Embryonic expression of the murine homologue of SALL1, the gene mutated in Townes--Brocks syndrome

SALL1 is one of three human homologues of the Drosophila region-specific homeotic gene spalt (sal). Mutations of SALL1 on chromosome 16q12.1 cause Townes--Brocks syndrome (TBS) which is characterized by defects in multiple organ systems including limbs, ears, kidneys and anus. Here, we have analyzed the expression of the mouse homologue of SALL1 (Sall1) during early embryogenesis. Sall1 expression is very prominent in the developing brain and the limbs. Other sites of expression include the meso- and metanephros, lens, olfactory bulbs, heart, primitive streak and the genital tubercle. Hence, Sall1 expression to a large degree reflects the structures affected in human TBS.     

Cloning and expression analysis of SALL4, the murine homologue of the gene mutated in Okihiro syndrome

SALL4 is one out of four human homologues of the DROSOPHILA region-specific homeotic gene SPALT(SAL). Heterozygous mutations of SALL4 on chromosome 20q13.13--> q13.2 cause the autosomal dominant Okihiro syndrome which is characterized by radial limb defects, Duane anomaly and hearing loss. We have partially cloned the murine homologue of this gene, named SALL4, and completed the coding sequence by comparison to available EST and genomic sequences in the GenBank database. This comparison also revealed the chromosomal location of SALL4 on mouse chromosome 2H3 and suggested that a predicted testis expressed gene TEX20 at the very same locus is most likely not a gene on its own but part of the SALL4 3' UTR. We analyzed the expression of SALL4 during early embryogenesis by whole mount in situ hybridization and in the adult mouse by Northern blotting. In adult tissues, SALL4 expression is only found in testis and ovary. During embryonic development, SALL4 expression is widespread in early embryos and becomes gradually confined to the head region and the primitive streak. Prominent expression in the developing midbrain, branchial arches and the limbs suggests an important function of SALL4 during development of these structures as expected from the observation in Okihiro syndrome patients.     

Murine homolog of SALL1 is essential for ureteric bud invasion in kidney development.

SALL1 is a mammalian homolog of the Drosophila region-specific homeotic gene spalt (sal); heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We have isolated a mouse homolog of SALL1 (Sall1) and found that mice deficient in Sall1 die in the perinatal period and that kidney agenesis or severe dysgenesis are present. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud; homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth, a failure of tubule formation in the mesenchyme and an apoptosis of the mesenchyme. This phenotype is likely to be primarily caused by the absence of the inductive signal from the ureter, as the Sall1-deficient mesenchyme is competent with respect to epithelial differentiation. Sall1 is therefore essential for ureteric bud invasion, the initial key step for metanephros development.     

A SALL4 zinc finger missense mutation predicted to result in increased DNA binding affinity is associated with cranial midline defects and mild features of Okihiro syndrome

Truncating mutations of the gene SALL4 on chromosome 20q13.13–13.2 cause Okihiro and acro-renal-ocular syndromes. Pathogenic missense mutations within the SALL4 or SALL1 genes have not yet been reported, raising the question which phenotypic features would be associated with them. Here we describe the first missense mutation within the SALL4 gene. The mutation results in an exchange of a highly conserved zinc-coordinating Histidine crucial for zinc finger (ZF) structure within a C2H2 double ZF domain to an Arginine. Molecular modeling predicts that this exchange does not result in a loss of zinc ion binding but leads to an increased DNA-binding affinity of the domain. The index patient shows mild features of Okihiro syndrome, but in addition cranial midline defects (pituitary hypoplasia and single central incisor). This finding illustrates that the phenotypic and functional effects of SALL4 missense mutations are difficult to predict, and that other SALL4 missense mutations might lead to phenotypes not overlapping with Okihiro syndrome. This study has received approval by the institutional review board (Ethics committee) of the Medical Faculty, University of Freiburg, Germany. Jan Miertus and Wiktor Borozdin have equally contributed to this work.     

Zinc finger protein sall2 is not essential for embryonic and kidney development

SALL/Sall is a mammalian homolog of the Drosophila region-specific homeotic gene spalt (sal), and heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We earlier reported that mice deficient in Sall1 die in the perinatal period and that kidney agenesis or severe dysgenesis are present. We have now generated mice lacking Sall2, another Sall family gene. Although Sall2 is expressed mostly in an overlapping fashion versus that of Sall1, Sall2-deficient mice show no apparent abnormal phenotypes. Morphology and gene expression patterns of the mutant kidney were not affected. Mice lacking both Sall1 and Sall2 show kidney phenotypes comparable to those of Sall1 knockout, thereby demonstrating the dispensable roles of Sall2 in embryonic and kidney development.     

