Genetically programmed retinoic acid deficiency during gastrulation phenocopies most known developmental defects due to acute prenatal alcohol exposure in FASD.

Fetal Alcohol Spectrum Disorder (FASD) arises from maternal consumption of alcohol during pregnancy affecting 2%-5% of the Western population. In Xenopus laevis studies, we showed that alcohol exposure during early gastrulation reduces retinoic acid (RA) levels at this critical embryonic stage inducing craniofacial malformations associated with Fetal Alcohol Syndrome. A genetic mouse model that induces a transient RA deficiency in the node during gastrulation is described. These mice recapitulate the phenotypes characteristic of prenatal alcohol exposure (PAE) suggesting a molecular etiology for the craniofacial malformations seen in children with FASD. Gsc +/Cyp26A1 mouse embryos have a reduced RA domain and expression in the developing frontonasal prominence region and delayed HoxA1 and HoxB1 expression at E8.5. These embryos also show aberrant neurofilament expression during cranial nerve formation at E10.5 and have significant FASD sentinel-like craniofacial phenotypes at E18.5. Gsc +/Cyp26A1 mice develop severe maxillary malocclusions in adulthood. Phenocopying the PAE-induced developmental malformations with a genetic model inducing RA deficiency during early gastrulation strongly supports the alcohol/vitamin A competition model as a major molecular etiology for the neurodevelopmental defects and craniofacial malformations seen in children with FASD.

Cloning of a human S-phase cell cycle gene: use of transient expression for screening.

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.

BMP controls nitric oxide-mediated regulation of cell numbers in the developing neural tube.

Balanced cell proliferation and cell death determines neural precursor cell numbers in early stages of neural tube (NT) development. We have previously shown that nitric oxide (NO) regulates cell numbers locally in the NT of eight to 12 somite embryos. Here, we demonstrate that bone morphogenetic protein-4 (BMP-4), which is expressed in the ectoderm and dorsal NT at these developmental stages, induces programmed cell death (PCD) and promotes entry into the S-phase, via nitric oxide synthase (NOS) activity. These effects can be reversed by BMP-4 antagonists, such as follistatin and noggin, or by specific NOS inhibitors, resulting in low NO levels that facilitate mitosis and reduce PCD. Ectopic BMP-4 induction of PCD is restricted to the dorsal NT, whereas promotion of the S-phase is evenly observed across the dorsal-ventral (D-V) axis. Prolonged exposure to either BMP-4 or NOS inhibitors, which results in high or low NO levels, respectively, causes NT defects. The results presented here throw new light on the BMP signaling pathway. The local presence of BMP-4 helps to regulate cell numbers in the developing NT by a NO-mediated pathway, which is essential for normal NT formation.

The chicken caudal genes establish an anterior-posterior gradient by partially overlapping temporal and spatial patterns of expression.

The caudal genes in vertebrates as in invertebrates assume a posterior position along the anterior-posterior axis and they appear to regulate the expression of the Hox genes. The third chicken caudal gene, Cdx-C, was cloned. Extensive comparisons of the sequence of this protein to the other known members of this homeobox family has lead to the suggestion that vertebrate genomes contain three members of the caudal homeobox family. A comparative study of the chicken Cdx-A and Cdx-C genes during gastrulation and neurulation revealed the differences between the genes. The caudal genes exhibit sequential activation in the newly formed neural plate and sequential extinction in axial midline structures during the primitive streak regression along the anterior-posterior axis. This pattern of expression suggests that the number and identity of caudal genes expressed along the anterior-posterior axis changes dynamically.

Patterning of the mesoderm involves several threshold responses to BMP-4 and Xwnt-8.

Two secreted signaling molecules, Xwnt-8 and BMP-4, play an essential role in the dorso-ventral patterning of the mesoderm in Xenopus. Here we investigate how the Wnt-8 and the BMP-4 pathways are connected and how they regulate target genes in the lateral and ventral marginal zone. BMP-4 regulates the transcription of Xwnt-8 in a threshold dependent manner. High levels of BMP-4 induce the expression of the Wnt antagonist sizzled in the ventral marginal zone, independent of Xwnt-8 signaling. Xwnt-8 induces the early muscle marker myf-5 in the lateral marginal zone in a BMP independent manner. The expression of the homeobox gene Xvent-1 can be modulated through both the BMP-4 and the Xwnt-8 pathways. The spatial distribution and the level of BMP-4 activity in the lateral and ventral marginal zone is reflected in the dynamic expression pattern of Xwnt-8. The data support the view that Xwnt-8 is involved in the specification of lateral (somitogenic) mesoderm and BMP-4 in the specification of ventral mesoderm.

