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.     

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.     

Sall3 plays essential roles in horizontal cell maturation through regulation of neurofilament expression levels

The region-specific homeotic gene spalt (sal) gene plays a critical role in Drosophila development. The mammalian Sal homologous genes contain four members, and Sall3 is mainly expressed in horizontal cells. In the developing retinas of Sall3 knockout (KO) mice until around birth, horizontal precursor cells developed with comparable numbers and position; the horizontal cell marker NF160 was expressed weakly and neurite-like structure had once formed. Since Sall3-KO mice die at postnatal day 1, subsequent retinal development was examined by in vitro retinal explant culture. In the Sall3-KO retina culture, the expression of NF160 was abrogated, and neurite extension was not observed. Furthermore, Sall3-KO horizontal precursors were initially localized at the appropriate horizontal positions, but eventually moved to an abnormal site in the outer nuclear layer. Overexpression of Sall3 in retinal progenitors did not induce differentiation of retinal progenitor cells into the horizontal cell-fate, but enhanced NF160 expression and neurite extension. In addition, differentiation into Müller glia was promoted, and rod cells were severely suppressed without perturbing proliferation. In conclusion, Sall3 may not be involved in horizontal cell-fate determination, but rather functions to instruct terminal differentiation of horizontal cells and to maintain NF160 expression.     

Enhanced oral tolerance in transgenic mice with hepatocyte secretion of IL-10

Several cytokines derived from Th3 and Tr1 cells, including IL-10, are believed to regulate oral tolerance, but direct evidence is lacking. We have explored the potential role of IL-10 by generating transgenic (TG) mice with sustained hepatocyte-specific expression of rat IL-10. TG mice expressed rat IL-10 downstream of a transthyretin promoter, which led to serum levels that were increased 10- to 100-fold compared with normal animals. Animals were orally administered 1 mg of whole OVA for 5 consecutive days, with control animals receiving PBS. There were six animal groups: Either OVA or PBS were fed orally to rat IL-10 TG mice, non-TG wild-type mice without IL-10 administration, and non-TG wild-type mice administered rat IL-10 systemically. On day 8, all mice were immunized with two injections of OVA, and then analyzed on day 18. T cell proliferation responses were reduced by 65.8 +/- 14.3% after feeding of OVA in rIL-10 TG animals, compared with 39.4 +/- 15.6% in the non-TG mice (p = 0.02). Anti-OVA titers were expressed as fold increase over naive non-TG mice. After feeding, titers decreased by approximately 33% (from 3- to 2-fold) in TG animals and, to a lesser extent, in non-TG animals. IFN-gamma secretion by cultured popliteal lymphocytes decreased in TG animals by 83% after feeding and by 69% in non-TG animals. IL-4 secretion increased 4-fold in TG-fed mice, but did not significantly change in non-TG OVA-fed animals. In contrast to hepatic TG expression of rIL-10, systemic administration of rIL-10 had only a modest effect on tolerance. IL-10, when transgenically expressed in the liver enhances mucosal tolerance to an oral Ag.     

Unusual cell specific expression of a major human cytomegalovirus immediate early gene promoter-lacZ hybrid gene in transgenic mouse embryos.

Transgenic mice carrying the human cytomegalovirus immediate early gene promoter driving the E. coli lacZ gene displayed an unusual cell specific expression of beta-galactosidase during development. LacZ expression was first detected in cells lining the apex of the neural fold of day 8.5 embryos. By day 10 of gestation, expression was prominent in the spinal ganglia, the ganglia of cranial nerves V, VII, VIII, IX, and X, in a line of cells marking the ventrolateral pathway adjacent to the dermamyotome, and in a column of differentiated cells in the entire ventrolateral neural tube posterior to the mesencephalon. Expression was also found in the myotomes. Neural tube explants from day 8.5 embryos cultured in vitro showed lacZ expression in cells migrating away from the explant. We conclude that the HCMV-IEP-lacZ transgene is expressed in a subpopulation of neural crest cells and its early derivatives.     

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.     

Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene.

The human papillomavirus type 16 (HPV-16) genome is commonly present in human cervical carcinoma, in which a subset of the viral genes, E6 and E7, are expressed. The HPV-16 E6 and E7 gene products can associated with and inactivate the tumor suppressor proteins p53 and Rb (the retinoblastoma susceptibility gene product), and in tissue culture cells, these viral genes display oncogenic properties. These findings have led to the hypothesis that E6 and E7 contribute to cervical carcinogenesis. This hypothesis has recently been tested by using transgenic mice as an animal model. HPV-16 E6 and E7 together were found to induce cancers in multiple tissues in which they were expressed, including squamous cell carcinoma, the cancer type most commonly associated with HPV-16 in the human cervix. We have extended these studies to investigate the in vivo activities of HPV-16 E7 when expressed in squamous epithelia of transgenic mice. Grossly, E7 transgenic mice had multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair on neonates, thickened ears, and loss of hair in adults. In lines of mice expressing higher levels of E7, we observed stunted growth and mortality at an early age, potentially caused by an incapacity to feed. Histological analysis demonstrated that E7 causes epidermal hyperplasia in multiple transgenic lineages with high penetrance. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and an expansion of the keratin 10-positive layer of cells and was associated with hyperkeratosis. Hyperplasia was found at multiple sites in the animals in addition to the skin, including the mouth palate, esophagus, forestomach, and exocervix. In multiple transgenic lineages, adult animals developed skin tumors late in life with low penetrance. These tumors arose from the squamous epithelia and from sebaceous glands and were characterized histologically to be highly differentiated, locally invasive, and aggressive in their growth properties. On the basis of these phenotypes, we conclude that HPV-16 E7 can alter epithelial cell growth parameters sufficiently to potentiate tumorigenesis in mice.     

