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Molecular and Cellular Biology, December 2003, p. 9046-9060, Vol. 23, No. 24
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.24.9046-9060.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Brookdale Center for Developmental and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029-6574
Received 2 July 2003/ Returned for modification 20 August 2003/ Accepted 18 September 2003
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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The homeobox gene Sax2 and its predicted translation product show high similarity to the Drosophila S59/slouch gene and the mouse Sax1 gene (11, 44, 45). Aside from mouse and Drosophila, members of this family have been described in chicken (41), Caenorhabditis elegans (19), flatworms (35), Xenopus (26), and humans (32). In mammals there appear to be only two Sax genes forming the NK1 gene family, which is part of the NKL gene cluster (20). Other gene families of this cluster include the Hmx, Msx, and other NK gene families. Expression patterns for the NK1 gene family have been described in Drosophila, mouse, and chicken. Drosophila S59/slouch gene expression occurs about 5 h after fertilization in mesoderm cells that develop into muscle founder cells. In addition, the Drosophila S59/slouch gene is expressed in the developing nervous system and to a lesser extent in the midgut (11). The mouse Sax1 gene is expressed early in the ectoderm lateral to the primitive streak, and later Sax1 expression is restricted to distinct areas of the spinal cord; it is especially high in rhombomere 1 and in the pretectum (45). It has been shown that Sax1 mRNA is most abundant in day 10 embryos and gradually decreases in day 12 and 14 embryos (44). The expression pattern of Sax1 suggests two different functions for the gene during development; an early involvement in the determination of the posterior neuroectoderm and a later involvement in the specification of specific subsets of neurons (45). The similarities in the expression pattern and the amino acid sequence among the Drosophila S59/slouch and the mouse Sax1 genes and the Sax2 gene led us to speculate that Sax2 may also play an important role in the development of the nervous system. We used RNA in situ hybridization on paraffin sections to determine the expression pattern for the Sax2 gene during embryogenesis. Here we show that the onset of Sax2 gene expression occurs as early as embryonic stage E10.5 in the ventral central nervous system and overlaps with the expression pattern of the Sax1 gene. To further investigate the role of Sax2 during development, we generated a Sax2 knockout mouse line by inserting the lacZ gene into the coding sequence and analyzed the expression pattern in the Sax2-lacZ null allele. ß-Galactosidase staining, like the RNA in situ hybridization results, revealed the expression of Sax2 in the limb, the eye, the ventral neural tube, and different specific subsets of cells in the developing rostral brain during embryogenesis.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ES cell culture and chimeric mouse production. To generate targeted gene disruption in embryonic stem (ES) cells, 10 µg of DNA of pRS14 was linearized by AseI and electroporated into 107 cells as described previously (53). Positive clones were selected by growing the ES cells in the presence of G418 at concentrations in the range of 150 to 450 µg/ml. Altogether, 192 G418-resistant clones were selected and analyzed by Southern blotting using a 5' external probe (SpeI-XhoI [see Fig. 2B]), resulting in a 6.6-kb band for the wild-type clone and an 8.8-kb band for the mutant clone. Positive ES cell lines were microinjected into C57BL/6J blastocysts, and the resulting male chimeras were backcrossed to C57BL/J6 females for a mixed genetic background. For ß-galactosidase staining, embryos were collected at stages E8.5 to E14.5 as previously described (14).
