A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Summary

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Abstract

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Protocol

Our goal here is to test the functional redundancy of two transcription factors for specification of cardiomyocyte progenitors. The factors are encoded by the two related genes gata5 and gata6 and we are using the zebrafish model to understand their relative functions¹. Our strategy is to block gene function using morpholinos². We will inject morpholinos for one or the other gene into fertilized eggs, and compare the embryonic phenotype with embryos derived from eggs injected simultaneously with both morpholinos. To simplify the evaluation of phenotypes, we use eggs derived from fish that carry a transgene that expresses GFP in ....

Discussion

In the experiment described here, we combined two morpholinos, each of which alone generate a distinct range of phenotypes, and found an entirely different phenotype when they were co-injected together. Of course, we needed a justification for doing this experiment in the first place. We suspected that the two genes might compensate each other for an earlier function, in this case cardiac specification. This is because the genes are highly related (and capable of binding to the same DNA sequences) and are expressed in ov.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fish breeder tanks and dividers	Aquatic Habitats
Fish nets	Aquatic Habitats
System water			60mg Instant Ocean” per liter dH2O
100mm Petri dishes	BD Biosciences	351029
Micropipette needle puller	Sutter Instrument Co.	P-97 Flaming/Brown
3.5" glass capillaries	Drummond Scientific	3-000-203-G/X
Razor blade	Personna Medical Care	94-120-71
Micro grinder	Narishige International	EG-4
Gridded microscope eyepiece	Premiere	MF-02
Micrometer	WARD’s Natural Science	94 W 9910
Ultrapure agarose	Invitrogen	15510-027
Spatula	Fisher Scientific	14-357Q
Scale	Mettler Toledo	AB54-S/FACT
Disposable weigh dishes	Fisher Scientific	2-202-B
Conventional microwave	GE Healthcare
Erlenmeyer flask	Corning	4980
Microscope slides	Fisher Scientific	12-552
Graduated cylinder	Fisher Scientific	08-572-6D
Automatic pipette man	BrandTech	2026333
70% ethanol wash solution	Tarr	801VWR
N2 tank	Tech Air
Microinjector	Harvard Apparatus	PLI-100
Micromanipulator	Micro Instruments	LTD
Dissecting microscope	Nikon Instruments	SMZ1500
Parafilm	American National Can	PM-992
Mineral oil	Sigma-Aldrich	M5904
Kimwipes	Kimberly-Clark Corporation	34155
gata5 splice site morpholino	Genetools, LLC		5’-TCTTAAGATTTTTACCTATACTGGA-3’
gata5 ATG Blocker	Genetools, LLC		5’-AAGATAAAGCCAGGCTCGAATACAT-3’
gata6 Long Isoform ATG Blocker	Genetools, LLC		5’-AGCTGTTATCACCCAGGTCCATCCA-3’
Disposable Pasteur Pipette	Fisher Scientific	13-678-20B
Tricaine Methanesulfonate	Argent Labs	MS-222
Methycellulose	ICN Biomedicals	155495
Heat Block	Fisher Scientific	11-718-4
Sharp forceps	Dumont		Size 55
Depression Microslides	VWR international	48339-009