cdx4/lacZ and cdx2/lacZ protein gradients formed by decay during gastrulation in the mouse

Expression of the mouse caudal genes cdx4 and cdx2 is examined by use of lacZ reporter constructs expressed in transgenic mouse embryos. During early gastrulation, up to at least 8.5 days of development, reporter mRNA distributions are apparently similar to those of endogenous cdx mRNAs. By 8.25 to 8.8 days, cdx/lacZ protein activities have become distributed as posterior-to-anterior gradients along the neural and mesoderm tissues. The gradients form by decay of activity as cells become distanced from the regressing tailbud. In situ hybridization studies indicate that the decay is primarily in cdx/lacZ protein activities rather than mRNAs. As gastrulation proceeds, the locations of the gradients regress progressively posteriorly along the growing axis. Our results indicate how cdx4 and cdx2 protein gradients might be generated by decay during normal development. The smoothness of the gradients that we detect shows that there cannot be extensive mixing of cells once they leave the tailbud to contribute to the growing axis. An enhancer element located in the first intron of the cdx4 gene is essential for correct transgene expression.     

Specification and maintenance of the spinal cord stem zone

Epiblast cells adjacent to the regressing primitive streak behave as a stem zone that progressively generates the entire spinal cord and also contributes to paraxial mesoderm. Despite this fundamental task, this cell population is poorly characterised, and the tissue interactions and signalling pathways that specify this unique region are unknown. Fibroblast growth factor (FGF) is implicated but it is unclear whether it is sufficient and/or directly required for stem zone specification. It is also not understood how establishment of the stem zone relates to the acquisition of spinal cord identity as indicated by expression of caudal Hox genes. Here, we show that many cells in the chick stem zone express both early neural and mesodermal genes; however, stem zone-specific gene expression can be induced by signals from underlying paraxial mesoderm without concomitant induction of an ambivalent neural/mesodermal cell state. The stem zone is a site of FGF/MAPK signalling and we show that although FGF alone does not mimic paraxial mesoderm signals, it is directly required in epiblast cells for stem zone specification and maintenance. We further demonstrate that caudal Hox gene expression in the stem zone also depends on FGF and that neither stem zone specification nor caudal Hox gene onset requires retinoid signalling. These findings thus support a two step model for spinal cord generation - FGF-dependent establishment of the stem zone in which progressively more caudal Hox genes are expressed, followed by the retinoid-dependent assignment of spinal cord identity.     

Expression of the mouse labial-like homeobox-containing genes, Hox 2.9 and Hox 1.6, during segmentation of the hindbrain.

The sequence of a mouse Hox 2.9 cDNA clone is presented. The predicted homeodomain is similar to that of the Drosophila gene labial showing 80% identity. The equivalent gene in the Hox 1 cluster is Hox 1.6 which shows extensive similarity to Hox 2.9 both within and outside the homeodomain. Hox 2.9 and Hox 1.6 are the only two mouse members of the labial-like family of homeobox-containing genes as yet identified. Hox 2.9 has previously been shown to be expressed in a single segmental unit of the developing hindbrain (rhombomere) and has been predicted to be involved in conferring rhombomere identity. To analyse further the function of Hox 2.9 during development and to determine if the other mouse labial-like gene Hox 1.6, displays similar properties, we have investigated the expression patterns of these two genes and an additional rhombomere-specific gene, Krox 20, on consecutive embryonic sections at closely staged intervals. This detailed analysis has enabled us to draw the following conclusions: (1) There are extensive similarities in the temporal and spatial expression of Hox 2.9 and Hox 1.6, throughout the period that both genes are expressed in the embryo (7 1/2 to 10 days). At 8 days the genes occupy identical domains in the neuroectoderm and mesoderm with the same sharp anterior boundary in the presumptive hindbrain. These similarities indicate a functional relationship between the genes and further suggest that the labial-like genes are responding to similar signals in the embryo. (2) By 9 days the neuroectoderm expression of both genes retreats posteriorly along the anteroposterior (AP) axis. The difference at this stage between the expression patterns is the persistence of Hox 2.9 in a specific region of the hindbrain, illustrating the capacity of Hox 2.9 to respond to additional positional regulatory signals and indicating a unique function for this gene in the hindbrain. (3) The restriction of Hox 2.9 expression in the hindbrain occurs at 8 1/2 days, approximately the same time as Krox 20 is first detected in the posterior adjoining domain. The mutually exclusive expression of Hox 2.9 and Krox 20 demarcated by sharp expression boundaries suggest that compartmentalisation of cells within the hindbrain has occurred up to 6 h before rhombomeres (morphological segments) are clearly visible. (4) Hox 2.9 expression is confined to the region of rhombomere 4 that shows cell lineage restriction and, unlike Krox 20, is expressed throughout the period that rhombomeres are visible (to 11 1/2 days).(ABSTRACT TRUNCATED AT 400 WORDS)     

