INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

The control of retinal determination (RD) in Drosophila requires eyeless, sine oculis, eyes absent, and dachshund, which function together as components of a network (1, 6, 30, 33). These genes are expressed prior to photoreceptor differentiation and are required for retinal morphogenesis (2, 7, 25, 31). Moreover, eyeless, eyes absent, and dachshund are sufficient for inducing retinal fates, as targeted misexpression results in ectopic eye formation from nonneuronal tissues. eyeless is a member of the Pax gene family of transcription factors (9). sine oculis encodes a protein possessing a homeodomain motif with DNA-binding activity (17). eyes absent and dachshund encode novel nuclear proteins lacking DNA-binding motifs but have been proposed to act as transcription cofactors (2, 6, 25). The relationships between the RD genes cannot be summarized completely by a simple, linear pathway. Although Eyeless activates transcription of eyes absent and sine oculis, which in turn appear to activate dachshund, there is evidence of extensive cross-regulation between the RD genes (1, 6, 14, 30, 33). For example, misexpression of Dachshund induces ectopic eyeless, eyes absent, and sine oculis expression (6, 33). Together these findings suggest that the Drosophila RD genes cooperate as components of a network to control cell fate decisions by regulating target gene expression.

Vertebrate homologues of eyeless (Pax6), eyes absent (Eya1 to Eya3), sine oculis (Six3 and Six6), and dachshund (Dach) that are expressed in the developing eye have been identified (3, 4, 10, 11, 15, 20, 21, 29, 31, 37, 41). Evidence of a functional role for some of these genes during eye development has also been demonstrated. Mutations in Pax6 and PAX6 result in the Small eye phenotype in mice and ANIRIDIA in humans, respectively (12, 13, 19, 31, 35, 36). Furthermore, misexpression of Pax6 results in ectopic eye formation in Xenopus (8). Similarly, Six3 misexpression leads to ectopic lens and retina formation in the teleost Medaka (24, 28). Finally, reminiscent of the regulatory relationship between eyes absent and eyeless in Drosophila (14), expression of mouse Eya1 and Eya2 in the lens placode is dependent on Pax6 (41). These data suggest that the Drosophila RD network has been conserved in vertebrates. However, it is not known whether all vertebrate homologues of the Drosophila RD genes function during eye development and if these genes operate in the same or parallel pathways.

A mouse homologue of dachshund, Dach (hereafter referred to as Dach1), has been cloned, and its embryonic expression pattern has been characterized (4, 11, 15, 21, 25). Comparison of Dachshund and Dach1 sequences reveals the presence of two highly conserved sequences, Dachshund domain 1 (DD1) and DD2 (11). The Dachshund N terminus, including DD1, autonomously activates transcription in yeast, and DD2 has been shown to physically interact with Eyes absent, suggesting that Dachshund regulates gene expression through protein-protein interactions facilitated by DD1 and DD2 (6). Expression analysis of Dach1 demonstrates that it is expressed in the developing retina, limbs, and nervous system, which is analogous to the expression pattern of Drosophila dachshund (4, 11, 15, 25). As dachshund mutant flies lack eyes, have truncated limbs, and display defects in brain development, the structural and expression similarities shared between Dach1 and dachshund suggest a similar role for Dach1 during vertebrate development (22, 25, 26).