Expression of the novel murine homeobox gene Sax-1 in the developing nervous system.

We have isolated the novel murine Sax-1 gene, a member of the NK-1 class of homeobox genes, and report its expression pattern in the developing central nervous system (CNS) in comparison to two other homeobox genes, Evx-1 and Pax-6. Sax-1 was found to be transiently expressed in the developing posterior CNS. First seen in the ectoderm lateral to the primitive streak, the signal later encompassed the neural plate. Posteriorly, the expression domain overlapped with the Evx-1 expression in the streak, while anteriorly it was delimited by the Pax-6 signal in the neural tube. This early phase starting at day 9.5 pc, Sax-1 was expressed in distinct areas of spinal cord, hindbrain and forebrain. Particularly strong signals were detected in rhombomere 1 and in the pretectum. In these areas, subsets of neurons may be marked and specified. In addition to the normal pattern of Sax-1 during development, the expression in different mouse mutants was analysed. In Brachyury curtailed homozygotes, the expression of Sax-1 was found to be reduced during neurulation and even lost at day 9.0 pc. Ventral shift and finally loss of the signal in the ventral spinal cord was observed in Danforth's short tail homozygotes.

The Xvex-1 antimorph reveals the temporal competence for organizer formation and an early role for ventral homeobox genes.

The organizer in vertebrate embryos has been shown to play a central role in their development by antagonizing ventralizing signals and promoting dorsal development. The ventral homeobox gene, Xvex-1, is capable of fulfilling some of the functions of BMP-4. By fusion to activation and repression domains, Xvex-1 was shown to function as a repressor of transcription. The activator version of Xvex-1, the antimorph, was made inducible by fusion to the ligand binding domain of the glucocorticoid receptor. The organizer genes, gsc and Otx-2, were identified as direct targets of Xvex-1. The XVEX-1 antimorph can induce the formation of secondary axes. Temporal analysis of secondary axis induction revealed that the competence to induce a secondary organizer ends with the onset of gastrulation. The same temporal competence window was exhibited by an inducible gsc construct. Partial loss of Xvex-1 activity was able to improve the efficiency of secondary axis induction by the dominant negative BMP receptor or Smad6. These observations together with the early widespread expression of Xvex-1 throughout the embryo prior to gastrulation encoding a homeodomain repressor protein, suggest that elements of the ventral signaling pathway play an important role during late blastula in restricting the formation of Spemann's organizer.

A role for the homeobox gene Xvex-1 as part of the BMP-4 ventral signaling pathway.

BMP-4 is believed to play a central role in the patterning of the mesoderm by providing a strong ventral signal. As part of this ventral patterning signal, BMP-4 has to activate a number of transcription factors to fulfill this role. Among the transcription factors regulated by BMP-4 are the Xvent and the GATA genes. A novel homeobox gene has been isolated termed Xvex-1 which represents a new class of homeobox genes. Transcription of Xvex-1 initiates soon after the midblastula transition. Xvex-1 transcripts undergo spatial restriction from the onset of gastrulation to the ventral marginal zone, and the transcripts will remain in this localization including at the tailbud stage in the proctodeum. Expression of Xvex-1 during gastrula stages requires normal BMP-4 activity as evidenced from the injection of BMP-4, Smad1, Smad5 and Smad6 mRNA and antisense BMP-4 RNA. Xvex-1 overexpression ventralizes the Xenopus embryo in a dose dependent manner. Partial loss of Xvex-1 activity induced by antisense RNA injection results in the dorsalization of embryos and the induction of secondary axis formation. Xvex-1 can rescue the effects of overexpressing the dominant negative BMP receptor. These results place Xvex-1 downstream of BMP-4 during gastrulation and suggest that it represents a novel homeobox family in Xenopus which is part of the ventral signaling pathway.