Factors affecting cellular mosaicism in the expression of a lacZ transgene in two-cell stage mouse embryos.

In the present study, we have analysed the expression pattern of a lacZ transgene (CMZ12) in preimplantation stage mouse embryos. The transgene is expressed at the two-cell stage, where it shows cellular mosaicism due to variable expressivity. The variable gene expression indicates a partial penetrance of the transgene. The extent of variation in expression is influenced by the genetic background of the oocyte. DBA/2 and CFLP genetic backgrounds promote high expression of the transgene, while Balb/c, C57BL/6, DDK, and F1 (C57BL/6 x CBA) genetic backgrounds give none or very little lacZ activity. In vitro culture of one-cell embryos to the two-cell stage induces the expression of lacZ in all strain backgrounds tested. The variation in CMZ12 expression is a transient phenomenon and does not affect later stage activity of the transgene. Nuclear transfer experiments and DNA methylation analysis suggests that a heritable modification of the transgene locus has not occurred.     

The use of lacZ gene fusions in the studies of mammalian development: developmental regulation of mammalian homeobox genes in the CNS

1. Mammalian cells can produce bacterial beta-galactosidase when they carry the lacZ gene under the control of mammalian regulatory elements. The single cell resolution of the beta-galactosidase histochemical detection method makes this molecule an excellent marker in studies of development at the cellular and molecular level. Different lacZ fusion genes can be engineered to study the histological diversification of cell lineages, the developmental regulation of isolated genes, or to recognize and clone genes with new expression profile. 2. Transgenic mice carrying lacZ gene fusions provide information on the cell type, developmental stage and spatial specificity of cis-acting regulatory regions linked to a mammalian homeobox gene. We describe our strategy for designing the gene fusions. 3. The scord region of the Hox 1.3 gene is sufficient to determine spatially restricted expression of a heterologous protein in the midgestational spinal cord. We propose to use this region to alter the expression pattern of other homeobox gene products. Developmental alterations due to a variant expression pattern would point to the function of the misexpressed gene.     

Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression

Polycomb group genes were identified as a conserved group of genes whose products are required in multimeric complexes to maintain spatially restricted expression of Hox cluster genes. Unlike in Drosophila, in mammals Polycomb group (PcG) genes are represented as highly related gene pairs, indicative of duplication during metazoan evolution. Mel18 and Bmi1 are mammalian homologs of Drosophila Posterior sex combs. Mice deficient for Mel18 or Bmi1 exhibit similar posterior transformations of the axial skeleton and display severe immune deficiency, suggesting that their gene products act on overlapping pathways/target genes. However unique phenotypes upon loss of either Mel18 or Bmi1 are also observed. We show using embryos doubly deficient for Mel18 and Bmi1 that Mel18 and Bmi1 act in synergy and in a dose-dependent and cell type-specific manner to repress Hox cluster genes and mediate cell survival of embryos during development. In addition, we demonstrate that Mel18 and Bmi1, although essential for maintenance of the appropriate expression domains of Hox cluster genes, are not required for the initial establishment of Hox gene expression. Furthermore, we show an unexpected requirement for Mel18 and Bmi1 gene products to maintain stable expression of Hox cluster genes in regions caudal to the prospective anterior expression boundaries during subsequent development.     

