The albino-deletion complex in the mouse defines genes necessary for development of embryonic and extraembryonic ectoderm

A detailed embryological analysis has been undertaken on embryos carrying the c4FR60Hd-, c5FR60Hg- or c2YPSj-albino deletions of mouse chromosome 7. Embryos homozygous for the c4FR60Hd deletion are abnormal at day 7.5 of gestation. The extraembryonic ectoderm does not develop, and primitive-streak formation and mesoderm production do not occur. In contrast, extensive development of the extraembryonic ectoderm, as well as mesoderm production, are observed in the c5FR60Hg- and c2YPSj-homozygous embryos. The mesoderm does not, however, organize into somites and the neural axis does not form. The embryos are grossly abnormal by day 8.5 of development. There are two other albino deletions (c6H and c11DSD) that are known to affect the embryo around the time of gastrulation (Niswander et al. 1988), and the lethal phenotype observed for the c4FR60Hd-homozygous embryos is similar to that described for c6H-homozygous embryos, whereas the c5FR60Hg- and c2YPSj-homozygous embryos display a phenotype that is similar to c11DSD-homozygous embryos. A detailed complementation analysis using these five deletions revealed that the c5FR60Hg, c2YPSj and c11DSD deletions could partially complement the phenotype produced by the c4FR60Hd and c6H deletions in any combination. Extensive development of the extraembryonic structures and production of mesoderm occurs in the compound heterozygotes. These results suggest that the distal breakpoints of the c5FR60Hg, c2YPSj and c11DSD deletions lie more proximal than the distal breakpoints of the c4FR60Hd and c6H deletions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular mapping of albino deletions associated with early embryonic lethality in the mouse

The albino-deletion complex consists of more than 37 deletions that remove an area of mouse chromosome 7 including the albino coat-color locus. Previous genetic and embryological studies with five of these deletions (C11DSD, c5FR60Hg, c4FR60Hd, c2YPSj, c6H) defined at least two genes required for normal development of the embryonic and extraembryonic ectoderm of early postimplantation embryos. A molecular genetic analysis of this region has been initiated using palb18, a genomic clone that defines the D7TM18 locus that maps to a region of chromosome 7 removed by the c11DSD deletion but not by the c5FR60Hg, c4FR60Hd, c2YPSj, or c6H deletions. palb18 was obtained by chromosomal microdissection and microcloning of the wild-type albino region. A genomic clone isolated with palb18 contains a repeat sequence localized primarily to the proximal region of the five deletions. The repeat sequence hybridizes differentially to the five deletion DNAs. The patterns of hybridization associated with these DNAs were used to define the order of the proximal breakpoints as centromere-c11DSD-c2YPSj-(c5FR60Hg-c4FR60Hd)- c6H. This order was confirmed by isolation of additional single-copy sequences. The molecular probes described here should allow for identification and isolation of the deletion breakpoints and thus provide immediate access to the distal side of the deletions where the genes affecting the development of the embryonic and extraembryonic ectoderm are located.     

The Albino-Deletion Complex of the Mouse: Molecular Mapping of Deletion Breakpoints That Define Regions Necessary for Development of the Embryonic and Extraembryonic Ectoderm

Previous complementation analyses with five (c11DSD, c5FR60Hg, c2YPSj, c4FR60Hd, c6H) of the mouse albino deletions defined at least two genes on chromosome 7, known as eed and exed, which are necessary for development of the embryonic and extraembryonic ectoderm, respectively, of early postimplantation embryos. The region of chromosome 7 containing these two genes has now been accessed at the molecular level by cloning two of the deletion breakpoint-fusion fragments. The c2YPSj breakpoints were isolated by cloning an EcoRI fragment containing a copy of an albino region-specific repeat unique to c2YPSj DNA. Similarly, the c11DSD breakpoints were isolated by cloning a c11DSD EcoRI fragment detected by a unique-sequence probe mapping proximal to the albino-coat-color locus. By mapping the cloned breakpoints relative to the remaining three deletions, the c11DSD distal breakpoint was found to define the distal limit of the region containing eed, whereas the c2YPSj and c6H distal breakpoints were found to define the proximal and distal limits, respectively, of the region containing exed.     

