Single Whole mount in situ hybridization of RNA

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The gene expression of pax gene family is very evident in a Danio rerio (Zebrafish) embryo. ISH (in situ hybridization) using DIG labeled antibodies were used to have tissue specific pax gene expression, the embryo was developed 32h p.f. The pax 3 gene was present in probe C; pax 3 protein activates the genes for specialized gene function in early embryonic development, it express in the neural crest cells which produce melanocytes. Pax 6 gene is also known as master sensory organ developer; it controls many specialized aspects of eye development. Pax6 protein activates the genes responsible for formation of eye, spinal cord and brain (CNS). The PCR product was analyzed using BLAST, and product was amplified for gene of interest to produce single strand RNA plasmid which acts complimentary to mRNA.

Introduction:

Genes controlling development are highly conserved among organisms, from nematodes to humans. In studying embryogenesis, the zebrafish (Danio rerio) is a critical animal model. The adults are highly fecund while the embryos with their small size, rapid development and exceptional optical clarity offer an edge to embryological studies (Kimmel et al. 1990). Whole mount ISH uses a labeled RNA strand (i.e. probe) to localize a specific RNA sequence in an entire tissue. In this experiment, ISH of digoxigenin (standard immunohistochemical marker for ISH)-labeled antisense RNA probes to whole mount zebrafish embryos was used. Embryos are fixed by permeabilized using BCIP/NBT before being soaked in the digoxigenin-labeled probe. Favoring conditions for specific hybridizations are used to complementary mRNA sequences in the tissues expressing desired gene. Alkaline phosphotase cleaves the probes which enables the covalent coupling of digoxigenin into antisense RNA probes. The probes are detected by immunohistochemistry using an alkaline phosphotase-conjugated antibody against digoxigenin and a chromogenic substrate (Thisse.c et al. 2008).One of the reasons to produce a PCR amplified product for gene of interest (PAX) is to produce single strand RNA plasmid which acts complementarily to mRNA.

The pax (paired box) genes are a family of tissue specific transcription factors differentially expressed during embryonic development (Krauss et al. 1991). The PCR products were analyzed via BLAST (Data not shown) to determine the sequence homology. The sequences of interest were found to be homologous with pax 1, pax 2A, pax 3A, pax 3B, and pax 6A. This experiment reports the embryonic expression pattern of the pax genes in zebrafish embryos during developmental stages ranging from 24hpf (hours post fertilization) to 36hpf.

The pax 6 is expression begins during early neurulation. The hindbrain and the ventral spinal cord extending to the posterior end of the CNS express pax-6. The expression of pax 6 gene is important during cell-to-cell signaling, which would result into induction of eye and regionalization of the brain and spinal cord. After 24 hp.f. Embryos express pax 6 in the brain including eye, the pituitary gland and the olfactory bulb

Material and Methods:

Embryo Culturing:

Freshly spawned fertilized eggs were recovered from the aquarium tanks, suspended in petri dishes in embryo culture medium (E3 medium) and cultured at 28.5 degrees C.

Embryo Harvesting:

Zebrafish embryos at 24hpf were manually dechorionated, stored in methanol, followed by washing with PBS.

Fixation:

1x fish fix was used for 20 mins at room temperature.

Washing:

PTW (PBS with 0.1% tween 20), PBT (PTW with 2% sheep or goat serum, 0.2% BSA)

In situ hybridization was undertaken as previously described.

Reagents:

Hyb Solution, pre-hyb Solution, Staining Buffer, NBT, BCIP, 0.1 PO buffer.

Antibody:

1:200 dilution of DIG-labeled probe (1 ul of probe in 50 ul water and 50 ulformamide).

Embryological Staging guides: kimmel et al 1995, Dahm, R. 2002

Results:

Pax genes govern development of sensory organs:

The pax genes are expressed outside nervous system in early stage of organ development. Pax 1 expressed in developing thymus, pax 2 and 8 expressed in kidney, pax 2 expressed in formation of eye, pax 6 expressed in eye (fig c) and the pancreas, pax 3 expressed in different sections of brain (fig a & b) (Mansouri et al. 1999).

Action of pax3:

Pax3 is expressed in the neural crest, neural tube and somite's (fig a & e), the pax 3 genes are active cells during embryonic development, which is known as neural crest cells.

Pax3 protein regulates the activity of genes such as MITF to form specialized tissues or cell types. Pax3 genes are involved in brain regionalization (mid brain, fore brain, hind brain) (fig. b & f)

Action of pax6:

Pax6 protein activates genes responsible for formation on eyes, spinal cord and brain (CNS) during embryonic development (fig c, d, g, & h), pax6 controls many specialized aspects of eye development, it also regulates the development of olfactory bulb (brain cells to process smell).

fore brain

Cerebellum

Lateral position

Lateral position

Dorsal position

Ventral position

eyes

hind brain

Optic tectum

somites

Mid brain

hind brain

Mid brain

Spinal cord

a

b

d

c

Fig: 32h p.f. zebrafish embryos treated with different RNA probes. a & b - probe C (pax 3a), c & d - probe E (pax 6a)

Note: Pictures taken from my samples

The analysis of the results was done by using the staging embryological guides. One of the most prominent staining was of brain, and optical vesicles. In probe C, cerebellum could be observed while in probe E, a spinal cord was observed. The somites in each embryo vary according to the stage of post fertilization.

h

g

f

e

Fig: 32h p.f. Danio rerio embryos. e & g - probe C (pax 3a), g & h - probe E (pax 6a)

Note: Pictures taken for reference by Dr. Vincent Cunliffe

Discussion:

Pax genes are critical to embryonic development as illustrated by their respective expression in various organs. Indeed, disorders such as aniridia in pax 6 mutants exemplify the importance of pax gene expression.

Pax 6 as a master controller of the sensory system

Both mice and zebrafish express a variant form of the pax-6 message, containing a 14 amino acid insertion in the paired domain (PUSCHEL et al, 1992). The conservation of this variant suggests its importance in developmental processes. Further, pax-6 has been described as a master controller for ocular and sensory development.

The involvement of other conserved genes such as Hedgehog gene and pax-6 in ocular development indicates an interaction between them.

Both copies of Pax-6 are required for ocular development

Heterozygous mutations of pax-6 cause aniridia (Yasuda et al., 2002) indicating that the heterozygous state is hypomorphic. It may be worthwhile to investigate such a state.

Pax-3 may assist the activity of pax-6

Pax-3 expression is also important for ocular development. Its contribution may be investigated by tissue specific knockdown of pax-3 while maintaining pax-6 expression.

The structural and functional conservation of these genes suggests that despite different kinds of cell movements during gastrulation and neurulation, vertebrates share a common genetic program of development (Dressier and Grass,1988; Kimmel, 1989).

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