The sacral autonomic outflow is sympathetic.

A kinship between cranial and pelvic visceral nerves of vertebrates has been accepted for a century. Accordingly, sacral preganglionic neurons are considered parasympathetic, as are their targets in the pelvic ganglia that prominently control rectal, bladder, and genital functions. Here, we uncover 15 phenotypic and ontogenetic features that distinguish pre- and postganglionic neurons of the cranial parasympathetic outflow from those of the thoracolumbar sympathetic outflow in mice. By every single one, the sacral outflow is indistinguishable from the thoracolumbar outflow. Thus, the parasympathetic nervous system receives input from cranial nerves exclusively and the sympathetic nervous system from spinal nerves, thoracic to sacral inclusively. This simplified, bipartite architecture offers a new framework to understand pelvic neurophysiology as well as development and evolution of the autonomic nervous system.

Neurodevelopment. Parasympathetic ganglia derive from Schwann cell precursors.

Neural crest cells migrate extensively and give rise to most of the peripheral nervous system, including sympathetic, parasympathetic, enteric, and dorsal root ganglia. We studied how parasympathetic ganglia form close to visceral organs and what their precursors are. We find that many cranial nerve-associated crest cells coexpress the pan-autonomic determinant Paired-like homeodomain 2b (Phox2b) together with markers of Schwann cell precursors. Some give rise to Schwann cells after down-regulation of PHOX2b. Others form parasympathetic ganglia after being guided to the site of ganglion formation by the nerves that carry preganglionic fibers, a parsimonious way of wiring the pathway. Thus, cranial Schwann cell precursors are the source of parasympathetic neurons during normal development.

Ventilatory response to hyperoxia in newborn mice heterozygous for the transcription factor Phox2b.

Heterozygous mutations of the transcription factor PHOX2B have been found in most patients with central congenital hypoventilation syndrome, a rare disease characterized by sleep-related hypoventilation and impaired chemosensitivity to sustained hypercapnia and sustained hypoxia. PHOX2B is a master regulator of autonomic reflex pathways, including peripheral chemosensitive pathways. In the present study, we used hyperoxic tests to assess the strength of the peripheral chemoreceptor tonic drive in Phox2b+/-newborn mice. We exposed 69 wild-type and 67 mutant mice to two hyperoxic tests (12-min air followed by 3-min 100% O2) 2 days after birth. Breathing variables were measured noninvasively using whole body flow plethysmography. The initial minute ventilation decrease was larger in mutant pups than in wild-type pups: -37% (SD 13) and -25% (SD 18), respectively, P<0.0001. Furthermore, minute ventilation remained depressed throughout O2 exposure in mutants, possibly because of their previously reported impaired CO2 chemosensitivity, whereas it returned rapidly to the normoxic level in wild-type pups. Hyperoxia considerably increased total apnea duration in mutant compared with wild-type pups (P=0.0001). A complementary experiment established that body temperature was not influenced by hyperoxia in either genotype group and, therefore, did not account for genotype-related differences in the hyperoxic ventilatory response. Thus partial loss of Phox2b function by heterozygosity did not diminish the tonic drive from peripheral chemoreceptors.

C21orf5, a novel human chromosome 21 gene, has a Caenorhabditis elegans ortholog (pad-1) required for embryonic patterning.

