Hoxd4 and Rarg interact synergistically in the specification of the cervical vertebrae.

We show that, relative to single null mutants, mice bearing mutations in both Hoxd4 and Rarg display malformations of the basioccipital bone, and first (C1) and second cervical vertebrae (C2) at increased penetrance and expressivity, demonstrating synergy between Hoxd4 and Rarg in the specification of the cervical skeleton. In contrast to Rarg mutants, retinoic acid (RA) treatment on embryonic day 10.5 of Hoxd4 single or Hoxd4;Rarg double mutants does not rescue normal development of C2. Somitic expression of Hoxd4 is not altered in wild-type or Rarg mutant animals before or after RA treatment on day 10.5, suggesting that Hoxd4 and Rarg act in parallel to regulate the expression of target genes directing skeletogenesis.

RARbeta mediates the response of Hoxd4 and Hoxb4 to exogenous retinoic acid.

One action of retinoids in development is the regulation of Hox gene expression. Hoxd4 and Hoxb4 expression in the embryonic hindbrain is anteriorized by retinoic acid (RA) treatment of mid-gestation mouse embryos. Here we demonstrate that retinoic acid receptor beta (Rarb) deficient mice present only a partial anteriorization of either Hoxd4 or Hoxb4 in response to RA treatment. Our results strongly suggest that RARbeta is a mediator of their RA-response, and reveal anteroposterior polarity within a single rhombomere (r). Additionally, we generated mice doubly mutated for Hoxd4 and Rarb in an attempt to identify common morphogenetic pathways between these two genes. We conclude that there are no synergistic interactions between Hoxd4 and Rarb in the specification of the cervical vertebrae.

Characterization and retinoic acid responsiveness of the murine Hoxd4 transcription unit.

We have characterized the transcription unit of a murine Hox gene in the fourth paralogous group, Hoxd4. We have identified two Hoxd4 transcription start sites by S1 analysis. The upstream promoter (P2) is 5.2 kilobase pairs upstream from the coding region, while the downstream promoter (P1) is 1.1 kilobase pairs distant. Both promoters bear a cluster of start sites. Multiple transcripts were identified by Northern blot, originating from both promoters and multiple polyadenylation signals. Expression of P1 transcripts in the neural tube shows an anterior border at the rhombomere 6/7 boundary, corresponding to previous reports (Gaunt, S. J., Krumlauf, R., and Duboule, D. (1989) Development 107, 131-141; Morrison, A., Moroni, M. C., Ariza-McNaughton, L., Krumlauf, R., and Mavilio, F. (1996) Development 122, 1895-1907). A more posterior boundary in the central nervous system was observed for P2 transcripts. We observed strong expression up to somite 6 and weak expression in somite 5, correlating with the phenotype of Hoxd4 null mutant mice (Horan, G. S. B., Nagy Kovàcs, E., Behringer, R. R., and Featherstone, M. S. (1995) Dev. Biol. 169, 359-372). In response to retinoic acid, expression from P1 in the hindbrain was anteriorized after 4 or 24 h of treatment. P2 transcripts seemed to be less responsive and/or to have an indirect response to retinoic acid. The long 5'-untranslated region found in all Hoxd4 transcripts suggests that translation does not occur by a classical ribosome scanning mechanism.

Effect of IL-3 and E. coli LPS on the proliferation of mouse bone marrow cells in vitro.

Bone marrow cells from adult BALB/c mice were cultured at 37 degrees C, with 5% CO2 in air, in RPMI 1640 medium complemented with fetal calf serum. The addition of IL-3 (5% of WEHI-3-conditioned medium) or E. coli lipopolysaccharides (LPS, 50 micrograms/ml) to the cultures stimulated cell proliferation (1.29- and 1.22-fold, respectively, relative to control culture), whereas the simultaneous addition of the two factors reduced the number of cells recovered by 38% relative to those from control cultures (which were around 2.83 x 10(5) cells for each 10(6) plated cells). The frequency of blasts and cells with surface Ig presented the same pattern of variation (0.07 and 0.02%, respectively, in control cultures). The inhibitory effect of IL-3+LPS on cell proliferation was evident from the first day of culture, but more apparent on day 3. Macrophage-colony stimulating factor (M-CSF, L929-conditioned medium) and LPS each given alone stimulated proliferation but reduced it when given together. In contrast, fetal liver cells were not affected by the simultaneous addition of IL-3 and LPS or by M-CSF and LPS. The mechanism of action of the cumulative effect of these two factors is unknown. Since crude cell-conditioned medium was used as the source of IL-3, it is possible that another factor present in this medium interacts with LPS to cause the inhibitory effect on cell proliferation.

Effect of IL-3 and E. coli LPS on the proliferation of mouse bone marrow cells in vitro

Bone marrow cells from adult BALB/c mice were cultured at 37-C, with 5% CO2 in air, in RPMI 1640 medium complemented with fetal serum. The addition of IL-3 (5% of WEHI-3-conditioned medium) or E. coli lipopolysaccharides (LPS, 50 µg/ml) to the cultures stimulated cell proliferation (1.29 and 1.22-fold, respectively, relative to control culture), whereas the simultaneous addition of the two factors reduced the number of cells recovered by 38% relative those from control cultures (which were around 2.83 x 10***5 cells for each 10***6 plated cells). The frequency of blasts and cells with surface Ig presented the same pattern of variation (o.07 and 0.02%, respectively, in control cultures). The inhibitory effect of IL-3+LPS on cell proliferation was evident from the first day of culture, but more apparent on day 3. Macrophage-colony stimulating factor (M-CSF, L929-conditioned medium) and LPS each given alone stimulated proliferation but reduced it when given together. In contrast, fetal liver cells were not affected by the simultaneous addition of IL-3 and LPS or by M-CSF and LPS. The mechanism of action of the cumulative effect of these two factors in unknown. Since crude cell-conditioned medium was used as the source of IL-3, it is possible that another factor present in this medium interacts with LPS to cause the inhibitory effect on cell proliferation

Application of a flowchart for the detection of lysosomal storage diseases in 105 high-risk Brazilian patients.

Lysosomal storage diseases (LSD) are a group of more than 40 disorders, many of them with overlapping phenotype, in which clinical diagnosis is often difficult. Definitive diagnosis is based on enzyme assays, a large number of such assays usually being necessary during the investigation of each patient. In addition, there will frequently be a need for tissue culture in order to provide enough material for analysis. Taking into account these difficulties, we designed a flowchart for the detection of LSD that is based on 2 sets of tests requiring only random urine and heparinized blood. Here we describe this routine and report the results of its application to 105 Brazilian patients in whom a LSD was suspected. We think that the application of this rationale represents a saving of work and costs, and should be of special interest to genetic centers in developing countries.