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RNA in situ hybridization. Sagittal, transverse, and coronal sections of wild-type and Sax2 null allele mutant embryos at stages E10.5 to E18.5 were prepared as previously described (51, 53). Embryo tissues were fixed in 4% paraformaldehyde overnight, washed in phosphate-buffered saline, dehydrated through graded ethanol, given by two changes of Americlear (Fisher), and embedded in Paraplast (Fisher) overnight under vacuum. Sections of 7 µm were cut and floated onto Plus+ slides (Fisher), dried, and stored at 4°C. As a probe, construct p1083 was linearized with the restriction enzyme SfiI and antisense RNA was synthesized in the presence of 35S UTP using T7 RNA polymerase for in situ hybridization on sections of wild-type embryos. Autoradiography was performed by dipping the slides in a 1:3 ratio of H2O:Kodak NBT2 emulsion, air drying, and exposing for 3 to 7 days. Slides were developed in Kodak D19 and counterstained with hematoxylin. For RNA in situ hybridization experiments comparing wild-type and Sax2 null allele mutants, we used the partial cDNA (RS19) and construct RS37. Construct RS37 consists of a 2.8-kb NsiI fragment, containing sequences starting 1 kb upstream of the first exon to the second intron, subcloned into Bluescript KS vector. RS19 and RS37 were linearized with BamHI and EcoRI, respectively, and RNA was synthesized using T7 RNA polymerase.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generating a loss-of-function Sax2 null allele by lacZ knock-in. The loss-of-function mutant for the Drosophila S59/slouch gene causes 90% death at different stages during embryogenesis (25). In contrast to the Drosophila mutant, the Sax1 loss-of-function null mice do not exhibit an overt phenotype (P. Gruss, personal communication). This stark discrepancy between the fly and mouse phenotypes might be explained by the overlapping expression patterns of murine Sax1 and Sax2 and a possible functional equivalence between these genes. To determine the role of Sax2 during development, we generated a loss-of-function mutant mouse line. Cloning and the chromosome localization of Sax2 were previously described (7). An 18-kb NotI fragment containing the Sax2 gene was cloned into the SalI site of the pTZ18U vector, resulting in construct p1083. Comparing a sequence of more than 6 kb obtained from construct p1083 with existing genomic databases, combined with our cDNA sequence analysis, predicted an mRNA of 1,074 bp derived from four exons containing a homeobox located in the third exon and an N-terminal conserved element, the EH1 motif, involved in transcriptional repression located in the second exon (44). The predicted mRNA was confirmed by a 744-bp cDNA clone obtained by RT-PCR using mRNA from embryos at stage E11.5 and specific primers corresponding to sequences in the second exon spanning 70 bp upstream of the cloning site for the targeting vector and downstream of the homeobox in the third exon (data not shown).
To generate the Sax2 targeting vector, we inserted an IRESlacZ/floxedneo cassette into the BspEI sites located in the second exon and second intron of the Sax2 gene (Fig. 2A). The targeting vector was electroporated into ES (R1) cells, and 27 of 192 ES clones were confirmed positive by Southern blotting. Four of these ES clones were microinjected into blastocysts to generate chimeras. Of 24 chimeric males, 16 transmitted the gene through the germ line.
Phenotype of Sax2 null mice. Sax2 heterozygous pups were indistinguishable from wild-type animals at all stages of development and postnatal life. Intermating of Sax2 heterozygous animals yielded homozygous mutant newborns in the predicted Mendelian ratios. At birth, homozygous pups were indistinguishable from their wild-type or heterozygous littermates up to 2 days postpartum; subsequently, homozygous pups were easily recognizable owing to their smaller size (Fig. 2C and D). The homozygous pups continued to grow much more slowly than their littermates, and 85% of the homozygous pups died within the first 3 weeks of birth (Fig. 2C and data not shown). Although homozygous mice were runted, they did not exhibit any obvious abnormal behavior or motor skills. All Sax2 null pups showed normal suckling behavior, and milk was found in their stomachs. At 2 or 3 days prior to their death, Sax2 null pups became lethargic and showed signs of wasting. The few homozygous animals surviving to adulthood were fertile, but all the offspring from homozygous intermatings died within 4 days postnatally.