Molecular cloning, chromosomal localization, and expression of the murine SALL1 ortholog Sall1

SALL1 has been identified as one of now three human homologs of the region specific homeotic gene spalt (sal) of Drosophila, which encodes a zinc finger protein of characteristic structure. Mutations of SALL1 on chromosome 16q12.1 cause Townes-Brocks syndrome (TBS, OMIM no. 107480). In order to facilitate functional studies of this gene in a model organism, we searched for the murine homolog of SALL1. Here we report the genomic cloning, chromosome mapping, and partial expression analysis of the gene Sall1. Sequence comparison, Northern blot hybridization as well as the conserved chromosome location on the homologous mouse chromosome indicate that we have indeed isolated the murine homolog of SALL1.     

Sall4 Is Transiently Expressed in the Caudal Wolffian Duct and the Ureteric Bud,but Dispensable for Kidney Development

The kidney, the metanephros, is formed by reciprocal interactions between the metanephric mesenchyme and the ureteric bud, the latter of which is derived from the Wolffian duct that elongates in the rostral-to-caudal direction. Sall1 expressed in the metanephric mesenchyme is essential for ureteric bud attraction in kidney development. Sall4, another member of the Sall gene family, is required for maintenance of embryonic stem cells and establishment of induced pluripotent stem cells, and is thus considered to be one of the stemness genes. Sall4 is also a causative gene for Okihiro syndrome and is essential for the formation of many organs in both humans and mice. However, its expression and role in kidney development remain unknown, despite the essential role of Sall1 in the metanephric mesenchyme. Here, we report that mouse Sall4 is expressed transiently in the Wolffian duct-derived lineage, and is nearly complementary to Sall1 expression. While Sall4 expression is excluded from the Wolffian duct at embryonic (E) day 9.5, Sall4 is expressed in the Wolffian duct weakly in the mesonephric region at E10.5 and more abundantly in the caudal metanephric region where ureteric budding occurs. Sall4 expression is highest at E11.5 in the Wolffian duct and ureteric bud, but disappears by E13.5. We further demonstrate that Sall4 deletion in the Wolffian duct and ureteric bud does not cause any apparent kidney phenotypes. Therefore, Sall4 is expressed transiently in the caudal Wolffian duct and the ureteric bud, but is dispensable for kidney development in mice.     

SALL4 is directly activated by TCF/LEF in the canonical Wnt signaling pathway

The SALL4 promoter has not yet been characterized. Animal studies showed that SALL4 is downstream of and interacts with TBX5 during limb and heart development, but a direct regulation of SALL4 by TBX5 has not been demonstrated. For other SAL genes, regulation within the Shh, Wnt, and Fgf pathways has been reported. Chicken csal1 expression can be activated by a combination of Fgf4 and Wnt3a or Wnt7a. Murine Sall1 enhances, but Xenopus Xsal2 represses, the canonical Wnt signaling. Here we describe the cloning and functional analysis of the SALL4 promoter. Within a minimal promoter region of 31bp, we identified a consensus TCF/LEF-binding site.The SALL4 promoter was strongly activated not only by LEF1 but also by TCF4E. Mutation of the TCF/LEF-binding site resulted in decreased promoter activation. Our results demonstrate for the first time the direct regulation of a SALL gene by the canonical Wnt signaling pathway.     

Identification of kidney mesenchymal genes by a combination of microarray analysis and Sall1-GFP knockin mice

SALL1, a causative gene for Townes-Brocks syndrome, encodes a zinc finger protein, and its mouse homolog (Sall1) is essential for metanephros development, as noted during gene targeting. In the embryonic kidney, Sall1 is expressed abundantly in mesenchyme-derived structures from condensed mesenchyme, S-, comma-shaped bodies, to renal tubules and podocytes. We generated mice in which a green fluorescent protein (GFP) gene was inserted into the Sall1 locus and we isolated the GFP-positive population from embryonic kidneys of these mice by fluorescein-activated cell sorting. The GFP-positive population indeed expressed mesenchymal genes, while the negative population expressed genes in the ureteric bud. To systematically search for genes expressed in the mesenchyme-derived cells, we compared gene expression profiles in the GFP-positive and -negative populations using microarray analysis, followed by in situ hybridization. We detected many genes known to be important for metanephros development including Sall1, GDNF, Raldh2, Pax8 and FoxD1, and genes expressed abundantly in the metanephric mesenchyme such as Unc4.1, Six2, Osr-2 and PDGFc. We also found groups of genes including SSB-4, Smarcd3, micro-Crystallin, TRB-2, which are not known to be expressed in the metanephric mesenchyme. Therefore a combination of microarray technology and Sall1-GFP mice is useful for systematic identification of genes expressed in the developing kidney.