PUBLICATION
The spatio-temporal patterning of Hoxa9 and Hoxa13 in the developing zebrafish enteric nervous system
- Authors
- Doodnath, R., Wride, M., and Puri, P.
- ID
- ZDB-PUB-111024-8
- Date
- 2012
- Source
- Pediatric surgery international 28(2): 115-121 (Journal)
- Registered Authors
- Keywords
- zebrafish, enteric nervous system, Hoxa9, Hoxa13
- MeSH Terms
-
- Animals
- Disease Models, Animal
- Enteric Nervous System/embryology*
- Enteric Nervous System/metabolism
- Gene Expression Regulation, Developmental*
- Hirschsprung Disease/genetics
- Hirschsprung Disease/metabolism
- Homeodomain Proteins/biosynthesis
- Homeodomain Proteins/genetics*
- In Situ Hybridization
- RNA/genetics*
- Zebrafish
- PubMed
- 21971947 Full text @ Pediatr. Surg. Int.
Citation
Doodnath, R., Wride, M., and Puri, P. (2012) The spatio-temporal patterning of Hoxa9 and Hoxa13 in the developing zebrafish enteric nervous system. Pediatric surgery international. 28(2):115-121.
Abstract
Background
Hirschsprung’s disease is characterised by the absence of ganglion cells in the distal bowel, a process which is controlled
by complex genetic pathways. Homeobox genes have a major role in gut development and this is depicted by the enteric Hox code
which describes the different spatial and temporal expression of Hox genes. Hoxa9 and Hoxa13 mutations have been discovered in patients with Hirschsprung’s disease (HD). The aim of this study was to determine the spatio-temporal
pattern of Hoxa9 and Hoxa13 in enteric nervous system (ENS) development using the zebrafish model.
Methods
Purified plasmids that contained the gene of interest were obtained and inoculated into culture medium to exponentially increase
the number of bacteria containing the plasmid. Cells were then harvested by centrifugation and plasmid DNA was extracted,
which was then linearised and precipitated. RNA digoxigenin-labelled probes were made by in vitro transcription reaction.
In situ hybridisation was carried out using these probes on zebrafish embryos which were collected from 24 to 120 h post fertilisation
(hpf), by which time the zebrafish intestine is fully developed. Embryos were then mounted in glycerol and imaged using an
Olympus B40 microscope and images were taken using an Olympus Super F1.8 digital camera.
Results
At 24 hpf, Hoxa9 expression is seen in the forebrain and hindbrain and also in the very distal myotome whereas Hoxa13 expression, however, is seen only at the forebrain and hindbrain. At 48 hpf, Hoxa9- and Hoxa13-labelled cells are seen migrating distally from the forebrain into the notochord and spinal cord. At 72 hpf, Hoxa9-labelled cells can be seen throughout the spinal cord whereas Hoxa13 positive cells are seen migrating down from the spinal cord and in the proximal gut. By 96 hpf, Hoxa9- and Hoxa13-labelled cells have migrated down the full length of the spinal cord and along the proximal and mid intestine. By 120 hpf,
Hoxa9 and Hoxa13 positive cells can be seen along the entire length of the zebrafish intestine.
Conclusions
These results show further evidence that Hoxa9 and Hoxa13 are involved in the early and organised patterning of ENS development in the zebrafish model.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping