZic (Hydra-Zic) with various Zic proteins and Gli proteins from other phyla. The Cysteine and Histidine residues of the C2H2 motifs are boxed in red. Identical amino acids or conservative changes in all sequences are boxed in black; sequences with >80% similarity are boxed in gray. Note, that HyZic contains a number of diagnostic residues of the Zic protein subfamily. (C) Phylogenetic tree of Zic, Gli and Macho zinc finger motifs using the maximum likelihood program TREE-PUZZLE (Schmidt et al., 2002). The analysis is based on CLUSTALW alignments. Numbers are percent statistical support. HyZic clusters showed >90% statistical support to the Zic protein family, when the more derived Ciona sequences were removed from the analysis (data not shown).

View larger version (65K): [in a new window]   Fig. 2. mRNA expression patterns of Hyzic (A), Cnash (B) and Nowa (C) in Hydra vulgaris whole mounts.

View larger version (66K): [in a new window]   Fig. 3. Cell cluster analysis of Hyzic expression. (A-E) Nests of 1, 2, 4, 8 and 16 Hyzic-positive nematoblasts, counter-stained with DAPI. (F) Quantitative analysis of the various nest sizes expressing either Hyzic, Cnash or Nowa. Data shown are mean±s.d. of 15-20 animals.

View larger version (88K): [in a new window]   Fig. 4. Hyzic expression during different stages of oogenesis. (A) Early stage 1 [stages according to Miller et al. (Miller et al., 2000)] of egg field formation, without a morphological sign of an oocyte but already devoid of Hyzic expression. (B) Stage 2 of oocyte development indicated by the swollen ectoderm in the upper part of the body column. (C) Stage 5, when most of the oocyte has already retracted from the eggfield. (D) Mature oocyte (stage 7). Note, that the former egg field is still completely devoid of Hyzic expressing cells.

View larger version (94K): [in a new window]   Fig. 5. Double in situ hybridization with probes against Hyzic, Cnash and Nowa, in all possible combinations. (A) Hyzic (red) and Cnash (blue) show no overlap in their expression patterns. (B) Similarly, Hyzic (red) and Nowa (blue) are expressed in different subpopulations of nematoblasts. (C) Only the messages of Cnash (blue) and Nowa (red) show a substantial co-localization, as demonstrated by the violet cell nests. (D) Quantitative analysis of the overlapping expression of Cnash and Nowa. Scale bars: 50 µm (A-C).

View larger version (74K): [in a new window]   Fig. 6. BrdU/in situ double labeling. (A-D) BrdU pulse labeling combined with in situ hybridization against Hyzic (A,D), Cnash (B) and Nowa (C). Gene expression is shown in black, whereas BrdU-positive nuclei appear in green. (A) A substantial fraction of Hyzic-expressing cells are BrdU positive. (B) No Nowa-expressing cell nests are positive for BrdU. (C) Cnash-expressing cell nests also show no BrdU signal. (D) Hyzic/BrdU double labeling further reveals that some BrdU-positive nests do not express Hyzic (arrows), indicating that not all proliferating cell nests express Hyzic. (E) Quantitative analysis of Hyzic, Cnash and Nowa expressing clusters of all sizes that were continuously labelled with BrdU at 0, 12, 24 and 48 hours.

View larger version (72K): [in a new window]   Fig. 7. Loss of Hyzic and Cnash expressing cells after treatment with hydroxyurea. (A-C) Hyzic expression after 0 (A), 12 (B) and 18 (C) hours of hydroxyurea (HU) treatment. (D-F) Cnash expression after 0 (D), 48 (E) and 72 (F) hours of HU treatment. (G) Quantitative analysis of loss of cell clusters expressing Hyzic, Cnash and Nowa. Very similar results were obtained by stem cell depletion of the temperature-sensitive mutant sf-1 (Terada et al., 1988). Data shown are mean±s.d. of 15-20 animals.

View larger version (23K): [in a new window]   Fig. 8. The role of Hyzic in neuronal differentiation. (A) Schematic of the stem cell proliferation, nerve and nematocyte differentiation pathway in Hydra [modified from David and Gierer (David and Gierer, 1974)]. Determination of stem cells to neuron or nematocyte differentiation can occur at any proliferating stage from 2 to 8 cells (white circles in the middle). The probability of a determination event is represented by the thickness of arrows (the thicker the arrow the more likely the event). After determination to nematocyte differentiation, nematoblasts undergo a variable number of cell divisions leading to nests of 2, 4, 8, 16 and 32 cells. At any of these stages, nematoblast cells can undergo a final mitosis and enter the differentiation program, which is mainly characterized by the development of the nematocyst (shown by the increasing black color). The red labeled circles represent Hyzic-expressing proliferating cells already committed to the nematocyte pathway. The onset of Hyzic expression is not sharply defined, so that additional proliferating nests could be detected that might also give rise to nerve cell precursors or enter the nematocyte pathway at later stages (see also Fig. 6D). Post-mitotic differentiating nematocytes express Cnash and Nowa, as indicated by the blue circles. (B) Genetic pathway leading to nematocyte differentiation. It is unclear whether Chordin also acts upstream of Hyzic in Hydra. (C) Genetic regulation of neural differentiation in vertebrates, showing that at least some homologous genes are involved in neural differentiation in both Hydra and vertebrates.

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