Liver growth in the embryo and during liver regeneration in zebrafish requires the cell cycle regulator, uhrf1

Sadler et al. 10.1073/pnas.0610774104.

Supporting Information

Files in this Data Supplement:

SI Figure 6
SI Table 1
SI Figure 7
SI Table 2
SI Text

Fig. 6. uhrf1 is mutated in hi²⁷² embryos. A. uhrf1 expression was determined in day 5 WT and MT embryos from hi²⁷² by standard PCR, compared to actin (Inset) and by Q-PCR with hi³⁰²⁰ and hi²⁷² embryos (gray bars). top2a levels were determined in day 5 embryos from hi²⁷². Expression levels relative to tbp were calculated and shown as fold change over phenotypically WT siblings. The experiment was run in triplicate; bars indicate the standard deviation. (B) Neighbor-joining tree illustrating the UHRF1 and UHRF2 relationships, corrected for multiple substitutions, gaps excluded, and bootstrapped. The bootstrap numbers are out of 1,000. There are two UHRF1 genes in mammals and only a uhrf1 homolog in the other species listed. (C) Domain map of UHRF1.

Fig. 7. uhrf1 is not required for fin regeneration. The terminal 3-4 mm of the tail fin of uhrf^+/+ and uhrf^+/- 6-month-old fish was resected, and the same fish was inspected for regrowth each day for 14 days. Representative images of each genotype (uhrf^+/+, fish 3; uhrf^+/-, fish 5 as marked in each picture on the left) are shown before the cut (uncut), immediately after the cut (t = 0) and 4 and 11 days of regeneration. By 11 days, »1 mm of regrowth is observed in each fish.

Table 1. Survival of adult zebrafish after PH

Table 2. Primer sequences

 

Primer name	Gene	Species	Application	Sequence 5'-3'
272-F1	uhrf1	D. rerio	Standard PCR	GACGGCATCTACAAGGTGGT
272-R1	uhrf1	D. rerio	Standard PCR	TGGTAGTTTTTGCCCAGGTC
actin-F	b-actin	D. rerio	Standard PCR	CATCAGCATGGCTTCTGCTCT
actin-R	b-actin	D. rerio	Standard PCR	GCAGTGTACAGAGACACCC
hUHRF1-F1q	UHRF1	H. sapiens	Standard PCR	GTAAAGTGGAGGAGACGTTCC
hUHRF1-R1q	UHRF1	H. sapiens	Standard PCR	TCTGCAGAGGCTGGTTCACC
mhGAPDH-Fq	Gapdh	H. sapiens R. norvegicus	Standard PCR	GCCAAAAGGGTCATCATCTC
mhGAPDH-Rq	Gapdh	H. sapiens R. norvegicus	Standard PCR	ATGGCATGGACTGTGGTCAT
rUHRF1-F1q	Uhrf1	R. norvegicus	Standard PCR	cttcctgagcaaggtgaagg
rUHRF1-R1q	Uhrf1	R. norvegicus	Standard PCR	ggtccaggtcatagcgacat
mcycA2-F1	Ccna2	M. musculus	Q-PCR	cttggctgcaccaacagtaa
mcycA2-R1	Ccna2	M. musculus	Q-PCR	atgactcaggccagctctgt
mCcne-Fq	Ccne	M. musculus	Q-PCR	TCTGTGCATTCTAGCCATCG
mCcne-Rq	Ccne	M. musculus	Q-PCR	ACAAAAGGCACCATCCAGTC
mcyclo-F1	cyclophilin	M. musculus	Q-PCR	ggccgatgacgagccc
mcyclo-R1	cyclophilin	M. musculus	Q-PCR	tgtctttggaactttgtctgcaa
muhrf1-F1	Uhrf1	M. musculus	Q-PCR	cttccaagacaggcaaaagc
muhrf1-R1	Uhrf1	M. musculus	Q-PCR	cctctttcaccttgctcagg
zccna2-F	ccna2	D. rerio	Q-PCR	CCAATAACTGAAGCCATAGCCTC
zccna2-R	ccna2	D. rerio	Q-PCR	TACAAATATCTGGCTGAATCAAGC
zccnd1-Fq1	ccnd1	D. rerio	Q-PCR	ctgtgcgacagacgtcaact
zccnd1-Rq1	ccnd1	D. rerio	Q-PCR	ggtgaggttctgggatgaga
zccne-F1q	ccne	D. rerio	Q-PCR	caactacaaccccgacaacc
zccne-R1q	ccne	D. rerio	Q-PCR	tgatccgacagtcgttcaga
zef1a-2	ef1a	D. rerio	Q-PCR	cgtctgccacttcaggatgtg
zef1a-3	ef1a	D. rerio	Q-PCR	ACTTGCAGGCGATGTGAGCAG
zfabp-Fq	fabp10	D. rerio	Q-PCR	cacctccaaaactcctggaa
zfabp-Rq	fabp10	D. rerio	Q-PCR	ttctgcagaccagctttcct
zfoxmll-Fq1	foxm1l	D. rerio	Q-PCR	tcagcctgtgacctcatctg
zfoxmll-Rq1	foxm1l	D. rerio	Q-PCR	aagagagtgctgtcggggta
zjun-Fq	jun	D. rerio	Q-PCR	aagaccctgaagtcgcaaaa
zjun-Rq	jun	D. rerio	Q-PCR	caaaatgtccttcggctctc
zmet-Fq	c-met	D. rerio	Q-PCR	aggaacaggcccagaaagat
zmet-Rq	c-met	D. rerio	Q-PCR	ttgtggctgaaacactctgc
zmyc-Fq	myca	D. rerio	Q-PCR	tgactgtggaaaagcgacag
zmyc-Rq	myca	D. rerio	Q-PCR	gctgctgttgatgctgtgat
ztbp-1F	tbp	D. rerio	Q-PCR	gatcacgcggatttgatctt
ztbp-1R	tbp	D. rerio	Q-PCR	ggggctattgggagacctac
ztop2a-Rq	top2a	D. rerio	Q-PCR	caaaccagcctctttcttcg
ztopo2a-Fq	top2a	D. rerio	Q-PCR	aagtgggcaaaccaaaagtg
zuhrf1-F	uhrf1	D. rerio	Q-PCR	GAAGTGGAGTGTCATTTTCACAC
zuhrf1-R	uhrf1	D. rerio	Q-PCR	AAAACCAGATCCAGGATCGACG

