Analysis of Upstream Elements in the HuC Promoter Leads to the Establishment of Transgenic Zebrafish with Fluorescent Neurons

Dbx homeodomain proteins are important for spinal cord dorsal/ventral patterning and the production of multiple spinal cord cell types. We have examined the regulation and function of Dbx genes in the zebrafish. We report that Hedgehog signaling is not required for the induction or maintenance of these genes; in the absence of Hedgehog signaling, dbx1a/1b/2 are expanded ventrally with concomitant increases in postmitotic neurons that differentiate from this domain. Also, we find that retinoic acid signaling is not required for the induction of Dbx expression. Furthermore, we are the first to report that knockdown of Dbx1 function causes a dorsal expansion of nkx6.2, which is thought to be the cross-repressive partner of Dbx1 in mouse. Our data confirm that the dbx1a/1b/2 domain in zebrafish spinal cord development behaves similarly to amniotes, while extending knowledge of Dbx1 function in spinal cord patterning. Developmental Dynamics 236:3472-3483, 2007.

Section: Effects On Dbx Gene Expression Lead To Changes In the Specifmentioning

confidence: 99%

Regulation and function of Dbx genes in the zebrafish spinal cord

Gribble

Nikolaus

Dorsky

2007

“…In particular, to understand morphogenesis fully, it will be important to follow cell movements in live embryos. Recently, many groups have reported successful zebrafish transgenesis by using a variety of promoters to drive expression of GFP (e.g., Motoike et al, 2000;Park et al, 2000;Koster and Fraser, 2001). Transgenic lines expressing GFP in cardiac precursors would allow realtime imaging of cardiac morphogenesis in wild-type and mutant embryos, bringing a new level of resolution to our understanding of the wild-type process and the precise roles of key players.…”

Section: Perspectives and Future Directionsmentioning

confidence: 99%

Cardiac patterning and morphogenesis in zebrafish

Yelon

2001

103

Development of the embryonic vertebrate heart requires the precise coordination of pattern formation and cell movement. Taking advantage of the availability of zebrafish mutations that disrupt cardiogenesis, several groups have identified key regulators of specific aspects of cardiac patterning and morphogenesis. Several genes, including gata5, fgf8, bmp2b, one-eyed pinhead, and hand2, have been shown to be relevant to the patterning events that regulate myocardial differentiation. Studies of mutants with morphogenetic defects have indicated at least six genes that are essential for cardiac fusion and heart tube assembly, including casanova, bonnie and clyde, gata5, one-eyed pinhead, hand2, miles apart, and heart and soul. Furthermore, analysis of the jekyll gene has indicated its important role during the morphogenesis of the atrioventricular valve. Altogether, these data provide a substantial foundation for future investigations of cardiac patterning, cardiac morphogenesis, and the relationship between these processes.

“…As a proof-of-principle for FAC-sorting and RNA-profiling fluorescent-proteinlabelled zebrafish neurons, we decided to isolate and compare GFP-labelled neurons from Tg(pax2a:GFP) embryos (Picker et al, 2002) and EGFP-labelled neurons from Tg(elav13:EGFP) embryos (Park et al, 2000). In addition, as a control comparison population, we isolated samples of all trunk cells.…”

Section: Resultsmentioning

confidence: 99%

“…Zebrafish (Danio rerio) were maintained on a 14-hr light/10-hr dark cycle at 28.5°C and embryos were obtained from natural, grouped spawnings of identified heterozygous or homozygous Tg(pax2a:GFP) (Picker et al, 2002) or Tg(elavl3:EGFP) (Park et al, 2000) adult fish. Embryos were allowed to develop normally and were staged according to prim staging (Kimmel et al, 1995).…”

Section: Fish Linesmentioning

confidence: 99%

“…Therefore, we reasoned that we could use this line to FAC-sort GFPlabelled CiAs and test whether we could obtain RNA from these cells of sufficient quality and quantity for expression-profiling. As a further proof-of-principle for FAC-sorting and RNA-profiling fluorescent-protein-labelled zebrafish neurons and as a comparison population for the Tg(pax2a:GFP) samples, we decided to FAC-sort EGFP-labelled neurons from Tg(elav13:EGFP) embryos, in which at least most post-mitotic neurons express EGFP (Park et al, 2000;Gribble et al, 2007). Finally, as a control and further comparison population, we also FACsorted all trunk cells.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

RNA profiling of FAC‐sorted neurons from the developing zebrafish spinal cord

Cerda

Hargrave

Lewis

2008

In this report, we describe a successful protocol for isolating and expression-profiling live fluorescentprotein-labelled neurons from zebrafish embryos. As a proof-of-principle for this method, we FAC-sorted and RNA-profiled GFP-labelled spinal CiA interneurons and compared the expression profile of these cells to those of post-mitotic spinal neurons in general and to all trunk cells. We show that RNA of sufficient quality and quantity to uncover both expected and novel transcription profiles via Affymetrix microarray analysis can be extracted from 5,700 to 20,000 FAC-sorted cells. As part of this study, we also further confirm the genetic homology of mammalian and zebrafish V1 interneurons, by demonstrating that zebrafish V1 cells (CiAs) express genes that encode for the transcription factors Lhx1a and Lhx5. This protocol for dissociating, sorting and RNA-profiling neurons from organogenesis-stage zebrafish embryos should also be applicable to other developing organs and tissues and potentially other model organisms. Developmental Dynamics 238:150 -161, 2009.