Figure 2 The Bicc1 protein contains 2 functional domains: KH domains and a SAM domain. Similarity is observed in the amino acid sequence alignment for Bicc1 functional domains using ClustalW.  The KH and SAM domains are shown. The top row depicts the mouse Bicc1 peptide sequence with zebrafish Bicc1 underneath.  Blocks of colored amino acids represent identical or conserved residues between the two species.  The overall percent identity between the mouse and zebrafish for each functional domain is indicated on the right. Green:  hydroxyl and amide residues; Blue:  small hydrophobic and aromatic residues; Red:  basic residues; Purple:  acidic residues; Orange:  Glycine;  Gold:  Proline; Teal:  Histidine.
Figure 4. Phenotype of morphants
Each panel depicts representative animals at 72 hours post fertilization. No gross abnormalities were detected in animals injected with the control MO. In the animals injected with the zbicc1 MO, edema (arrowheads) and pronounced dorsal curvature of the trunk (asterisk) were noted.
Body curvature and edema are consistent with other cystic models.
If mouse and zebrafish Bicc1 have equivalent functions in the kidney,  addition of the mouse Bicc1 gene into zbicc1 knockdown zebrafish embryo will rescue the PKD phenotype.
Polycystic Kidney Disease (PKD) is an inherited disorder affecting nearly 600,000 Americans.  Clinical signs include renal enlargement, progressive cyst formation and ultimately end-stage renal disease.  Mutations in Bicc1 result in PKD in the mouse. In this experiment a zebrafish model for PKD is established by using morpholinos (MO) to knockdown expression of Bicc1.
Insertion of the Mouse Bicc1 Gene Into zbicc1 Knockdown Zebrafish Embryos
Will Rescue the PKD Phenotype
B. Victoria Nelson*1, Denise J. Bouvrette1, Anand Chandrasekhar2, Elizabeth C. Bryda1
1Department. of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO.
2Department of Biological Sciences, University of Missouri, Columbia, MO
•Bicc1 is highly conserved across species, especially within the functional domains.
•Morpholino knockdown of zbicc1 results in an abnormal phenotype.
•It is predicted that mouse Bicc1 can rescue the cystic phenotype in zbicc1 morphants.
•Success of this experiment will support the use of the zbicc1 zebrafish model as an alternative to the mouse model to better understand the role of Bicc1 in kidney development and renal cyst formation.
Patented by Higuchi et al., 1993, Japan
Figure 3. Injection of embryos with either:
•Morpholino to knockdown endogenous zbicc1 mRNA and Phenol red
•Control morpholino and Phenol Red
23
40
37
53
81
87
91
92
93
95
90
amino acid
% identity
8
47
45
24
69
77
84
86
86
92
85
nucleotide
% homology
?
Caenorhabditis elegans
3R
Anopheles gambiae
2L
Drosophila melanogaster
?
Danio rerio
?
Xenopus laevis
6
Gallus gallus
28
Bos taurus
4
Canis familiaris
10
Pan trogodytes
20
Rattus norvegicus
10
Homo sapiens
Chromosome #
10
Mus musculus
vs.
Table 1.  Conservation of Bicc1 at both the amino acid and nucleotide levels across species. ? = chromosomal location unknown.
C
RNA binding
Protein interactions
N
 SAM
KH1
KH2
KH3
Figure 1.  Functional domains within the predicted Bicc1 protein.
Bicc1 Protein
Step 1: Clone mouse Bicc1 gene into zebrafish expression
vector
pCS2+
+
Hypothesis
Introduction
Rescue of zbicc1 Morphant With mBicc1
Acknowledgements
Conclusions
Step 3: Observe phenotype up to 6 days post fertilization.
Predicted results:
Step 2: Inject zbicc1 morpholino + mBicc1 construct into
2 to 4 cell stage zebrafish embryos
zbicc1 MO
+mBicc1
construct
 Control MO
No
injection
zbicc1 MO
This work was supported in part by a grant to E.C.B. from the American Society of Nephrology (M. James Scherbenske Grant). The University of Missouri Veterinary Research Scholars Program was supported by funds from Merck-Merial, Pfizer and the MU College of Veterinary Medicine.
Development of zbicc1 Model for PKD
Species Conservation
Figure 5. Cross section of kidney caudal to pectoral fin. Histological analysis of morphant phenotype. A: Wild type control embryo, 6 days old, 40X
B: zbicc1 morphant, 6 days old, 40X
NOTE: Cysts lined with flattened epithelium C: Wild type control embryo, 6 days old, 100X D. zbicc1 morphant, 6 days old, 100X NOTE: Cysts lined with flattened epithelium
Pd = pronephric duct
G   = glomerulus
T   = pronephric tubule
Nc = notochord
  *   = cyst
Embryos were fixed in 2% glutaraldehyde/2% paraformaldehyde/0.1M calcium codylate buffer, dehydrated in a graded ethanol series, and dorsally embedded in JB4 resin.  Embryos were cross-sectioned at 4mm, mounted on slides and counter-stained with toluidine blue.  Shown here are representative samples of control embryos and zbicc1 morphants (n=4). 
PKD (cysts)
Normal
Normal
Normal
RESCUE
mBicc1
pCS2+
mBicc1