Gene targeting and expression analysis of mouse Tem1/endosialin using a lacZ reporter

TEM1 (endosialin) expression is increased in the stroma and tumor vasculature of several common human cancers. The exact physiological role of TEM1 is still unknown since Tem1-deficient mice are viable and show only a lower rate of abdominal site-specific tumor invasion in tumor transplantation experiments. Previous studies have reported Tem1 expression in mouse embryos and adults, but did not determine the timing or location of the earliest expression, and did not examine all organ systems. Using the highly sensitive Bluo-Gal staining method for detecting temporal and spatial Tem1-lacZ activity in lacZ knock-in (+/lacZ) mice, we found that Tem1 gene expression was initially detectable in the dorsal aortic wall, the heart, the umbilical vessels, the first branchial arch, and the cephalic mesenchyme at E9.5. From E10.5 to E14.5, Tem1 gene expression was additionally seen mainly in the genital tubercle, the mesonephros, the whisker follicles, the mesenchymal tissues around the eye, and the lung. Remarkably, the kidney expressed abundant Tem1-lacZ starting from E16.5. Postnatally, Tem1 expression decreased in most organs but elevated expression persisted in the renal glomerulus and the uterus, where the expression pattern varied at different estrous cycle stages. Co-localization studies indicated that most vimentin-positive cells co-expressed Tem1-lacZ, while a large portion of CD31- or desmin-positive cells were also positive for Tem1-lacZ. Taken together, our observations suggest that Tem1 is expressed throughout embryonic and adult development in several types of mesenchymal cells closely related to blood vessels.     

Unexpected position-dependent expression of H-2 and beta 2-microglobulin/lacZ transgenes.

In order to study sequences involved in the developmentally regulated and tissue-specific expression of the class I Major Histocompatibility Complex (MHC) genes, we have constructed several H-2/lacZ transgenic lines in which the 5' regulatory sequences of the H-2Kb gene are linked to the Escherichia coli beta-galactosidase (lacZ) gene. In five H-2/lacZ lines, the pattern of lacZ expression, detected histochemically varied greatly from line to line. None of the H-2/lacZ transgenes were transcribed in cells normally expressing a high level of endogenous H-2 molecules, although these H-2 regulatory sequences have been shown to be sufficient to drive tissue-specific expression of other reporter genes. Interestingly, when constructs containing 5' beta 2-microglobulin (beta 2m) regulatory sequences linked to lacZ were used to derive transgenic lines, similar results were obtained. A survey of lacZ labeling in H-2/lacZ and beta 2m/lacZ transgenic mice strongly suggests that these transgenes are very sensitive to position effect, lacZ expression being controlled by endogenous chromosomal regulatory elements specific for each insertion site. Here we describe the complex pattern of lacZ expression in the different transgenic lines during development; we discuss the unusual properties of these transgenes and underline their potential use for developmental studies and characterization of genomic sequences involved in spatiotemporal gene expression.     

Acquisition of Hox codes during gastrulation and axial elongation in the mouse embryo

Early sequential expression of mouse Hox genes is essential for their later function. Analysis of the relationship between early Hox gene expression and the laying down of anterior to posterior structures during and after gastrulation is therefore crucial for understanding the ontogenesis of Hox-mediated axial patterning. Using explants from gastrulation stage embryos, we show that the ability to express 3' and 5' Hox genes develops sequentially in the primitive streak region, from posterior to anterior as the streak extends, about 12 hours earlier than overt Hox expression. The ability to express autonomously the earliest Hox gene, Hoxb1, is present in the posterior streak region at the onset of gastrulation, but not in the anterior region at this stage. However, the posterior region can induce Hoxb1 expression in these anterior region cells. We conclude that tissues are primed to express Hox genes early in gastrulation, concomitant with primitive streak formation and extension, and that Hox gene inducibility is transferred by cell to cell signalling. Axial structures that will later express Hox genes are generated in the node region in the period that Hox expression domains arrive there and continue to spread rostrally. However, lineage analysis showed that definitive Hox codes are not fixed at the node, but must be acquired later and anterior to the node in the neurectoderm, and independently in the mesoderm. We conclude that the rostral progression of Hox gene expression must be modulated by gene regulatory influences from early on in the posterior streak, until the time cells have acquired their stable positions along the axis well anterior to the node.