CHox E, a chicken homeogene of the H2.0 type exhibits dorso-ventral restriction in the proliferating region of the spinal cord.

CHox E is a novel chicken homeogene that belongs to the H2.0 family of homeodomains. Its homeobox sequence is interrupted by an intron between amino acids 44 and 45. Expression of CHox E during embryogenesis is localized to the central nervous system. The anterior boundary of CHox E expression can initially be localized to rhombomere number 1, later in development this boundary reaches up to the rhombencephalic isthmus. CHox E expression in the spinal cord localizes dorso-ventrally to the dorsal half of the basal plate. CHox E expression is always restricted to the proliferating region, the ventricular zone. As the ventricular zone becomes restricted laterally, so does the CHox E expressing region. Once this region of the ventricular zone ceases to exist, CHox E specific transcripts become undetectable. The site and time of CHox E expression suggest a very early function in the differentiation of the cells derived from that region of the ventricular zone.

The dorsalizing and neural inducing gene follistatin is an antagonist of BMP-4.

Specific signaling molecules play a pivotal role in the induction and specification of tissues during early vertebrate embryogenesis. BMP-4 specifies ventral mesoderm differentiation and inhibits neural induction in Xenopus, whereas three molecules secreted from the organizer, noggin, follistatin and chordin dorsalize mesoderm and promote neural induction. Here we report that follistatin antagonizes the activities of BMP-4 in frog embryos and mouse teratocarcinoma cells. In Xenopus embryos follistatin blocks the ventralizing effect of BMP-4. In mouse P19 cells follistatin promotes neural differentiation. BMP-4 antagonizes the action of follistatin and prevents neural differentiation. In addition we show that the follistatin and BMP-4 proteins can interact directly in vitro. These data provide evidence that follistatin might play a role in modulating BMP-4 activity in vivo.

Nested expression and sequential downregulation of the Xenopus caudal genes along the anterior-posterior axis.

Expression of the Xenopus Xcad-1 and Xcad-2 genes initiates during early gastrulation exhibiting a dorsoventral asymmetry in their domains of transcription. At mid-gastrulation the ventral preference becomes stronger and the caudal genes take up a posterior localization in their expression, which they will maintain until their downregulation along the dorsal midline. Comparison of the three Xenopus caudal genes revealed a temporal and spatial nested set of expression patterns. The transcription of the caudal genes is sequentially downregulated with the one expressed most caudally (Xcad-2) being shut down first, this sequence is most evident along the dorsal midline. This pattern of expression suggests a role for the caudal genes as posterior determinants along the anteroposterior axis. In chicken, mouse, man and Xenopus three members of the caudal family have been identified in the genome. Even though in Xenopus the Xcad-3 gene has been previously described, in order to obtain a better insight on the role of the caudal genes a comparative study of all three frog genes was performed.

The Xcad-2 gene can provide a ventral signal independent of BMP-4.

Patterning of the marginal zone in the Xenopus embryo has been attributed to interactions between dorsal genes expressed in the organizer and ventral-specific genes. In this antagonistic interplay of activities, BMP-4, a gene that is not expressed in the organizer, provides a strong ventralizing signal. The Xenopus caudal type homeobox gene, Xcad-2, which is expressed around the blastopore with a gap over the dorsal lip, was analyzed as part of the ventral signal. Xcad-2 was shown to efficiently repress during early gastrula stages the dorsal genes gsc, Xnot-2, Otx-2, XFKH1 and Xlim-1, while it positively regulates the ventral genes, Xvent-1 and Xvent-2, with Xpo exhibiting a strong positive response to Xcad-2 overexpression. Xcad-2 was also capable of inducing BMP-4 expression in the organizer region. Support for a ventralizing role for Xcad-2 was obtained from co-injection experiments with the dominant negative BMP receptor which was used to block BMP-4 signaling. Under lack-of-BMP-signaling conditions Xcad-2 could still regulate dorsal and ventral gene expression and restore normal development, suggesting that it can act downstream of BMP-4 signaling or independently of it. Xcad-2 could also inhibit secondary axis formation and dorsalization induced by the dominant negative BMP receptor. Xcad-2 was also shown to efficiently reverse the dorsalizing effects of LiCl. These results place Xcad-2 as part of the ventralizing gene program which acts during early gastrula stages and can execute its ventralizing function in the absence of BMP signaling.