We examined whether the mutant mice exhibited gross behavioral or neurological differences by performing several tests including the contact righting test and reaching reflex test for neurological reflexes, the jar-circling test for hyperactivity and adaptation to a new environment, the gait test for motor abilities, and the forelimb grip test for neuromuscular abnormalities. In all cases, with the exception of the forelimb grip test, the mutants did not show any statistically significant differences in their behavior or performance. The grip test revealed that the Sax2 null mutants were able to grip the suspension bar but could not hold on as long as the wild-type pups. While wild-type mice could hang from the bar for up to 90 s, the maximum time for the mutants never exceeded 30 s. From our observations, it is unclear at present whether this difference in behavior is due to neuromuscular abnormalities or is related to a secondary effect owing to an evident malnutrition of the Sax2 null animals.
To exclude the notion that the phenotype we obtained was influenced by the presence of the PKGneo selection cassette, we intermated Sax2 heterozygous animals with a Cre recombinase-expressing mouse line (34) that exerts Cre activity as early as the zygote-blastomere stage of mouse embryogenesis. Loss of the neo gene in the resulting heterozygote offspring was confirmed by Southern blotting. Sax2 heterozygotes segregating free of the Cre transgene were selected in subsequent generations. Intermatings of Sax2 heterozygous animals carrying only the IRESlacZ reporter (Fig. 2A) produced offspring exhibiting the same phenotype as described above (data not shown), indicating that the PGKneo cassette did not contribute to the observed phenotype.
Analysis of lacZ gene expression in heterozygous and homozygous Sax2 embryos. To further examine the expression pattern of Sax2 and to analyze possible effects of the loss-of-function mutation, we compared the lacZ expression pattern of heterozygous and homozygous embryos by ß-galactosidase staining. Embryos were collected at stages E8.5 to 14.5 for ß-galactosidase staining as previously described (14). Similar to the RNA in situ hybridization results, ß-galactosidase staining revealed expression of the Sax2 gene in the ventral neural tube, the hindbrain, the midbrain, the eyes, and the apical ectodermal ridge (AER) (Fig. 3). lacZ gene expression was first detected at stage E10 in the AER of the forelimb (data not shown). At embryonic stage E10.5, ß-galactosidase staining was also detected in the AER of the hindlimb (Fig. 3B and D) and to a lesser degree in the ventral neural tube (Fig. 3B). The same expression pattern was observed in embryos at stage E11.5 (Fig. 3E to H). At embryonic stage E13.5, lacZ gene expression still persisted in the AER remnant, but at that time it was found only in the cells distal to the region of the developing digits and not in the interdigital region (Fig. 3I and J, inserts). Sax2-directed lacZ expression in the neural tube occurred in two distinct columns located on either side of the midline of the neural tube (Fig. 3K and L). Expression of lacZ in the lens and hindbrain was first seen at embryonic stage E12.5 (data not shown) and showed its highest expression level at stage E13.5 (Fig. 3K to P). lacZ gene expression in the limb and neural tube was stronger in homozygous than in heterozygous embryos, which would be expected owing to gene dosage effects; expression in the midbrain-hindbrain boundary region and especially in the eye was strikingly down regulated in the Sax2 homozygous nulls (Fig. 3K, L, O, and P). In addition to lacZ expression in the ventral neural tube, we detected ß-galactosidase staining in the ventral midbrain of Sax2 homozygous null embryos but not in the heterozygous embryos (Fig. 3I and J). Unlike in the eye and midbrain-hindbrain boundary region, where lacZ gene expression was down regulated in the Sax2 null embryos, in the ventral midbrain lacZ expression was up regulated in the Sax2 null homozygous embryo. ß-Galactosidase staining was still detected at embryonic stage E14.5 and later in all the tissues described above (data not shown) in a manner similar to the RNA in situ hybridization results for Sax2.
