Damjanovski S et al. (2000), Multiple stage-dependent roles for histone deac...

XB-ART-9798

Int J Dev Biol 2000 Oct 01;447:769-76.

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Multiple stage-dependent roles for histone deacetylases during amphibian embryogenesis: implications for the involvement of extracellular matrix remodeling.

Damjanovski S , Sachs LM .

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Histone acetylation has long been implicated in the regulation of gene expression. Recently, a number of histone acetyltransferase and histone deacetylase genes have been identified and cloned. Molecular studies have shown that these enzymes influence transcriptional regulation as components of cofactor complexesthat interact with diversetranscription factors. However, relatively little is known about their function during development. Here, we make use of the ability to manipulate Xenopus laevis embryos in vitro to study the role of histone deacetylases in development. We first demonstrate that the histone deacetylase Rpd3 and its associated co-repressor Sin3A are coordinately expressed during embryogenesis. Rpd3 and Sin3A are known to be part of at least one large corepressor complex, which is involved in transcriptional regulation by many transcription factors, suggesting that deacetylase activity is important for embryogenesis through transcriptional regulation. Indeed, treating developing embryos with a specific histone deacetylase inhibitor, trichostatin A (TSA), leads to embryonic lethality with severe defects in the head and tail regions. Furthermore, the effects of TSA are stage-dependent with the severity of the defects decreasing when treatment is initiated at later stages. On the other hand, a sharp bend (kink) develops in the tail even when TSA treatment begins at tadpole hatching. We provide evidence that this tail defect may be in part due to the TSA-dependent inhibition of the expression of the matrix metalloproteinase gene stromelysin-3, which has been implicated in tail development through extracellular matrix remodeling.

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Species referenced: Xenopus laevis
Genes referenced: hdac1 hdac3 lrp5 mmp11 mmp13 ncr3 rpl8 shh sin3a

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	Fig. 1. Coordinated spatial and temporal expression of histone deacetylase Rpd3 and corepressor Sin3. Digoxigenin labeled Rpd3 and Sin3 antisense mRNA was used as probes in whole mount analysis of their developmental expression in albino Xenopus embryos. (A,B,C,D) Rpd3 and (E,F,G,H) Sin3 expression was examined at the end of neuralation (stage 18/21, A,E), tailbud (stage 31/32,B,F), hatching stages (stage 35/36, C,G), and at the onset of feeding (stage 45, D,H). Both Rpd3 and Sin3 were abundantly ex- pressed, particularly in dorsal axial structures and in head and anterior structures. Their expression diminished by tadpole feeding. Their expression profiles overlap extensively. Note that Rpd3 was also expressed in the dorsal region of the embryo in the area of the proctodeal channel (boxed area, which was enlarged in the insert in B), where stromelysin-{remaining fig caption text missing from publication}
	Fig. 2. Blocking histone deacetylases leads to stage-dependent developmen- tal defects. TSA was added to embryo or tadpole-rearing water at a concentration of 100 nM at various developmental stages as indicated and phenotypic effects were subsequently assayed when control ani- mals reached indicated stages. All em- bryos within a treatment group developed essentially identical phenotypes (also see Table 1). In general all treatment groups were delayed in their development, and treatment of embryos up to and including stage 26 resulted in developmental arrest and death when control embryos reached feeding stage. All treatments, except when started at stage 41 or later, resulted in kinked tails. Earlier treatments (starting prior to stage 26) also resulted in embryos with eye and head defects, ventral swell- ing, and impaired swimming ability.
	Fig. 3. TSA treatment prior to and at stage 20 results in severe head and eye defects. Embryos treated with 100 nM TSA beginning at stage 18 displayed severe head and tail defects when examined when control sibling reached stages 35 (A and B) and 41 (C). The two sides of the same four embryos were pictured in A and B to demonstrate the pres- ence of only one pigmented structure, pre- sumable the rudiment of the eye, on only one side of the embryo (A, arrow) but not on the other (B). Note also the irregularly shaped swollen head, protruding cement gland, swol- len tail tip, and the lack of a prominent tail fin. By stage 41 (C), the swelling in the head is more pronounced and in addition to the single pigmented area present in earlier embryos, often an additional pigmented structure devel- ops on the same side (arrowhead).
	Fig. 4. TSA treatment causes numerous tissue defects in embryos. Embryos treated without (control) or with TSA begin- ning at stage 20 were fixed when control embryos reached stage 35 and sectioned for histological examination. (A,C) Longitu- dinal and (B,D) cross sections were exam- ined of control (A,B,) and TSA treated (C,D) embryos. (A,B) Control embryos revealed well defined structures such as eye (ey), branchial arches (ba), otic vesicle (o), heart (h), well organized somites (s), tail fin (f), and narrow proctodeal channel (pc). (C) Mid trunk cross sections revealed a well- organized neural tube (nt), notochord (n) epidermis (e) and somites (s). (D,E) TSA treatment resulted in swelling in the head (he) and thorax (ve), a poorly organized eye (ey), poorly organized somites (s), and a wide and irregularly shaped tail and proctodeal channel (pc). In cross section (D) the neural tube (nt) and the notochord (n) were seen to be irregular, the epidermis appears thickened (e), and the somites (s) were disorganized. Bar represents 1 mm in A and C and 0.4 mm in B and D.
	Fig. 5. Stromelysin-3 (ST3) expression, but not hedgehog (xhh) or collagenase-3 (Col3) expression is altered by TSA. Albino embryos with- out (control, top of each panel ) or with (bottom of each panel) 100 nM TSA treatment from stage 20/21 were fixed when control embryos reached stage 33-35 (A,B,C), or stage 41 (D,E,F). ST3 (A, D), xhh (B, E) and Col3 (C, F) mRNA expression was examined by whole mount in situ hybridization on fixed animals. ST3 was largely expressed in the head and the dorsal/proctodeal endoderm region (A, D single arrow) of control animals. Following treatment with TSA, the expression of ST3 was largely unchanged with the exception of the reduction in dorsal/ proctodeal endoderm region (A, D arrow). Despite the TSA induced changes in phenotype, the expression patterns of xhh and Col3 were essentially unchanged (compared the top to bottom animal in B, C, E, F). Note that the kink in the tail of TSA-treated embryos was not very obvious compared to that in Fig. 2. This is due to the whole mount in situ procedure. The protease digestions, high temperatures, and dehydration process caused all embryos to be dorsally curved, thereby diminishing the kinked tail phenotype associated with TSA.
	mmp11 (matrix metallopeptidase 11) gene expression in Xenopus laevis embryos, NF stage 33-35, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	mmp11 (matrix metallopeptidase 11) gene expression in Xenopus laevis embryos, NF stage 41, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	mmp13 ( matrix metallopeptidase 13 (collagenase 3) ) gene expression in Xenopus laevis embryos, NF stage 33-35, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	mmp13 ( matrix metallopeptidase 13 (collagenase 3) ) gene expression in Xenopus laevis embryos, NF stage 41, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	Fig. 6. RT-PCR analysis confirms the reduction in ST3 expression in embryonic tails by TSA treatment. Embryos were treated without (control, -) or with (+) TSA beginning at stage 20. When control embryos reached stage 35, RNA was isolated from total embryos or just the tail region (the posterior 25% of the embryo). The RNA was used for semi- quantitative comparison of the levels of expression of ST3 and the control gene encoding the ribosomal protein L8, rpl8, whose expression did not vary due to TSA treatment in the whole embryo or the tail section. ST3 expression in the total embryo was not affected by TSA but there was a dramatic decrease in ST3 expression in the tail.