To contribute to the development of the transcription map of human chromosome 21 (HC21), we isolated a new transcript, C21orf5 (chromosome 21 open reading frame 5), encoding a predicted 2298-amino-acid protein. Analysis of the genomic DNA sequence revealed that C21orf5 consists of 37 exons that extend over 130 kb and maps between the CBR3 (carbonyl reductase 3) and the KIAA0136 genes. Northern blot analyses showed a ubiquitously expressed RNA species of 8.5 kb. RNA in situ hybridization on brain sections of normal human embryos revealed a strong labeling in restricted areas of the cerebral cortex. In silico analysis of the deduced C21orf5 protein revealed several highly probable transmembrane segments but no known protein domains or homology with known proteins. However, there were significant homologies to several hypothetical Caenorhabditis elegans proteins and Drosophila melanogaster genomic sequences. To investigate the function of C21orf5, we isolated the cDNA of the C. elegans ortholog and performed double-stranded RNA-mediated genetic interference experiments. The major phenotype observed in the progeny of injected animals was embryonic lethality. Most of the tissues of the embryo failed to undergo proper patterning during gastrulation, and morphogenesis did not occur; thus we termed the ortholog pad-1, for patterning defective 1. These results indicated that pad-1 is essential for the development and the survival of C. elegans. This study provides the first example of the use of C. elegans as a model to study the function of genes on human chromosome 21 that might be involved in Down syndrome.

Transcriptional map of the 2.5-Mb CBR-ERG region of chromosome 21 involved in Down syndrome.

The region of chromosome 21 between genes CBR and ERG (CBR-ERG region), which spans 2.5 Mb on 21q22.2, has been defined by analysis of patients with partial trisomy 21. It contributes significantly to the pathogenesis of many characteristics of Down syndrome, including morphological features, hypotonia, and mental retardation. Cosmid contigs covering 80% of the region were constructed and EcoRI maps produced. These cosmids were used for exon trapping and cDNA selection from three cDNA libraries (fetal brain, fetal liver, and adult skeletal muscle). Isolated exons and cDNAs were mapped on the EcoRI map, organized into contigs, sequenced, and used as probes for Northern blot analysis of RNA from fetal and adult tissues. We identified 27 genuine or highly probable transcriptional units evenly distributed along the CBR-ERG region. Eight of the transcriptional units are known genes.

Molecular mapping of 21 features associated with partial monosomy 21: involvement of the APP-SOD1 region.

We compared the phenotypes, karyotypes, and molecular data for six cases of partial monosomy 21. Regions of chromosome 21, the deletion of which corresponds to particular features of monosomy 21, were thereby defined. Five such regions were identified for 21 features. Ten of the features could be assigned to the region flanked by genes APP and SOD1: six facial features, transverse palmar crease, arthrogryposis-like symptoms, hypertonia, and contribution to mental retardation. This region, covering the interface of bands 21q21-21q22.1, is 4.7-6.4 Mb long and contains the gene encoding the glutamate receptor subunit GluR5 (GRIK1).

3.6-Mb genomic and YAC physical map of the Down syndrome chromosome region on chromosome 21.

The Down syndrome chromosome region (DCR) on chromosome 21 has been shown to contain a gene(s) important in the pathogenesis of Down syndrome. We constructed a long-range restriction map of the D21S55-D21S65 region covering the proximal part of the DCR. Pulsed-field gel electrophoresis of lymphocyte DNA digested with three rare cutting enzymes. NotI, NruI, and MluI, was used to establish two physical linkage groups of 5 and 7 markers, respectively, spanning 4.6 Mb on the NotI map. Mapping analysis of 40 YACs allowed the selection of 13 YACs covering 95% of the D21S55-D21S65 region and spanning 3.6 Mb. The restriction maps of these YACs and their positioning on the genomic map allowed 19 markers to be ordered, including 4 NotI linking clones, 9 polymorphic markers, the CBR gene, and the AML1 gene. The distances between markers could also be estimated. This physical map and the location of eight NotI sites between D21S55 and D21S17 should facilitate the isolation of previously unidentified genes in this region.

Mapping of the Down syndrome phenotype on chromosome 21 at the molecular level.