Genetic analysis of the exed region in mouse

The extraembryonic ectoderm development (exed) mutant phenotype was described in mice homozygous for the c(6H) deletion, a radiation-induced deletion in the tyrosinase region of mouse Chromosome 7. These mutants fail to gastrulate and die around embryonic day 8.0. Several genes including, for example, embryonic ectoderm development (eed), are deleted in the c(6H) mutants; however, the portion of the chromosome responsible for the more severe exed phenotype is localized to a 20-kb region called the "exed-critical region." To understand the genetics behind the exed phenotype, we analyzed this region in two ways. First, to determine whether the 20-kb exed-critical region alone causes the mutant phenotype, we removed it from a wild-type chromosome. The resulting mice homozygous for this deletion were viable and fertile, indicating that the 20-kb exed-critical region by itself is not sufficient to cause the phenotype when deleted. We then sequenced the 20-kb exed-critical region and no expressed exons were found. Several short matches to GenBank Expressed Sequence Tag (EST) databases were identified; however, none of these ESTs mapped to the region. Taken together, these results indicate that the exed phenotype may either be a position effect on a distal gene caused by the c(6H) breakpoint or the result of composite effects of nullizygosity of multiple genes in the deletion homozygotes.     

X chromosome inactivation revealed by the X-linked lacZ transgene activity in periimplantation mouse embryos

Using H253 mouse stock harboring X-linked HMG-lacZ transgene, we examined X chromosome inactivation patterns in sectioned early female embryos. X-gal staining patterns were generally consistent with the paternal X inactivation in the trophectoderm and the primitive endoderm cell lineages and random inactivation in the epiblast lineages. The occurrence of embryonic visceral endoderm cells apparently at variance with the paternal X chromosome inactivation in 7.5 dpc embryos was explained by the replacement of visceral endoderm cells with cells of epiblast origin. The frequency of cells negative for X-gal staining in 4.5-5.5 dpc XmXp* embryos fluctuated considerably especially in the extraembryonic ectoderm and the primitive endoderm, whereas it was less variable in the embryonic ectoderm. We could not, however, determine whether it is a normal phenomenon revealed for the first time by the use of HMG-lacZ transgene or an abnormality caused by the multicopy transgene.     

Genetic rescue of lethal genotypes in the mouse

Deletions of gene sequences in chromosome 7 of the mouse are known to interfere with biochemical and cellular development differentiation with lethal effects in homozygotes. The presence of the corresponding wild-type alleles in Cattanach's translocation (chromosomes 7 to X) is able to “rescue” potentially lethal females if they are made heterozygous for the translocation-carrying X chromosome. This holds true for those chromosome 7 deletions with perinatally lethal effects, whereas “rescue” is not readily accomplished with the deletions that cause early embryonic lethality. Females homozygous for the relevant deletion sequences and heterozygous for the translocation-carrying X chromosome are mosaics of two cell types: those in which the wild-type alleles included in the translocated piece complement the depleted sequences, resulting in a normal cellular phenotype, and those with the ordinary X chromosome expressing the lethal phenotype. The developmental interactions between the two cell types and their role in the mechanisms responsible for survival of females homozygous for lethal deletions are discussed. The failure of “rescue” of embryonic lethals reflects as yet unknown temporal and functional aspects of X-inactivation early embryogenesis.     

Tetraploid embryos rescue the early defects of tw5/tw5 mouse embryos

tclw5 is a t-complex recessive lethal mutation of the tw5-haplotype. Since tw5/tw5 embryos die soon after implantation, the tclw5 gene is thought to play an important role in early embryogenesis. Previous histological studies have demonstrated that tw5 homozygotes do not survive past the gastrulation stage due to extensive death of the embryonic ectoderm, whereas the extraembryonic tissues were less affected. In the present study, we demonstrate that tw5/tw5 embryos may be distinguished from wildtype littermates at embryonic (E) day 5.5. At this stage, the visceral endoderm of tw5/tw5 embryos appeared to be different, possessing smaller and fewer vacuoles compared to normal littermates. This led us to hypothesize that the visceral endoderm may be affected by tclw5. Confirmation was provided by the rescue of tw5/tw5 embryos following aggregation with tetraploid embryos. However, rescued embryos did not survive past E9.0 and displayed an underdeveloped posterior region. This would indicate that the actions of tclw5 extend beyond the midgestation stage.     

Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation in the mouse.

Lim1 is a homeobox gene expressed in the extraembryonic anterior visceral endoderm and in primitive streak-derived tissues of early mouse embryos. Mice homozygous for a targeted mutation of Lim1 lack head structures anterior to rhombomere 3 in the hindbrain. To determine in which tissues Lim1 is required for head formation and its mode of action, we have generated chimeric mouse embryos and performed tissue layer recombination explant assays. In chimeric embryos in which the visceral endoderm was composed of predominantly wild-type cells, we found that Lim1(-)(/)(-) cells were able to contribute to the anterior mesendoderm of embryonic day 7.5 chimeric embryos but that embryonic day 9.5 chimeric embryos displayed a range of head defects. In addition, early somite stage chimeras generated by injecting Lim1(-)(/)(-) embryonic stem cells into wild-type tetraploid blastocysts lacked forebrain and midbrain neural tissue. Furthermore, in explant recombination assays, anterior mesendoderm from Lim1(-)(/)(-) embryos was unable to maintain the expression of the anterior neural marker gene Otx2 in wild-type ectoderm. In complementary experiments, embryonic day 9.5 chimeric embryos in which the visceral endoderm was composed of predominantly Lim1(-)(/)(-) cells and the embryo proper of largely wild-type cells, also phenocopied the Lim1(-)(/)(-) headless phenotype. These results indicate that Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation and that its inactivation in these tissues produces cell non-autonomous defects. We discuss a double assurance model in which Lim1 regulates sequential signaling events required for head formation in the mouse.     

Deletion mapping of the T/t complex: evidence for a second region of critical embryonic genes

The developmental effects of three different deletion mutations of the T/t complex of the mouse have been studied. The three mutations, TOak Ridge (OR), TOrleans (TOrl), and THair pin (THp), each produce a unique homozygous lethal phenotype: THp homozygotes fail to develop normally past the morula stage, TOrl homozygotes past the blastocyst stage, and TOR homozygotes past the egg cylinder stage. In compound embryos (TX/TY), the lethal phenotype observed corresponds to the shared length of deleted chromosome. This interaction allows the regions of chromosome 17, containing genetic information critical to early mammalian development, to be mapped.     

An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome

Deletion of chromosome 22q11, the most common microdeletion detected in humans, is associated with a life-threatening array of birth defects. Although 90% of affected individuals share the same three megabase deletion, their phenotype is highly variable and includes craniofacial and cardiovascular anomalies, hypoplasia or aplasia of the thymus with associated deficiency of T cells, hypocalcemia with hypoplasia or aplasia of the parathyroids, and a variety of central nervous system abnormalities. Because ablation of neural crest in chicks produces many features of the deletion 22q11 syndrome, it has been proposed that haploinsufficiency in this region impacts neural crest function during cardiac and pharyngeal arch development. Few factors required for migration, survival, proliferation and subsequent differentiation of pharyngeal arch neural crest and mesoderm-derived mesenchyme into their respective cardiovascular, musculoskeletal, and glandular derivatives have been identified. However, the importance of epithelial-mesenchymal interactions and pharyngeal endoderm function is becoming increasingly clear. Fibroblast growth factor 8 is a signaling molecule expressed in the ectoderm and endoderm of the developing pharyngeal arches and known to play an important role in survival and patterning of first arch tissues. We demonstrate a dosage-sensitive requirement for FGF8 during development of pharyngeal arch, pharyngeal pouch and neural crest-derived tissues. We show that FGF8 deficient embryos have lethal malformations of the cardiac outflow tract, great vessels and heart due, at least in part, to failure to form the fourth pharyngeal arch arteries, altered expression of Fgf10 in the pharyngeal mesenchyme, and abnormal apoptosis in pharyngeal and cardiac neural crest. The Fgf8 mutants described herein display the complete array of cardiovascular, glandular and craniofacial phenotypes seen in human deletion 22q11 syndromes. This represents the first single gene disruption outside the typically deleted region of human chromosome 22 to fully recapitulate the deletion 22q11 phenotype. FGF8 may operate directly in molecular pathways affected by deletions in 22q11 or function in parallel pathways required for normal development of pharyngeal arch and neural crest-derived tissues. In either case, Fgf8 may function as a modifier of the 22q11 deletion and contribute to the phenotypic variability of this syndrome.     