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Analysis of Sax2 gene expression in the developing eye. Sax2 gene expression in the developing eye is first detected at embryonic stage E12.5 (data not shown), coinciding with the onset of primary lens fiber cell differentiation (13), and Sax2 expression becomes highest at embryonic stage E13.5, as determined by ß-galactosidase staining (Fig. 3M to P). Furthermore, transverse sections of paraffin-embedded ß-galactosidase stained embryos at stage E13.5 revealed that lacZ expression occurs in the proximal region of the lens fibers (Fig. 4A and B), with a high expression level in heterozygous embryos and a very weak expression level in homozygous embryos. Performing RNA in situ hybridization using a probe corresponding to the 5' end of the Sax2 mRNA (which is present in both the wild-type and Sax2 null alleles), we confirmed the expression pattern and the down regulation of Sax2 gene expression in the developing Sax2 null eye (Fig. 4C and D), suggesting a positive autoregulation mechanism for Sax2 gene expression in these cells. To further determine whether other genes involved in the development of the eye, in particular in lens development, are affected by the Sax2 loss-of-function mutation, we analyzed several genes involved in the development of the eye, specifically in the development of the lens fibers by RNA in situ hybridization, as shown in Fig. 4E to P. While Alk6 (13), Bmp4 (15), and Fgf1 (9) were expressed predominantly in the lens fiber cells (Fig. 4E to J), Hmx1 (53), Ncadherin (55), and Pax6 (8) were also expressed in the neural retina (Fig. 4K to P). All the genes used for the RNA in situ hybridization show an overlapping expression pattern with the Sax2 gene in the lens fiber cells, but, surprisingly, none exhibits any change in expression in the Sax2 null embryos, suggesting that Sax2 lies either downstream or in a separate developmental pathway from the genes investigated here.
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As we have shown in Fig. 1c, panels A to F, Sax2 is expressed in close proximity to the ST and Pet1 genes, two marker genes for serotonergic neurons. Among the functions ascribed to serotonergic neurons are the regulation of anxiety, affection, aggression, drug abuse, and food uptake (reviewed in reference 16). Because of the close proximity of Sax2 gene expression to serotonergic neurons, it is possible that the loss of the Sax2 gene product might affect food uptake and therefore contribute to the observed phenotype. We performed an RNA in situ hybridization analysis on transverse paraffin sections of embryos at stage E16.5 to further examine the cells expressing Sax2 and to explore the possibility of an interaction between Sax2 and these serotonergic genes. Sax2 gene expression occurred in the midbrain-hindbrain boundary adjacent to cells expressing ST (Fig. 6U to X). While Sax2 gene expression was down regulated in the homozygous null brain compared to the wild type (which concurs with the lacZ expression data shown in Fig. 3K and L), there was no significant difference in the level of expression of the ST gene in the Sax2 nulls. These data suggest that Sax2 is not directly interacting with ST; however, they do not exclude the possibility that there is an interaction between Sax2-expressing cells and serotonergic neurons, since it is possible that the decrease in the level of Sax2 protein in the homozygous mutant in the midbrain-hindbrain boundary region acts downstream from ST or possibly has an effect on other factors involved in the sensory neuron pathway.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutants with mutations of the growth hormone receptor/binding protein gene (56), Pax7 gene (46), VGF gene (18), and Twist genes (49) exhibited a similar phenotype of reduced postnatal growth rate. Mice lacking the growth hormone receptor/binding protein gene were born indistinguishable from their littermates, but by 3 weeks after birth the homozygous mice were significantly smaller. Aside from their regressing growth rate, these mice were viable and fertile. Growth retardation in this case might result from the increased levels of growth hormones in serum in the mutant mice (56). Mice lacking the basic loop-helix transcription factor Twist are similar to the Sax2 mutants, exhibiting postnatal growth retardation and dying within 3 to 4 weeks after birth. The phenotype appears to be caused by the loss of a negative-feedback mechanism associating Twist protein with the NF-
B subunit p65 and the repression of the cytokine promoters (49). At birth, cytokine expression, especially the expression of the cytokines tumor necrosis factor alpha and interleukin-1ß is very high, possibly reflecting an immune response to the stress of delivery and changes in the environment after birth (40). The Twist gene product down regulates this high expression rate of cytokines, and cytokine levels remain unnaturally high in Twist null mutants, leading to premature death (49). It is possible that Sax2 gene expression is required in a similar way to Twist gene expression in order to adjust the expression of target genes to the changed metabolism and/or environment after birth.