Phenotypic and molecular analysis of individuals with partial trisomy 21 can be used to determine which regions of chromosome 21 are involved in the pathogenesis of specific features of Down's Syndrome. Using dosage analysis of 27 sequences we defined, at the molecular level, the extent of the chromosome 21 duplication in ten individuals with partial trisomy 21. Phenotype-genotype correlations led to the definition of minimal regions, the duplications of which are linked to the expression of 23 clinical features of Down's Syndrome. The D21S55 region or Down's Syndrome Chromosome Region 1 (DCR1) (1/20 of the long arm), on 21q22.2-21q22.3 proximal, is involved in four cardinal features of the disease: mental retardation, growth retardation, muscular hypotonia and joint hyperlaxity, and in eight of the 18 more common morphological anomalies of the face, hands and feet. Overlapping the DCR1, the D21S55-MX1 region or DCR2 (1/10 of the long arm), spanning 21q21.2 down to the 1/4th proximal part of 21q22.3, is involved in the features defined by the DCR1 plus congenital heart defect and five additional morphological anomalies. Thus, our results indicate that duplication of a relatively small region of chromosome 21 plays a critical role in the pathogenesis of the Down's phenotype.

Molecular mapping of twenty-four features of Down syndrome on chromosome 21.

To determine which regions of chromosome 21 are involved in the pathogenesis of specific features of Down syndrome, we analysed, phenotypically and molecularly, 10 patients with partial trisomy 21. Six minimal regions for 24 features were defined by genotype-phenotype correlations. Nineteen of these features could be assigned to just 2 regions: short stature, joint hyperlaxity, hypotonia, major contribution to mental retardation and 9 anomalies of the face, hand and foot to the region D21S55, or Down syndrome chromosome region (DCR), located on q22.2 or very proximal q22.3, and spanning 0.4-3 Mb; 6 facial and dermatoglyphic anomalies to the region D21S55-MX1, including the DCR and spanning a maximum of 6 Mb on q22.2 and part of q22.3. Thus, the complex phenotype that constitutes Down syndrome may in large part simply result from the overdosage of only one or a few genes within the DCR and/or region D21S55-MX1.

No significant effect of monosomy for distal 21q22.3 on the Down syndrome phenotype in "mirror" duplications of chromosome 21.

Three Down syndrome patients for whom karyotypic analysis showed a "mirror" (reverse tandem) duplication of chromosome 21 were studied by phenotypic, cytogenetic, and molecular methods. On high-resolution R-banding analysis performed in two cases, the size of the fusion 21q22.3 band was apparently less than twice the size of the normal 21q22.3, suggesting a partial deletion of distal 21q. The evaluation of eight chromosome 21 single-copy sequences of the 21q22 region--namely, SOD1, D21S15, D21S42, CRYA1, PFKL, CD18, COL6A1, and S100B--by a slot blot method showed in all three cases a partial deletion of 21q22.3 and partial monosomy. The translocation breakpoints were different in each patient, and in two cases the rearranged chromosome was found to be asymmetrical. The molecular definition of the monosomy 21 in each patient was, respectively, COL6A1-S100B, CD18-S100B, and PFKL-S100B. DNA polymorphism analysis indicated in all cases a homozygosity of the duplicated material. The duplicated region was maternal in two patients and paternal in one patient. These data suggest that the reverse tandem chromosomes did not result from a telomeric fusion between chromosomes 21 but from a translocation between sister chromatids. The phenotypes of these patients did not differ significantly from that of individuals with full trisomy 21, except in one case with large ears with an unfolded helix. The fact that monosomy of distal 21q22.3 in these patients resulted in a phenotype very similar to Down syndrome suggests that the duplication of the genes located in this part of chromosome 21 is not necessary for the pathogenesis of the Down syndrome features observed in these patients, including most of the facial and hand features, muscular hypotonia, cardiopathy of the Fallot tetralogy type, and part of the mental retardation.

New chromosome 21 DNA markers isolated by pulsed field gel electrophoresis from an ETS2-containing Down syndrome chromosomal region.

To generate new chromosome 21 markers in a region that is critical for the pathogenesis of Down syndrome (D21S55-MX1), we used pulsed field gel electrophoresis (PFGE) to isolate a 600-kb NruI DNA fragment from the WA17 hybrid cell line, which has retained chromosome 21 as the only human material. This fragment, which contains the oncogene ETS2, was used to construct a partial genomic library. Among the 14 unique sequences that were isolated, 3 were polymorphic markers and contained sequences that are conserved in mammals. Five of these markers mapped on the ETS2-containing NruI fragment and allowed us to define an 800-kb high-resolution PFGE map.

Gene-dosage mapping of 30 DNA markers on chromosome 21.

Using a slot-blot method for the dosage of single-copy sequences, the copy numbers of 30 chromosome 21 markers were assessed in the blood DNA of 11 patients with partial trisomy or monosomy 21 and in the DNA of a patient-derived human-hamster hybrid cell line carrying a microduplication of chromosome 21. The physical order of these markers on chromosome 21 was thereby determined.

Slot blot method for the quantification of DNA sequences and mapping of chromosome rearrangements: application to chromosome 21.

As an alternative to the methods of gene dosage based on either RFLP studies or Southern blots using specific and reference probes, we designed a "slot blot" method for the evaluation of the copy number of unique chromosome 21 sequences. Varying amounts of denatured DNA from a normal control, a trisomy 21 patient, and the subject to be analyzed were loaded on the same membrane. Successive hybridizations with reference probes and chromosome 21 probes were then carried out. Intensities of the signals on autoradiograms were quantified by densitometric scanning. Graphic and statistical analysis of the linear regressions between reference and chromosome 21 probe signals were performed, and the conclusion that the DNA from the studied subject had two or three copies for a given chromosome 21 sequence was assessed by statistical comparison of the slopes. As a test for the validation of this method, 10 coded blood DNAs from five normal controls and from five trisomy 21 patients were analyzed, by using two reference (COL1A1 and COL1A2) and two chromosome 21 (D21S11 and D21S17) probes. Among the 10 DNAs analyzed, it was possible to diagnose, with 100% accuracy, normal controls and trisomic 21 individuals. Application of this methodology to the mapping of partial chromosome 21 rearrangements is presented.

Down syndrome critical region around D21S55 on proximal 21q22.3.

We have analysed the DNA of 2 patients with many manifestations of Down syndrome and partial duplication of distinct regions of chromosome 21, respectively, q11.205----q22.300 and q22.300----qter (Rahmani et al.: Proceedings of the National Academy of Sciences of the United States of America 86:5958-5962, 1989). Assessment of the copy number of five chromosome 21 sequences (SOD1, D21S17, D21S55, ETS2, and D21S15) has shown that D21S55 was duplicated in both cases. The size of the common duplicated region can be estimated between 400 and 3,000 Kb, after the results of pulsed-field gel analysis and from the knowledge of regional mapping of the probes D21S17, D21S55, and ETS2. This region, located on the proximal part of 21q22.3, is postulated to contain genes the overexpression of which plays a major role in the pathogenesis of Down syndrome.

Critical role of the D21S55 region on chromosome 21 in the pathogenesis of Down syndrome.

The duplication of a specific region of chromosome 21 could be responsible for the main features of Down syndrome. To define and localize this region, we analyzed at the molecular level the DNA of two patients with partial duplication of chromosome 21. These patients belong to two groups of Down syndrome patients characterized by different partial trisomies 21: (i) duplication of the long arm, proximal to 21q22.2, and (ii) duplication of the end of the chromosome, distal to 21q22.2 We assessed the copy number of five chromosome 21 sequences (SOD1, D21S17, D21S55, ETS2, and D21S15) and found that D21S55 was duplicated in both cases. By means of pulsed-field gel analysis and with the knowledge of regional mapping of the probes D21S17, D21S55 and ETS2, we estimated the size of the common duplicated region to be between 400 and 3000 kilobases. This region, localized on the proximal part of 21q22.3, is suspected to contain genes the overexpression of which is crucial in the pathogenesis of Down syndrome.