PMID- 16642204
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20211020
IS  - 1076-9188 (Print)
IS  - 1076-9188 (Linking)
VI  - 19
IP  - 2
DP  - 2000 Apr
TI  - THE EFFECTS OF 5-AZA-2'-DEOXYCYTIDINE (D-AZA) ON SONIC HEDGEHOG EXPRESSION IN 
      MOUSE EMBRYONIC LIMB BUDS.
PG  - 125-133
AB  - 5-Aza-2'-deoxycytidine (d-AZA) causes temporally-related defects in the mouse. At 
      1.0 mg/kg on gestational day (GD) 10, d-AZA causes hindlimb phocomelia. Sonic 
      hedgehog (Shh) plays a significant role in the normal development of limbs in 
      rodent species. Sonic hedgehog peptides, found in the posterior mesenchyme of 
      limb buds, are involved in patterning functions and in the regulation of both 
      anterior-posterior polarity and proximal-distal outgrowth of the limb. The 
      objective of the present study was to analyze alterations in Shh expression 
      subsequent to d-AZA exposure. Pregnant mice were treated with d-AZA via 
      intraperitonlal injection on GD 10. Controls were untreated. The reverse 
      transcription-polymerase chain reaction (RT-PCR), whole mount in situ 
      hybridization (ISH), and whole mount immunohistochemistry (WMI) were used to 
      analyze expression patterns of Shh . For RT-PCR, embryonic hindlimb buds (buds) 
      were taken 0, 4, 8, 12, or 24 hr after exposure. Cyclophilin was used as the 
      baseline monitor. RNA was transcribed to cDNA and used as template with Shh 
      specific primers for amplification. Whole embryos were collected 12 and 24 hr 
      posttreatment for ISH. An antisense primer specific for Shh was used in an 
      oligo-based ISH protocol. Whole embryos were collected 36 and 48 hr posttreatment 
      for WMI. The antibody corresponding to the amino terminal subunit of the Shh 
      peptide was used. There was a treatment related up-regulation of Shh transcripts 
      by 12 and 24 hr posttreatment. The protein response of up-regulation was 
      detectable by 36 and 48 hr posttreatment. Our data suggest that 
      5-aza-2'-deoxycytidine-induced hindlimb defects may be associated with 
      alterations in the level of Shh expression. This may be part of a cascade of 
      signaling events involved in d-AZA-induced hindlimb defects. Work is ongoing to 
      determine the relationship of other gene species that may cooperate with Shh in 
      the induction of the hindlimb defects.
FAU - Branch, Stacy
AU  - Branch S
AD  - Department of Toxicology, North Carolina State University, Raleigh, North 
      Carolina, USA.
FAU - Smoak, Ida W
AU  - Smoak IW
LA  - eng
GR  - R29 ES008452-06/ES/NIEHS NIH HHS/United States
GR  - R29 ES008452-04/ES/NIEHS NIH HHS/United States
GR  - R29 ES008452-05/ES/NIEHS NIH HHS/United States
GR  - R29 ES008452/ES/NIEHS NIH HHS/United States
GR  - R29 ES008452-02/ES/NIEHS NIH HHS/United States
GR  - R29 ES008452-03/ES/NIEHS NIH HHS/United States
PT  - Journal Article
PL  - United States
TA  - Toxic Subst Mech
JT  - Toxic substance mechanisms
JID - 9512523
PMC - PMC1447672
MID - NIHMS8197
EDAT- 2006/04/28 09:00
MHDA- 2006/04/28 09:01
PMCR- 2008/01/26
CRDT- 2006/04/28 09:00
PHST- 2006/04/28 09:00 [pubmed]
PHST- 2006/04/28 09:01 [medline]
PHST- 2006/04/28 09:00 [entrez]
PHST- 2008/01/26 00:00 [pmc-release]
AID - 10.1080/10769180052008904 [doi]
PST - ppublish
SO  - Toxic Subst Mech. 2000 Apr;19(2):125-133. doi: 10.1080/10769180052008904.