Three distinct keratinocyte subtypes identified in human oral epithelium by their patterns of keratin expression in culture and in xenografts

We have characterized the cells that form the human oral epithelia by analyzing their patterns of keratin expression in culture and in transplants. Keratinocytes of all oral regions synthesized high levels of keratins K5/K14 and K6/K16,K17, as expressed by cells of all stratified squamous epithelia in culture. However, cells from different regions varied in their expression in culture of retinoid-inducible (K19 and K13) and simple epithelial (K7, K8 and K18) keratins. By these criteria, all oral cells could be classified as belonging to one of three intrinsically distinct subtypes: "keratinizing" (gingiva, hard palate), "typical nonkeratinizing" (inner cheek, floor of mouth, ventral tongue) and "special non-keratinizing" (soft palate), all of which differed from the epidermal keratinocyte subtype. Cells from fetal floor of mouth expressed a pattern of keratins in culture markedly different from that of adult floor of mouth cells but identical to that of the adult "special nonkeratinizing" subtype and similar to that of several oral squamous cell carcinoma lines. When cultures of oral keratinocytes were grafted to the dermis of nude mice, they formed stratified epithelial structures after 10 days. In some areas of the stratified structures, the basal layer recapitulated the K19 expression pattern of the oral region from which they had originated. Thus, regional differentiation of the oral epithelium is based on an intrinsic specialization of regional keratinocyte stem cells. Additionally, oral cell transformation either frequently involves reversion to the fetal keratin program or else oral cells that express this keratin program are especially susceptible to transformation.     

Establishment, development and fecundity of Taenia crassiceps in the intestine of prednisolone-treated Mongolian gerbils and inbred mice

Worm establishment, development and fecundity of Taenia crassiceps in the intestine of prednisolone (PTBA)-treated, 15- and 4-week-old Mongolian gerbils and 9-week-old inbred mice of 4 strains (AKR/J, BALB/cAn, B10D2/oSn and C57BL/KsJ) were investigated following oral administration of metacestodes. Gerbils were divided into 5 groups of 4 animals each according to the host age and commencement day of PTBA-treatment (day -13, -7 or 0 relative to infection). Worm recovery from the intestine on day 35 postinfection was not affected by host age, but fewer worms were recovered the earlier the commencement of PTBA-treatment. Worm size, determined by wet weight, total length and proglottis number, correlated inversely with worm burden, suggesting they were affected by the crowding effect. Proglottides were released normally in the faeces but were markedly depressed in all groups except for that of young gerbils. Furthermore, egg production and its development in gravid proglottides were markedly depressed in all groups. In PTBA-treated mice of 4 strains, sexually mature but not gravid worms were recovered from all mice of the AKR/J strain on days 20-32 postinfection, but none or few worms from the intestine of the others. PTBA-treatment did not inhibit all protective host defence mechanism(s) in the unnatural or alternative rodent definitive host of T. crassiceps.     

A mouse line expressing Sall1‐driven inducible Cre recombinase in the kidney mesenchyme

Sall1 is expressed in the metanephric mesenchyme in the developing kidney, and mice deficient in Sall1 show kidney agenesis or dysgenesis. Sall1 is also expressed elsewhere, including in the limb buds, anus, heart, and central nervous system. Dominant‐negative mutations of Sall1 in mice and humans lead to developmental defects in these organs. Here, we generated a mouse line expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the endogenous Sall1 promoter. Upon tamoxifen treatment, these mice showed genomic recombination in the tissues where endogenous Sall1 is expressed. When CreER^T2 mice were crossed with the floxed Sall1 allele, tamoxifen administration during gestation led to a significant decrease in Sall1 expression and small kidneys at birth, suggesting that Sall1 functions were disrupted. Furthermore, Sall1 expression in the kidney was significantly reduced by neonatal tamoxifen treatment. The Sall1CreER^T2 mouse is a valuable tool for in vivo time‐dependent and region‐specific knockout and overexpression studies. genesis 48:207–212, 2010. © 2010 Wiley‐Liss, Inc.