vhnf1, the MODY5 and familial GCKD-associated gene, regulates regional specification of the zebrafish gut, pronephros, and hindbrain - PubMed (original) (raw)

vhnf1, the MODY5 and familial GCKD-associated gene, regulates regional specification of the zebrafish gut, pronephros, and hindbrain

Z Sun et al. Genes Dev. 2001.

Abstract

Mutations in the homeobox gene vHnf1 are associated with human diseases MODY5 (maturity-onset diabetes of the young, type V) and familial GCKD (glomerulocystic kidney disease). In an insertional mutagenesis screen in zebrafish, we isolated mutant alleles of vhnf1. Phenotypes of these mutants include formation of kidney cysts, underdevelopment of the pancreas and the liver, and reduction in size of the otic vesicles. We show that these abnormalities arise from patterning defects during development. We further provide evidence that vhnf1 regulates the expression of key patterning genes for these organs. vhnf1 is required for the proper expression of pdx1 and shh (sonic hedgehog) in the gut endoderm, pax2 and wt1 in the pronephric primordial, and valentino (val) in the hindbrain. Complementary to the loss-of-function phenotypes, overexpression of vhnf1 induces expansion of the val expression domain in the hindbrain. We propose that vhnf1 controls development of multiple organs through regulating regional specification of organ primordia. The similarity between vhnf1-associated fish phenotypes and human symptoms suggests a correlation between developmental functions of vhnf1 and the molecular etiology of MODY5 and GCKD.

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Figures

Figure 1

Figure 1

The zebrafish vhnf1 gene. (A) Alignment of amino acid sequences of human (hu) and zebrafish (zf) vHnf1 by the Jotun Hein method. Black boxes show identical residues. Human vHnf1 sequence is from NM 00458. The region C-terminal to the homeodomain is the transactivation domain. The dimerization domain, homeodomain, and the ADII region of the transactivation domain are defined as before (Rey-Campos et al. 1991; Tronche and Yaniv 1992). (B) Diagram of proviral insertion sites in vhnf1. Only relevant exons are shown. I and II show two transcripts resulted from exon-skipping events in hi548 mutant embryos. (C) Disruption of vhnf1 expression by proviral insertions. Day 3 embryos from cross of wild-type TAB fish (WT) or phenotypic embryos from heterozygous crosses of hi548 (548), hi1843 (1843), and hi2169 (2169) were analyzed by RT–PCR for vhnf1 expression. RT–PCR for the β-actin gene was performed as loading control. In lane 2 of the upper panel, two bands migrated closely to each other.

Figure 1

Figure 1

The zebrafish vhnf1 gene. (A) Alignment of amino acid sequences of human (hu) and zebrafish (zf) vHnf1 by the Jotun Hein method. Black boxes show identical residues. Human vHnf1 sequence is from NM 00458. The region C-terminal to the homeodomain is the transactivation domain. The dimerization domain, homeodomain, and the ADII region of the transactivation domain are defined as before (Rey-Campos et al. 1991; Tronche and Yaniv 1992). (B) Diagram of proviral insertion sites in vhnf1. Only relevant exons are shown. I and II show two transcripts resulted from exon-skipping events in hi548 mutant embryos. (C) Disruption of vhnf1 expression by proviral insertions. Day 3 embryos from cross of wild-type TAB fish (WT) or phenotypic embryos from heterozygous crosses of hi548 (548), hi1843 (1843), and hi2169 (2169) were analyzed by RT–PCR for vhnf1 expression. RT–PCR for the β-actin gene was performed as loading control. In lane 2 of the upper panel, two bands migrated closely to each other.

Figure 2

Figure 2

vhnf1 expression during embryogenesis. (A) Time course of vhnf1 expression as indicated by RT–PCR. Samples were collected at indicated stages. (B_–_F) Whole-mount in situ hybridization shown in dorsal views, except for D. Anterior is to the left. (B) vhnf1 expression in tailbud-stage embryos. (C,D) vhnf1 (blue) expression in 4-somite-stage embryos. Embryos were double stained with a krox20 (red) probe. D is in a lateral view to show the intermediate mesoderm (arrow). (E,F) vhnf1 expression in the gut (arrow), the pronephric tubules, and ducts (arrow head) in embryos at 26 hpf (hour postfertilization; E) and 38 hpf (F).

