Schematic representation of the HMX1 predicted promoter region in zebrafish, human and mouse genomes (A). Note the presence of complete Hmx1 binding sites (CAAGTG) and the minimal binding core (CAAG). Activity of HMX1 on human UHRF1 promoter (B). The pGL3-Uhrf1 reporter plasmid was cotransfected with pcDNA3.1 empty, pcDNA3.1-Hmx1 expression vector, and pcDNA3.1-Hmx1 del SD1, del SD2, or del HD. HMX1 inhibits UHRF1 transcriptional activity by 40%. HMX1 del SD1, HMX1 del SD2 and HMX1 del HD had no statistical significant effect. Luciferase activity was normalized against β-galactosidase activity. Data are expressed as mean of three or more experiments. *; P<0.05 (ANOVA test).

(A-D) Quantitative RT-PCR from RNA isolated from zebrafish at 5 dpf. (A) Expression of hmx1 in wildtype zebrafish without or with heat shock. (B) Expression of hmx1 in transgenic zebrafish without or with heat shock. (C) Expression of uhrf1 in wildtype zebrafish without or with heat shock. (D) Expression of uhrf1 in transgenic zebrafish without or with heat shock. Heat shock treatment induced expression of Hmx1 in hsp70:Hmx1 but not in wildtype zebrafish. Uhrf1 expression increased in hsp70:Hmx1 zebrafish embryos at 5dpf. (E) Lateral view of hsp70:Hmx1 zebrafish embryos at 5 dpf. In situ hybridization was performed with antisense RNA probe specific for uhrf1. Uhrf1 expression increased in response to ubiquitous hmx1 heat activation, but remained limited to the dorsal, eye and branchial arches regions. Bar, E 100 μm.

(A) In situ hybridization specific for uhrf1. Hmx1 heatshock experiments induced uhrf1 expression in the retina and branchial arches region. Quantification of RNA transcript isolated from zebrafish retinas at 5 dpf. (B) Uhrf1 (b) and dnmt1 (c) expression increased in response to the activation of hmx1 in hsp70:Hmx1 embryos. Bar, A 75 μm.

(A) Wildtype and hmx1 mutant sequence analysis. Hmx1^mut150 mutant embryos carried a 170 bp deletion and a 21 bp insertion, while Hmx1^mut10 embryos reported a 10 bp deletion. (B,D) ExPasy translation tool depicted the disruption of the encoding sequence of the homeodomain and the downstream region composed of SD1 domains in exon2. C. Agarose gel of PCR amplified DNA products. The DNA isolated from WT, hmx1^mut10 and hmx1^mut150 embryos was amplified by PCR.

(A) Tracking of the growth of wildtype, hmx1^mut10 and hmx1^mut150 eye from 1 dpf to 3 dpf. Hmx1^mut10 and hmx1^mut150 eyes are reduced in size compared to wildtype embryos at 2 and 3 dpf stages. (B) Lens growth expressed as ratio lens/whole eye. hmx1^mut10 and hmx1^mut150 lenses are smaller at 3 dpf. (C) Wildtype, hmx1^mut10 and hmx1^mut150 eyes at 5 dpf. Wildtype and hmx1^mut10 present a fully developed lens (5C, a-b), while hmx1^mut150 embryos exhibit two opposing phenotypes (5C, c); on the left side a recovered lens is presented, while on the right a smaller lens presenting a clear phenotype called microphakia. (5C, d) graphic representation of lens/eye ratio; hmx1^mut150 presents a wide range of distribution of the lens size. Red arrows indicate the lens at 5 dpf. Data are expressed as mean of three to 5 experiments. *; P<0.05 (ANOVA test). Bar, C 100 μm.

(A) uhrf1 is expressed in the dorsal region and in the eye field region at 1 dpf in both wildtype and hmx1 mutant embryos (a,b). c,d: uhrf1 expression is reduced in the eye field, in the forebrain and branchial region (c,d). White arrow, retina and branchial arches; asterisk, forebrain. e, eye; fb, forebrain; ba, branchial arches. RT-PCR quantification of uhrf1 transcript isolated from the cranial region. (B) Uhrf1 expression is reduced at 5 dpf (b) but not at 24 hpf (a). Bar, A 100 μm.