The chicken homeo box genes CHox1 and CHox3: cloning, sequencing and expression during embryogenesis.

Several Drosophila genes involved in the control of segmentation and segment identity share a 183-bp conserved sequence termed homeo box. Homeo box sequences have been detected and cloned from the genomes of insects like Drosophila to vertebrates such as mouse and man. Two chicken homeo box genes CHox1 and CHox3, are described. Cloning of the CHox1 and CHox3 homeo boxes was performed using Drosophila and murine homeo box sequences as probes under low-stringency conditions. Analysis of both chicken homeo box sequences revealed them to be homeo boxes that have diverged from the Antennapedia class with homologies to homeo boxes of other organisms in the range of 75-42% at the nucleotide level and 69-41% at the protein level. Analysis of CHox3 expression during early embryo development showed that the gene codes for five transcripts 1.3, 1.9, 2.6, 5.6 and 7.9 kb in size. Three of the transcripts (1.3, 1.9 and 5.6 kb) are also recognized by a flanking non-homeo box containing probe. The levels of the different transcripts changed during the first five days of development. The most abundant transcripts (1.3 and 1.9 kb) are already present at the time the egg is laid. Their transcription peaks at day 1 of incubation and then decreases. The CHox1 transcripts are present at very low levels between days 2.5 and 4 of development. These two chicken genes represent bona fide Hox genes in a branch of vertebrates that evolved parallel to mammals.

A chicken homeo box gene with developmentally regulated expression.

Several homeo box sequences were isolated from a chicken (Gallus domesticus) genomic library. One phage, lambda GG7, was chosen for further study and the single homeo box sequence it contains was characterized. The sequence of the homeo box revealed it to be a distant member of the Antp class of homeo boxes. Expression of the CHox7 gene results in four transcripts, 1.4, 1.9, 2.4 and 3.5 kb in size. Transcript accumulation of the CHox7 gene peaks twice during embryogenesis, once with the 1.4 kb transcript during early somitogenesis and the second time with the 1.9 kb transcript during organogenesis.

The two Xenopus Gbx2 genes exhibit similar, but not identical expression patterns and can affect head formation.

Gbx2 homeobox genes are important for formation and function of the midbrain/hindbrain boundary, namely the isthmic organizer. Two Gbx2 genes were identified in Xenopus laevis, differing in 13 amino acids, including a change in the homeodomain. Xgbx2a is activated earlier during gastrulation and reaches higher levels of expression while Xgbx2b is expressed later, at lower levels and has an additional domain in the ventral blood islands. Their overexpression results in microcephalic embryos with shortened axes and defects in brain and notochord formation. Both genes encode functionally homologous proteins, which differ primarily in their temporal and spatial expression patterns.

Analysis of a Chinese hamster temperature-sensitive cell cycle mutant arrested in early S phase.

E36 ts24 is a temperature-sensitive cell cycle mutant which has been derived from the Chinese hamster lung cell line E36. This mutant is arrested in phase S when incubated at the restrictive temperature (40.3 degrees C) for growth. At this temperature, proliferation of the mutant cells ceases after 10 h. About 2 h earlier, DNA synthesis is arrested. These kinetic studies indicate that the execution point of the mutant cells is in early S phase well beyond the G1/S boundary. The pattern of replication bands in E36 ts24 cell grown for 9 h at 40.3 degrees C strengthen the kinetic studies and map the execution point to early S phase. The exact point of arrest of the mutant cells in phase S was mapped in early S phase near the execution point. At the point of arrest the cells continue to synthesize DNA at at a high rate but practically all of the newly synthesized DNA is degraded. This high rate of DNA degradation is limited to nascent DNA at the point of arrest. In the presence of 5-bromodeoxyuridine (5-BudR), the last E36 ts24 cells which reach mitosis at the restrictive temperature for growth show asymmetric replication bands which illustrate DNA degradation and resynthesis occurring in these cells at 40.3 degrees C.

Genomic organization and expression during embryogenesis of the chicken CR1 repeat.