Here we present the construction of a Dach1 knockout allele and characterize the effect of this mutation on Dach1 expression and mouse development. Despite the abrogation of the wild-type embryonic expression pattern, gross and histological analyses of the homozygous mutant eyes, limbs, and brains revealed no detectable malformations. At the time of birth, the frequency of the homozygous mutant and wild-type alleles are roughly Mendelian. However, this mutation is associated with postnatal lethality, a failure to suckle, and respiratory distress, although additional analyses failed to reveal a cause for this phenotype.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Generation of a Dach1 knockout allele. A 600-bp SmaI-EcoRI fragment of human DACH cDNA clone 381801RG (Research Genetics) was used to screen a 129/SvEv genomic phage library. Three Dach1 genomic clones were isolated and mapped relative to each other and Dach1 cDNA sequences. The genomic clones contained the first coding exon: 218 bp of 5' untranslated region, the putative translational start codon, 827 bp of open reading frame, and a splice site that is conserved between mouse Dach1 and Drosophila dachshund (11). These sequences encode amino acids 1 to 276, a region which includes most (98 of 107 amino acids) of the conserved DD1 domain. Dach1 genomic fragments flanking exon 1 were subcloned into pUSEFUL for assembly of a targeting vector (Allan Bradley, Baylor College of Medicine). Specifically, a 5' recombination arm (3.7-kb SalI-SacI fragment) and a 3' recombination arm (5.0-kb KpnI-SalI fragment) were subcloned into pUSEFUL flanking PGK-Hprt sequences (see Fig. 1A). The targeting vector was linearized with NotI and electroporated into the embryonic stem (ES) cell line AB2.1. Since homologous replacement abolishes a SacI site upstream of exon 1 (see Fig. 1A), SacI-digested Hprt⁺/herpes simplex virus thymidine kinase-negative AB2.1 colony DNA was screened by Southern analysis using 5' and 3' probes (flanking the recombination arms and not included in the targeting vector). As a consequence of recombination, PGK-Hprt sequences replace 319 bp upstream of exon 1, Dach1 exon 1, and approximately 2 kb of intron 1 (see Fig. 2A). From separate electroporations, two independent replacement events were isolated (ES cell lines B and G). In addition to the expected replacement, additional sequences derived from the targeting vector were inserted at the Dach1 locus (see Fig. 1A). A PCR-based assay was also developed for detecting the Dach1 mutant allele. Primers pl (5'-ACATGCACATACGCACACTTT) and p3 (5'-AAGAGGTCAAGACAGGAACATCA) amplify a 265-bp product from the wild-type allele, and primers p3 and p2 (5'-AGGCCACTTGTGTAGCGCCAA) produce a 381-bp amplicon from the mutant allele. The positions of these primers are shown in Fig. 1A.

In situ hybridization. A Dach1 riboprobe was prepared as previously described (11). A Dachl EcoRI cDNA fragment (amino acid 365 to the stop codon and 386 bp of 3' untranslated region) was cloned into the EcoRI site of pBluescript KS(+). These cDNA sequences are downstream of exon 1 and therefore are not deleted in the Dachl mutant allele. An antisense riboprobe was synthesized using T7 RNA polymerase. In situ hybridization of whole-mount embryos was performed as described elsewhere (32). Digoxigenin-labeled tissues were detected using antidigoxigenin antibody (FAb fragments; Boehringer Mannheim) coupled to alkaline phosphatase and nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate development (Boehringer Mannheim).

Immunohistochemistry. Dach1 cDNA sequences encoding amino acids 461 to 538 (11) were subcloned into pGEX4T to create a glutathione S-transferase (GST)-Dach1 expression construct. GST-Dach1 fusion protein was purified from Escherichia coli using glutathione-agarose beads and injected into rabbits (Cocalico Biologicals, Inc.). The serum isolated from rabbits was then used for immunohistochemistry. Embryos were fixed in 4% paraformaldehyde-phosphate-buffered saline (PBS) for 3 h at 4°C. After several washes in PBS, the embryos were equilibrated in 30% sucrose-PBS and transferred to OCT embedding medium. Tissue sections (8 to 10 µM) were incubated in blocking solution (1% goat serum, 0.1% Triton X-100, PBS) for at least 30 min, washed briefly in PBS, and then incubated with 1/500 dilution of anti-Dach1 antiserum in blocking solution overnight at 4°C. The sections were then washed several times in PBS and incubated with Cy3 secondary antibody in blocking solution for 1 h at room temperature. Sections were then washed, mounted on slides, and visualized.

Histology. Embryos and tissues were dissected, washed in cold PBS, and fixed in fresh 4% paraformaldehyde-PBS overnight at 4°C with gentle agitation. For central nervous system histology, newborn heads and necks were fixed in 10% formalin followed by decalcification in 10% formic acid. Tissues were washed twice in PBS for 30 min and then dehydrated with a PBS-distilled dH₂O-ethanol (EtOH) series. In preparation for paraffin embedding, dehydrated tissues were washed once in 1:1 xylene-EtOH for 15 min and once in xylene for 30 min. Tissues were immediately washed in a 1:1 mixture of xylene and molten (59°C) paraffin (Paraplast tissue embedding medium; Oxford Labware) for 15 min, washed three times in molten paraffin for 2 h and then embedded in paraffin using Biopsy Uni-Cassettes (Tissue Tek). Embedded tissues were sectioned using a Leica RM2165 microtome, stained with hematoxylin and eosin (H & E), and mounted.

Skeleton preparations. Skin and internal organs were carefully removed from euthanized newborn mice. Carcasses were fixed overnight in 95% EtOH and then stained with 0.015% alcian blue-20% acetic acid (Sigma) overnight. The samples were washed in 95% EtOH for at least 3 h and transferred to 2% KOH for 24 h, and any remaining soft tissue was removed carefully with forceps. Skeletons were stained overnight in 0.005% alizarin red sodium sulfate-1% KOH (Sigma) and then cleared in 1% KOH-20% glycerol for at least 2 days. Skeletons were subsequently stored in a 1:1 mixture of glycerol and 95% EtOH and photographed.