Sax2 gene expression and different mechanisms of autoregulation depending on cell type. Correct spatiotemporally regulated expression of transcription factors is crucial during development. Misexpression of developmental control genes can lead to severe defects and premature death. Analysis of Sax2 gene expression revealed different regulatory mechanisms in different tissues during embryogenesis, suggesting an important function for Sax2 during development. While the level of Sax2 gene expression does not change in the limbs and the neural tube of Sax2 mutants, Sax2 expression is down regulated in the Sax2 null midbrain-hindbrain boundary region and most obviously in the Sax2 null eyes. In contrast, no Sax2 gene expression is observed in the ventral midbrain in wild-type mice, but a very high Sax2 expression level is observed at both the Sax2 RNA and the ß-galactosidase levels in the null mutant, confined to two distinct areas of the ventral midbrain. Both these expression patterns suggest autoregulation of the Sax2 gene by a negative-feedback (repression) mechanism in the ventral midbrain and a positive-feedback (activation) mechanism in the midbrain-hindbrain boundary region and the eyes. The specific regulation of Sax2 gene expression in multiple tissues implies diverse roles for the Sax2 gene during development.
Autoregulation in either a positive- or negative-feedback manner has been shown for several homeobox genes in Drosophila and mammals. The organizer-specific homeobox gene goosecoid is one example of autoregulation by a negative-feedback mechanism. goosecoid contains two palindromic goosecoid binding elements, DE and PE, in its promoter region. It was shown in Xenopus embryos and mouse P19 teratocarcinoma cells that goosecoid protein binds the more proximal element and represses its own transcription. It is suggested that specific cofactors are required to inhibit repressor activity and lead to increased transcription (10). Several members of the Hox gene family are regulated in a positive-feedback mechanism. Hox genes are very important in the patterning of the anteroposterior axis and in the development of the nervous system. Different Hox genes are expressed in a specific spatiotemporal order to establish the segmentation of the hindbrain, and it has been shown, especially for Hoxa3 and Hoxa4, that cross- and auto-regulation of these genes is required for their function (29, 36). Further studies are required to determine the cis-acting DNA elements involved in the positive- and negative-feedback mechanisms for the Sax2 gene.
Repressive role for Sax2 during development of the central nervous system. Homeobox genes are critical transcription factors involved in the regulation of many processes during development by either activation or repression of target genes (reviewed in reference 28). The specification of their regulatory function depends on interactions with specific cofactors. As we have shown above, Sax2 is regulating its own expression in a positive- and negative-feedback mechanism depending on the specific cell type examined. How does Sax2 gene regulation relate to its function, and how does it modulate the expression of other genes? The Sax2 gene, as well as the Sax1 gene, contains, in addition to the homeobox, a repressor element, the EH1 motif (44), upstream of the homeobox. This motif is also present in all members of the Engrailed, Msh/msx, NK2, and goosecoid homeoprotein classes (48). It was shown that the EH1 motif plays an important role in the patterning of the ventral neural tube. The ventral neural tube is divided into five neural subgroups that are determined by two classes of homeobox genes; class I is repressed by specific Shh protein concentrations, while class II requires Shh protein for its activation. Cross-regulatory interactions between corresponding pairs of class I and class II genes determine the borders of the neural subgroups (reviewed in references 5, 27, and 38). Most of these homeobox genes contain an EH1 motif that is bound by the corepressors Groucho/TLE, which are expressed in the ventral neural tube at the time when neural patterning occurs (33). The presence of an EH1 motif in the Sax2 gene indicates a possible function as a transcription repressor but does not exclude an additional activator function. Homeobox genes can play a dual role as repressors or activators of transcription of their target genes depending on their interaction with specific cofactors (reviewed in reference 28).
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ACKNOWLEDGMENTS |
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This work was supported in part by grants AR46471 and DE13741 from the NIH to T.L.
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FOOTNOTES |
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