SI Text

Histology. Embryos were processed for H&E staining as described . Apoptosis was detected using the Terminal deoxynucleotidyl Transferase Nick End Labeling (TUNEL) method according to the In Situ Cell Death Detection Kit (Roche Biochemicals, Pleasanton, CA), except that the TUNEL enzyme was reduced to 1:20, and the enzyme labeling step was followed by a 2-hour incubation with a 1:100 dilution of CY3-SA.

Animals were used according to guidelines approved by the Animal Use Committee at Children's Hospital, Boston. Euthanized adult zebrafish were fixed in Bouin's fixative for 24 hours, followed by washing and dehydration through a graded series of ethanol and embedded in paraffin. Four-micromolar sections were cut in three equidistant sagittal steps and processed for hematoxylin and eosin (H&E) staining or for immunohistochemistry. Slides were deparaffinized and rehydrated through a graded ethanol series to water. Endogenous peroxidase activity was blocked by a 10-minute incubation in 3.2% H₂O₂/MeOH. Antigen unmasking was carried out in 0.1 M citrate, pH 7.4 at 80°C for 1 hour, followed by extensive washing, blocking with 10% normal horse serum/PBS, and incubation with 1:1,000 mouse anti-PCNA (P8825; Sigma, St. Louis, MO). Primary antibody was washed off with PBS, followed by incubation with 1:1,000 anti-mouse antibodies conjugated to horseradish peroxidase (Promega; Madison, WI) for 1 hour, washed extensively and developed with a 0.83 mg/ml 3,3-diaminobenzidine tetrahydrochloride PBS with 0.03% H₂O₂ for 6 minutes, counterstained with Harris hematoxylin, dehydrated, and coverslipped.

RNA Extraction, cDNA Preparation, and PCR. All RNA were isolated by using the Qiagen RNeasy kit. For zebrafish embryos, 10 WT and 10 mutant (MT) embryos on day 5 pf from two clutches were collected. Zebrafish adult tissue samples were dissected from one male. cDNA was prepared from 0.5-1 mg of RNA in a 20-ml reaction, using oligo dT nucleotides and the Invitrogen SuperScript II cDNA kit according to the manufacturer's protocol.

Standard PCR on cDNA was carried out as described in ref. 30 by using 0.5-2 ml of cDNA, and 0.2-0.5 mM of each primer listed in SI Table 2, for 30-36 cycles, and run on a 2% agarose gel. Q- PCR was carried out using 1 ml of cDNA, 1X FastStart DNA Master^PLUS SYBR Green I mix (Roche) and 0.1-0.5 mM of each primer, in triplicate using a Roche Light Cycler as follows: 95° C for 15 seconds, 60°C for 15 seconds, and 72°C for 15 seconds for 45 cycles. Expression levels were determined by the DC_T method by using the references tbp for embryos, ef1a for zebrafish adult livers, and cyclophilin for mouse.

30. Sadler KC, Amsterdam A, Soroka C, Boyer J, Hopkins N (2005) Development (Cambridge, UK) 132:3561-3572.

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