A Sall4 mutant mouse model useful for studying the role of Sall4 in early embryonic development and organogenesis

Abstract

SummarySALL4 is a homologue of the Drosophila homeotic gene spalt, a zinc finger transcription factor, required for inner cell mass proliferation in early embryonic development. It also interacts with other transcription factors to control the development of the anorectal region, kidney, heart, limbs, and brain. Truncating mutations in SALL4 cause Okihiro syndrome, manifest as Duane anomaly, radial ray defects and sensorineural and conductive deafness. We report the characterization of a novel murine Sall4 null allele created by bacterial recombineering in ES cells. Homozygous mutant mice exhibit early embryonic lethality. Heterozygous mutant mice recapitulate phenotypic features of Okihiro syndrome including deafness, lower anogenital tract abnormalities, renal hypoplasia, anencephaly, Hirschprung's disease, and skeletal defects. This phenotype shows important differences in cardiac and ear manifestations to previously characterized Sall4 mutant alleles and should prove useful for the investigation of the influence of modifier alleles and protein interactions on the transcriptional regulatory function of Sall4.Keywords bacterial recombineering; Sall4; Okihiro syndrome; mouse; organogenesis; embryonic stem cells SALL4 is a member of the SALL gene family of zinc finger transcription factors (Kohlhase et al., 1996), containing multiple C2H2 double zinc finger domains, homologous to the Drosophila spalt (sal) homeotic gene important in pattern formation and cell specification (de Celis et al., 1996(de Celis et al., , 1999Jurgens, 1988;Kuhnlein et al., 1994). Murine Sall4 (Kohlhase et al., 2002a) is required for inner cell mass proliferation in early embryonic development (Sakaki-Yumoto et al., 2006). It also interacts with other transcription factors, such as Sall1 and the Tbx family of transcription factors to control the development of the anorectal region, kidney, heart, limbs and brain, by acting on downstream targets including Fgf10 (Kiefer et al., 2003;Koshiba-Takeuchi et al., 2006;Sakaki-Yumoto et al., 2006). Mutations in SALL1 are associated with Townes-Brock syndrome (OMIM 107480) (Kohlhase, 2000;Powell and Michaelis, 1999) RESULTSTo inactivate Sall4 we generated a targeting vector (Sall4-TV1) by bacterial recombineering which was designed to delete exons 2-4 ( Fig. 1a). This deletion removes all zinc finger domains of the protein and is expected to result in a null allele. The Sall4-TV1 was used to target the locus in AB2.2 ES cells. Targeted clones were obtained at a rate of 15% (Fig. 1b). Two of these targeted clones were used to establish the Sall4 Brdm1 allele in mice (Fig. 1c).PCR analysis of E3.5 blastocysts identified all three genotypes to be present (Fig. 1d). RT-PCR was performed on E8.5 embryos (Fig. 1e). A reduction of mRNA transcript was evident in Sall4 m1/+ embryos compared with Sall4 +/+ embryos. Whole mount analysis of Sall4 m1/+ embryos at E10.5 revealed that Sall4 mRNA was expressed with the expected tissue distribution, namely midbrain, branchial arch, and limb bud mesenchyme (Kohlhas...

Diagnostic utility of SALL4 in primary germ cell tumors of the central nervous system: a study of 77 cases

“…[20][21][22][23] SALL4 forms a regulatory circuit with OCT4, NANOG, and SOX2 to maintain embryonic stem cell pluripotency and self-renewal. [24][25][26][27][28][29] In this self-stabilizing network, SALL4 regulates OCT4 transcription, suggesting acting upstream of OCT4. 28 In this study we have shown that SALL4 has a broader expression pattern than OCT4 in primary germ cell tumors of the CNS.…”

SALL4 is a novel sensitive and specific marker for metastatic germ cell tumors, with particular utility in detection of metastatic yolk sac tumors

“…32 In mice, Sall4 is essential to early embryogenesis, and homozygous mutant mice exhibit early embryonic lethality. 33,34 In humans, SALL4 is located on chromosome 20q13.13-13.2. 35 As in other species, SALL4 is essential to human development, and mutations in SALL4 lead to acro-renal-ocular and Okihiro syndromes.…”

Regulation and function of Spalt proteins during animal development

“…Zygotic transcription occurs after the four-cell stage, after which Sall4 mRNA levels continue to increase to the blastocyst stage . The effects of Sall4 deficiency were studied using knockout mice and knockdown embryos Elling et al, 2006;Sakaki-Yumoto et al, 2006;Koshiba-Takeuchi et al, 2006;Warren et al, 2007). Homozygous mutant mice die during peri-implantation stages, due to lack of proliferation of the inner cell mass.…”

“…Therefore, some phenotypes observed in SALL1 truncations could be explained by the reduction of SALL4 function. Homozygous Sall4 mutant mice dye during peri-implantation stages due to lack of proliferation of the inner cell mass Elling et al, 2006;SakakiYumoto et al, 2006;Warren et al, 2007). Interestingly heterozygous Sall4 mice reproduce most of the features of the OS.…”