Blood analysis. Serum was isolated from decapitated newborn mice using Microtainer serum separator tubes (Becton Dickinson). These samples were taken from pups with no obvious milk in their stomachs. Samples were processed and analyzed using a Vitros 950 chemistry analyzer (Ortho-Clinical Diagnostics, Johnson & Johnson), except for the analysis of blood glucose levels (see below). Averages and standard deviations are shown in Table 1. Measurements of sodium and potassium were performed on individual samples from three separate homozygous mutant and heterozygous newborns. To make these measurements, serum (8 to 10 µl) was diluted 1/11 in urine diluent. In control experiments, measurements of serial dilutions demonstrated values that were linearly proportional to the dilution factor. Measurements of alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, calcium, and triglycerides were performed on two different pooled samples from a total of 15 homozygous mutant and 19 heterozygous newborns diluted 1/2.5 in 0.9% NaCl. Measurements of creatine and phosphate were performed on two pools of four homozygous mutant and three pools for four heterozygous newborns. Measurements of glucose levels were performed on five individual homozygous mutant and 15 heterozygous newborns within 10 min of birth, using an Accu-Chek glucometer (Roche). Abdomens of newborn pups (less than 12 h old) were examined for the presence of milk. Approximately twofold higher levels of triglycerides were present in heterozygotes with milk than without milk.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Generation of Dach1 mutant mice. Utilizing a standard homologous knockout strategy, we generated a mutation in Dach1 by replacing exon 1 and flanking genomic sequences with PGK-Hprt (Fig. 1A). Southern blot analysis using a 3' genomic probe was used to identify two independent replacement events in ES cell lines and follow the Dach1 mutant alleles during the establishment of mouse lines B and G (Fig. 2A, right). Consistent with a homologous replacement event, an exon 1-specific probe demonstrated that exon 1 is deleted in the homozygous mutant newborns by Southern analysis (Fig. 2A, left). Associated with the replacement event was the insertion of additional sequences from the targeting construct at the Dach1 locus in lines B and G (Fig. 1A). However, the inserted sequences are not associated with a dominant phenotype, as heterozygotes appear indistinguishable from wild-type littermates. We also developed a PCR assay to genotype embryos, newborns, and adults (Fig. 1B). Genotype analysis of 151 2-h-old newborns from 21 intercrosses demonstrated that the frequency of the wild-type, heterozygote, and mutant homozygote genotypes were roughly Mendelian (Fig. 1C). These results demonstrate that there is no pronounced embryonic or fetal lethal phenotype associated with the Dach1 mutant allele.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Dach1 knockout strategy and genotype analysis. (A) Dach1 targeting strategy. The top line shows the wild-type 5' Dach1 region with exon 1 (white box) and intron 1 sequences (thin line) flanked by 5' and 3' recombination arms (grey boxes). The middle line shows the Dach1 knockout construct designed for homologous replacement of a SacI site, exon 1, and a 5' portion of intron 1 with PGK-Hprt sequences. The resultant Dach1 mutant allele is illustrated on the bottom line. Positions of genotyping primers p1, p2, and p3 are shown as arrows below the wild-type and mutant allele diagrams. The line below the asterisk represents additional sequences, derived from the targeting vector, inserted at the Dach1 locus which were associated with two independently isolated homologous replacement events (Materials and Methods). pBS, pBluescript; HSV tk, herpes simplex virus thymidine kinase gene. (B) PCR genotype analysis of tail DNA. An ethidium bromide-stained agarose gel containing amplification products from wild-type (+/+), heterozygote (+/), and homozygote (/) tail DNA is shown. Primer combination p1-p3 detects wild-type alleles, while primers p2 and p3 detect mutant alleles (Materials and Methods). (C) Genotype analysis of newborn mice. Tail DNA was collected from 151 newborn mice (21 intercross litters) and analyzed by PCR. The numbers and corresponding percentages of wild-type (+/+), heterozygote (+/), and homozygote (/) mutant animals are shown. Tails from these mice were taken less than 2 h after birth. Homozygotes may be slightly underrepresented since they may be eaten prior to detection.