Figure 2

Figure 2

vhnf1 expression during embryogenesis. (A) Time course of vhnf1 expression as indicated by RT–PCR. Samples were collected at indicated stages. (B_–_F) Whole-mount in situ hybridization shown in dorsal views, except for D. Anterior is to the left. (B) vhnf1 expression in tailbud-stage embryos. (C,D) vhnf1 (blue) expression in 4-somite-stage embryos. Embryos were double stained with a krox20 (red) probe. D is in a lateral view to show the intermediate mesoderm (arrow). (E,F) vhnf1 expression in the gut (arrow), the pronephric tubules, and ducts (arrow head) in embryos at 26 hpf (hour postfertilization; E) and 38 hpf (F).

Figure 3

Figure 3

Effects of vhnf1 inactivation on gut development. Anterior is to the left. (A,B) Longitudinal sections comparing the liver (arrow) in embryos at 72 hpf (hour postfertilization). (C_–_L) Whole-mount in situ hybridization in dorsal views. (C,D) insulin expression (arrow) in 48-hpf embryos. Insets show islets at higher magnifications. (E,F) hhex expression in the presumptive liver primordium (arrow) and the pancreatic bud (arrowhead) of 30-hpf embryos. (G,H) axl expression in the anterior foregut (arrow) of embryos at 26-hpf. axl is also present in more anterior endoderm (pe). (I,J) pdx1 expression in the duodenal endoderm (arrow) and the pancreatic bud (arrowhead) of 36-hpf embryos. (K,L) shh expression in the foregut (arrow) of 30-hpf embryos. Wild-type embryo, wt; hi548 mutant embryo, 548; floor plate, fl; pharyngeal endoderm, pe.

Figure 4

Figure 4

Effects of vhnf1 inactivation on pronephros development. Anterior is to the left, except for D and E. (A,B) Embryos raised in fish water containing PTU (1-phenyl-2-thiourea; Westerfield 1993) on day 4 of development showing kidney cyst (arrow) and pericardiac edema (arrowhead) in lateral views. (C) Schematic diagram of the zebrafish pronephros in a dorsal view. Posterior parts of the ducts are not shown. (D,E) Cross-sections comparing the proneprhic tubules (arrowheads) and glomeruli (arrows) in embryos at 54 hpf (hour postfertilization). (F,G) Whole-mount antibody staining with α6F on 38 hpf embryos in dorsal views. Pronephric tubules (arrowheads) started to form at the tip of pronephric ducts (arrows). (H_–_K) Whole-mount in situ hybridization on 38-hpf embryos in dorsal views. (H,I) pax2.1 expression in pronephric tubules (arrows) and ducts. (J,K) wt1 expression in the future glomeruli (arrows). Wild-type embryo, wt; hi548 mutant embryo, 548; notochord, nc; neural tube, nt.

Figure 5

Figure 5

Hindbrain patterning is affected by vhnf1 inactivation. Anterior is to the left. (A,B) Otic vesicles (arrows) in embryos at the 25-somite stage in lateral views. (C_–_H) Whole-mount in situ hybridization in dorsal views. In C, D, G, and H embryos were double stained with a pax2.1 probe (blue) to show relative positions. (C, D) krox20 (red) expression in 8-somite stage embryos. (E,F) hoxb1 expression in 4-somite stage embryos. (G,H) valentino expression (red) in embryos at the 4-somite stage. Wild-type embryo, wt; hi2169 mutant embryo, 2169.

Figure 6

Figure 6

Effects of vhnf1 overexpression on hindbrain patterning. All show whole-mount in situ hybridization on embryos at the 4-somite stage in dorsal views. Anterior is to the left. (A,B) val expression. (C,D) hoxb1 expression. (E,F) krox20 expression. LacZ,embryo injected with LacZ mRNA; vhnf1, embryo injected with vhnf1 mRNA.

Figure 7

Figure 7

Models for vhnf1 functions in the development of the gut endoderm, the pronephros, and the hindbrain. (A) vhnf1 activates the pdx1 expression and subsequently regulates the regional specification of the foregut endoderm via the _shh_-pdx1 network. (B) vhnf1 regulates regional specification of the pronephric primordia through the pax2–wt1 network. (C) vhnf1, during hindbrain development, establishes the anterior border of the val expression, subsequently activates the r5 krox20 expression, and maintains the posterior border of the r4 hoxb1 expression.

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