CR1 is one of the middle repetitive sequence elements present in the chicken genome. One such repetitive element (GG1-CR1) was found upstream of the chicken CHox E homeobox gene. Sequencing of GG1-CR1 demonstrated that it is one of the longest CR1 elements analyzed. Detailed comparison of all the CR1 sequences published has revealed three subfamilies of CR1 repeats containing various parts of the consensus sequence. We prepared DNA fragments from GG1-CR1 and used them to probe Southern blots and genomic and cDNA library lifts. The results confirm the division of CR1 into three subelements, two of which occur independently in many places in the genome. Northern blot analysis of the CR1 to chicken embryo RNA showed that the CR1 repeat can be part of poly(A)+ transcripts. These results suggest that the CR1 can be transcribed by readthrough from the promoter of the neighboring gene without detrimental effects on the expression of the gene itself. The level of CR1 containing transcripts rises during the first 5 days of embryonic development and then decreases.

Sequence analysis of the murine Hox-2.2, -2.3, and -2.4 homeo boxes: evolutionary and structural comparisons.

We have determined the nucleotide sequences and deduced the amino acid sequences of three tandemly arranged murine boxes of the Hox-2 homeo box gene complex on mouse chromosome 11 (Hox-2.2, -2.3, and -2.4). The type and position of differences with other sequenced homeo boxes were analyzed. Hox-2.2 is nearly identical with its cognate human homeo box Hu-2. Hox-2.3 shares 59 of 61 amino acids with the Antennapedia homeo domain of Drosophila and the MM-3 homeo domain of Xenopus and shows 60 of 61 amino acid identity with human HuC1. Hox-2.3, MM-3, and HuC1 also share a stretch of six glutamic acid residues followed by a stop codon 15-20 amino acids 3' of the homeo domain. Hox-2.4 is relatively divergent from most of the other homeo boxes sequenced to date; however, it matches the Hox-3.1 murine homeo domain at 60 of 61 positions. Sequence comparisons with other murine homeo domains, together with previous studies of their genomic organization and chromosomal location, provide support for the hypothesis of a large-scale duplication resulting in the two major murine homeo box gene complexes Hox-1 and Hox-2.

Expression of the murine homeo box gene Hox 1.5 during embryogenesis.

The spatial pattern of expression of the murine homeo box-containing gene Hox 1.5 was studied during embryogenesis. In situ hybridization of single-stranded RNA probes to mouse embryo sections revealed a specific spatial distribution of the Hox 1.5 transcripts in mouse embryos 8.5 to 12.5 days postcoitum (p.c.). Analysis of mouse embryos 8.5 days p.c. showed that the gene is expressed in a spatially restricted manner. Expression appears to be limited to the central nervous system with an anterior boundary in the hindbrain and extending posteriorly through caudal regions of the spinal cord. The same spatial pattern of expression was observed in embryos 9.5 to 12.5 days p.c. These results show that the murine Hox 1.5 gene is expressed in a spatially restricted manner during embryonic development similar to the patterns observed in Drosophila homeotic genes.

On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo.

Bone morphogenetic protein 4 (BMP-4) is expressed in the ventral marginal zone of the gastrulating embryo. At late gastrula stage this gene is expressed in the ventral-most part of the slit blastopore and in tissues that derive from it. At tailbud stages BMP-4 is expressed in the spinal cord roof plate, neural crest, eye and auditory vesicle. The interactions of BMP-4 with dorsal genes such as goosecoid (gsc) and Xnot-2 were studied in vivo. In embryos ventralized by UV irradiation and suramin treatment, BMP-4 zygotic transcripts accumulate prematurely and the entire marginal zone expresses this gene. The patterning effect of BMP-4 on ventro-posterior development can be revealed by a sensitive assay involving the injection of BMP-4 mRNA in the ventral marginal zone of embryos partially dorsalized with LiCl, which leads to the complete rescue of trunk and tail structures. The experiments presented here argue that BMP-4 may act in vivo as a ventral signal for the proper patterning of the marginal zone, actively interacting with dorsal genes such as gsc and Xnot-2. A model is proposed in which the timing of expression of various marginal zone-specific genes plays a central role in patterning the mesoderm.