Targeted deletion of fatty acid transport protein-4 results in early embryonic lethality

Fatty acid transport protein-4 (FATP4) is the major FATP in the small intestine. We previously demonstrated, using in vitro antisense experiments, that FATP4 is required for fatty acid uptake into intestinal epithelial cells. To further examine the physiological role of FATP4, mice carrying a targeted deletion of FATP4 were generated. Deletion of one allele of FATP4 resulted in 48% reduction of FATP4 protein levels and a 40% reduction of fatty acid uptake by isolated enterocytes. However, loss of one FATP4 allele did not cause any detectable effects on fat absorption on either a normal or a high fat diet. Deletion of both FATP4 alleles resulted in embryonic lethality as crosses between heterozygous FATP4 parents resulted in no homozygous offspring; furthermore, no homozygous embryos were detected as early as day 9.5 of gestation. Early embryonic lethality has been observed with deletion of other genes involved in lipid absorption in the small intestine, namely microsomal triglyceride transfer protein and apolipoprotein B, and has been attributed to a requirement for fat absorption early in embryonic development across the visceral endoderm. In mice, the extraembryonic endoderm supplies nutrients to the embryo prior to development of a chorioallantoic placenta. In wild-type mice we found that FATP4 protein is highly expressed by the epithelial cells of the visceral endoderm and localized to the brush-border membrane of extraembryonic endodermal cells. This localization is consistent with a role for FATP4 in fat absorption in early embryogenesis and suggests a novel requirement for FATP4 function during development.     

Karyotypes and X chromosome inactivation in segregants of a murine X-autosome translocation, T(X;4)37H

Karyotypes and X chromosome inactivation were studied in embryos obtained from female mice carrying T(X;4)37H translocation on day 6 to 8 of gestation by a BrdU-acridine orange method. A total of 18 different karyotypes were found in 477 embryos examined: 90.0% embryos were products expected from 2:2 alternate or adjacent 1 disjunction. 3:1 and adjacent 2 disjunctions accounted for approximately 8.0% and 0.7% conceptuses, respectively. In the embryo proper of balanced T37H/ + conceptuses, inactivation was random with respect to the normal X and the larger translocation X (4x) chromosome. In all the cells with the 4x inactive, the late replication apparently did not spread to the attached autosomal portion, although black/brown coat variegation implies spreading of inactivation into the autosomal region. The X chromosome segment deprived of the inactivation center remained active in all the cells examined and it exerted deleterious effects on embryonic or fetal development. Observation in embryos having two maternally derived X chromosomes showed that they were indeed resistant to inactivation in early extraembryonic cell lineages, and two copies of active X chromosomes in the trophectoderm fatally affected embryonic development due to inability to form the extraembryonic ectoderm and ectoplacental cone from the polar trophectoderm. In unbalanced X aneuploids the X chromosomes with the deletion were preferentially inactivated due to strong selection against nullisomy X.     