View larger version (58K): [in this window] [in a new window]   FIG. 2.   Targeted disruption of Dach1 exon1 is associated with abrogated RNA and protein expression. (A) Southern analysis of the Dach1 locus. Wild-type (+/+), heterozygote (+/), and homozygote (/) newborn tail DNA was digested with SacI, subjected to gel electrophoresis, and transferred to a nylon membrane. The filter was hybridized to a Dach1 exon 1 probe (Fig. 1A), washed, and exposed to film. Subsequently, the filter was stripped and hybridized to a 3' Dach1 probe (Materials and Methods). (B and C) Whole-mount in situ hybridization of E10 wild-type and homozygous mutant embryos. A Dach1 antisense riboprobe hybridizes to transcripts in the eye, limbs, neural tube, and brain in wild-type embryos (B), while this pattern is not detected in homozygous mutants (C) (11). This riboprobe corresponds to coding sequences located downstream of exon 1 and therefore is not deleted in the mutant allele. Tails were removed from the animals for PCR genotyping after color development. (D and E) Immunohistochemical analysis of E12.5 wild-type and homozygous mutant eye sections. A Dach1 antibody demonstrates protein expression within the peripheral retina, retinal pigmented epithelium, and developing cornea in wild-type eyes (D), while Dach1 staining is reduced to background levels in the homozygous mutant eyes (E). Lens and surface ectoderm staining is due to background staining of the secondary antibody.

Dach1 expression analysis in mutant embryos. To determine the effect of the knockout mutation on Dach1 RNA expression, whole-mount in situ hybridization analysis was performed. Embryos were hybridized in batch to a Dach1 antisense riboprobe and processed together through the color development step. PCR analysis of embryo tail DNA was then performed to compare genotypes with hybridization patterns (Fig. 2B). Unlike wild-type embryos that demonstrated expression in the brain, retina, and developing limbs, the Dach1 expression pattern in the homozygous mutants was dramatically reduced to background levels. Since the Dach1 antisense riboprobe used in these experiments is derived from coding sequences downstream of exon 1, the absence of Dach1 transcripts reflects changes in gene expression, presumably due to premature termination of nascent transcripts within PGK-hprt or the deletion of necessary regulatory elements.

Analysis of newborn Dach1 mutant mice. Observations of newborn litters demonstrated postnatal lethality associated with the Dach1 mutant allele (see below). To characterize phenotypes prior to death, we focused our analysis on progeny less than 2 h old, when lethality was infrequent. Although 20% of these newborns were homozygous mutant (Fig. 1C), the external anatomy of the newborns appeared to be indistinguishable from that of control littermates (see below). This was also observed in intercrosses between Dach1 mutant lines B and G in 129/SvEv and 129/SvEv/C57BL/6J backgrounds. In addition, comparison of the weights and lengths of wild-type, heterozygote, and homozygous mutant newborns revealed no differences, consistent with the absence of a pronounced developmental phenotype (Table 1 and data not shown, respectively).

View larger version (136K): [in this window] [in a new window]   FIG. 3.   Gross and histological analyses of newborn wild-type (+/+) and Dach1 homozygous mutant (/) mice. (A and B) H&E staining of eye sections. The retina, optic nerve, lens, retinal pigmented epithelium, cornea, and ciliary margin are present and appear to be normal in newborn Dach1 homozygous mutant eyes. (C and D) High magnification of H&E-stained retinas. Dach1 mutant retinas exhibit a similar degree of layering and cell numbers as newborn wild-type retinas. (E and F) The external dimensions and morphology of wild-type and Dach1 mutant legs and digits are similar. (G and H) Alcian blue-alizarin red staining of wild-type and homozygous mutant skeletons does not reveal any detectable differences in either bone or cartilage morphology. The homozygote right forelimb was detached to reveal the structure of the limb. (I and J) H&E-stained coronal sections of newborn brains showing cortical layers, hippocampi, and trigeminal ganglia (arrow) in both wild-type and homozygous mutant animals. (K and L) High magnification of H&E-stained neocortex from newborn mice. The marginal zone, cortical plate, subplate, and intermediate zone of the cortex can be seen in newborn Dach1 mutant brains. (M and N) Lungs of wild-type and homozygous mutant newborn animals do not show any detectable differences in airway branching, alveolar septation, spacing, and cellular composition. (O and P) Whole-mount in situ hybridization of newborn lungs using a Clara cell-specific riboprobe, CC10. Epithelial cells lining the terminal bronchioles with similar branching patterns are found in both wild-type and homozygous mutant lungs.

The Dach1 mutant allele is associated with postnatal lethality. Genotype analysis of intercross progeny revealed that Dach1 mutant homozygotes did not survive to weaning. Postnatal lethality was observed in intercrosses between Dach1 mutant lines B and G in 129/SvEv and 129/SvEv/C57BL/6J backgrounds. Analysis of 2 h-old litters demonstrated that 93% (29 of 31) of homozygous mutants were alive (Fig. 1C). Thus, most Dach1 mutants die between this time and 3 weeks of age.