Persistent Nucleoli in Early Postimplantation of Rat Embryos

Ultrastructural changes of the nucleolus in mitotic embryonic ectodermal cells of 7 1/2-day and 7 2/3-day rat embryos were examined. It was found that the nucleolus was broken down into small fragments during late prophase and metaphase, and that some of these fragments persisted in the cytoplasm of telophase cell (persistent nucleoli). No interphase embryonic ectodermal cells contained persistent nucleoli. Persistent nucleoli were also found in telophase cells of extraembryonic ectoderm, extraembryonic visceral endoderm and parietal endoderm of the embryos, but they disappeared in interphase cells. Persistent nucleoli in telophase cells tended to decrease in size with embryonic age, and they had almost completely disappeared in neuroectodermal cells of the telencephalon in 14 1/2-day embryos. They were concluded to be remnants of disappearing nucleoli in embryonic cells that were cycling too rapidly to permit their nucleoli to disappear completely.     

BMP receptor IA is required in the mammalian embryo for endodermal morphogenesis and ectodermal patterning

BMPRIA is a receptor for bone morphogenetic proteins with high affinity for BMP2 and BMP4. Mouse embryos lacking Bmpr1a fail to gastrulate, complicating studies on the requirements for BMP signaling in germ layer development. Recent work shows that BMP4 produced in extraembryonic tissues initiates gastrulation. Here we use a conditional allele of Bmpr1a to remove BMPRIA only in the epiblast, which gives rise to all embryonic tissues. Resulting embryos are mosaics composed primarily of cells homozygous null for Bmpr1a, interspersed with heterozygous cells. Although mesoderm and endoderm do not form in Bmpr1a null embryos, these tissues are present in the mosaics and are populated with mutant cells. Thus, BMPRIA signaling in the epiblast does not restrict cells to or from any of the germ layers. Cells lacking Bmpr1a also contribute to surface ectoderm; however, from the hindbrain forward, little surface ectoderm forms and the forebrain is enlarged and convoluted. Prechordal plate, early definitive endoderm, and anterior visceral endoderm appear to be expanded, likely due to defective morphogenesis. These data suggest that the enlarged forebrain is caused in part by increased exposure of the ectoderm to signaling sources that promote anterior neural fate. Our results reveal critical roles for BMP signaling in endodermal morphogenesis and ectodermal patterning.     

Fine structural analysis of the effect of trypan blue on the visceral endoderm of the mouse egg cylinder

The effects of a maternal injection of trypan blue on primitive streak mouse embryos were studied with electron microscopy. Commercial trypan blue was purified by descending paper chromatography, and pregnant females received an intraperitoneal injection of the collected blue fraction on the evening of the 7th day of gestation. Ultrastructurally, the changes in the visceral endoderm were apparent 10 min after the injection and included an increase in the number of fuzzy-coated vesicles in the apical cytoplasm. By 20 min the apical cytoplasm of the extraembryonic visceral endodermal cells was filled with many fuzzy-coated vesicles and numerous vacuoles of various size and electron density. 30 min after the injection, the extraembryonic visceral endodermal cells were relatively smooth lacking a microvillous border and evidence of endocytic activity was rare. Many embryonic visceral endodermal cells were observed in various stages of degeneration although the underlying embryonic ectoderm appeared unaffected. Morphologically, it appears that trypan blue exerts its first effect by altering the endocytic activity of the visceral endoderm.     

Indian hedgehog signaling in extraembryonic endoderm and ectoderm differentiation in ES embryoid bodies

We previously demonstrated that a member of the Hedgehog gene family, Indian hedgehog (Ihh), is expressed in the visceral endoderm of EC and ES cell embryoid bodies and mouse embryos. Overexpression studies suggested that Ihh was involved in visceral endoderm differentiation. We now provide evidence for a Hh response in the embryoid body core and in the mesothelial layer of the visceral yolk sac. We also demonstrate that treatment of ES embryoid bodies with the Hh antagonists cAMP and forskolin results in downregulation of the Hh response and altered embryoid body differentiation. The outer endoderm layer undergoes a transition to parietal endoderm while formation of an embryonic ectoderm layer surrounding a cavity is inhibited. These treatments also result in a decrease in the expression of markers for the mesoderm derivatives, blood and endothelial cells. We present a model to explain how Ihh and BMP signaling may regulate extraembryonic endoderm and embryonic ectoderm differentiation.