CX3CR1 expression is associated with the commitment of CSF-1R+ myeloid precursors to the macrophage/dendritic cell (DC) lineage. However, the relationship of the CSF-1R+ CX3CR1+ macrophage/DC precursor (MDP) with other DC precursors and the role of CX3CR1 in macrophage and DC development remain unclear. We show that MDPs give rise to conventional DCs (cDCs), plasmacytoid DCs (PDCs), and monocytes, including Gr1+ inflammatory monocytes that differentiate into TipDCs during infection. CX3CR1 deficiency selectively impairs the recruitment of blood Gr1+ monocytes in the spleen after transfer and during acute Listeria monocytogenes infection but does not affect the development of monocytes, cDCs, and PDCs.

The expression of the FMR-1 gene, which is implicated in fragile-X syndrome was investigated in human fetuses by in situ hybridization. In 8 and 9 week-old fetuses, FMR-1 mRNAs are expressed in proliferating and migrating cells of the nervous system, in the retina, and in several non-nervous tissues. In the brain of 25 week-old fetuses, FMR-1 mRNAs are produced in all nearly differenciated structures, with the highest level in cholinergic neurons of the nucleus basalis magnocellularis and in pyramidal neurons of hippocampus. The early transcription of FMR-1 gene and the distribution of FMR-1 mRNAs in human fetuses suggest that alterations of FMR-1 gene expression may contribute to the pathogenesis of fragile-X syndrome and especially the mental retardation.

Studies of genetically engineered flies and mice have revealed the role that orthologs of the human LIM homeobox LHX4 have in the control of motor-neuron-identity assignment and in pituitary development. Remarkably, these mouse strains, which bear a targeted modification of Lhx4 in the heterozygous state, are asymptomatic, whereas homozygous animals die shortly after birth. Nevertheless, we have isolated the human LHX4 gene, as well as the corresponding cDNA sequence, to test whether it could be involved in developmental defects of the human pituitary region. LHX4, which encodes a protein 99% identical to its murine counterpart, consists of six coding exons and spans >45 kb of the q25 region of chromosome 1. We report a family with an LHX4 germline splice-site mutation that results in a disease phenotype characterized by short stature and by pituitary and hindbrain (i.e., cerebellar) defects in combination with abnormalities of the sella turcica of the central skull base. This intronic mutation, which segregates in a dominant and fully penetrant manner over three generations, abolishes normal LHX4 splicing and activates two exonic cryptic splice sites, thereby predicting two different proteins deleted in their homeodomain sequence. These findings, which elucidate the molecular basis of a complex Mendelian disorder, reveal the fundamental pleiotropic role played by a single factor that tightly coordinates brain development and skull shaping during head morphogenesis.

No abstract

Cystinosis is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The causative gene, CTNS, encodes cystinosin, a seven-transmembrane-domain protein, which we recently showed to be a lysosomal cystine transporter. The most severe and frequent form of cystinosis, the infantile form, appears around 6 to 12 months, with a proximal tubulopathy (de Toni-Debré-Fanconi syndrome) and ocular damage. End-stage renal failure is reached by 10 years of age. Accumulation of cystine in all tissues eventually leads to multisystemic disease. Treatment with cysteamine, which reduces the concentration of intracellular cystine, delays disease progression but has undesirable side effects. We report the first Ctns knockout mouse model generated using a promoter trap approach. We replaced the last four Ctns exons by an internal ribosome entry site-␤gal-neo cassette and showed that the truncated protein was mislocalized and nonfunctional. Ctns ؊/؊ mice accumulated cystine in all organs tested, and cystine crystals, pathognomonic of cystinosis, were observed. Ctns ؊/؊ mice developed ocular changes similar to those observed in affected individuals, bone defects and behavioral anomalies. Interestingly, Ctns ؊/؊ mice did not develop signs of a proximal tubulopathy, or renal failure. A preliminary therapeutic trial using an oral administration of cysteamine was carried out and demonstrated the efficiency of this treatment for cystine clearance in Ctns ؊/؊ mice. This animal model will prove an invaluable and unique tool for testing emerging therapeutics for cystinosis.

Ocular mal-development results in heterogeneous and frequently visually disabling phenotypes that include coloboma and microphthalmia. Due to the contribution of bone morphogenetic proteins to such processes, the function of the paralogue Growth Differentiation Factor 3 was investigated. Multiple mis-sense variants were identified in patients with ocular and/or skeletal (Klippel-Feil) anomalies including one individual with heterozygous alterations in GDF3 and GDF6. These variants were characterized, individually and in combination, through integrated biochemical and zebrafish model organism analyses, demonstrating appreciable effects with western blot analyses, luciferase based reporter assays and antisense morpholino inhibition. Notably, inhibition of the zebrafish co-orthologue of GDF3 accurately recapitulates patient phenotypes. By demonstrating the pleiotropic effects of GDF3 mutation, these results extend the contribution of perturbed BMP signaling to human disease and potentially implicate multi-allelic inheritance of BMP variants in developmental disorders.

Apoptosis plays a major role in the development of the central nervous system. Previous studies of apoptosis induction during retinal development are difficult to interpret, however, because they explored different mouse strains, different developmental periods, and used different assays. Here, we first established a comprehensive sequential pattern of cell death during the whole development of the C57BL/6J mouse retina, from E10.5 to postnatal day (P) 21 by using the terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP)-biotinylated nick end labeling (TUNEL) assay. We confirmed the existence of three previously described apoptotic peaks and identified another, later peak at P15, in both the outer nuclear layer, in which the photoreceptors differentiate, and the ganglion cell layer. Comparison of wild-type C57BL/6 mice, gld mice, defective in the death ligand fasL, and bax-/-mice, defective in the pro-apoptotic BAX protein, revealed a minor role for FAS ligand but a crucial role for BAX in both apoptosis and normal retinal development. The lack of BAX resulted in thicker than normal inner neuroblastic and ganglion cell layers in adults, with larger numbers of cells and an impaired electroretinogram response related to a decreased number of responsive cells. Our findings indicate that cell death during normal retinal development is important for the modeling of a functional vision organ and showed that the pro-apoptotic BAX protein plays a crucial role in this process. Developmental Dynamics 228:231-238, 2003.

Proteins of the bone morphogenetic protein (BMP) family are known to have a role in ocular and skeletal development; however, because of their widespread expression and functional redundancy, less progress has been made identifying the roles of individual BMPs in human disease. We identified seven heterozygous mutations in growth differentiation factor 6 (GDF6), a member of the BMP family, in patients with both ocular and vertebral anomalies, characterized their effects with a SOX9-reporter assay and western analysis, and demonstrated comparable phenotypes in model organisms with reduced Gdf6 function. We observed a spectrum of ocular and skeletal anomalies in morphant zebrafish, the latter encompassing defective tail formation and altered expression of somite markers noggin1 and noggin2. Gdf6(+/-) mice exhibited variable ocular phenotypes compatible with phenotypes observed in patients and zebrafish. Key differences evident between patients and animal models included pleiotropic effects, variable expressivity and incomplete penetrance. These data establish the important role of this determinant in ocular and vertebral development, demonstrate the complex genetic inheritance of these phenotypes, and further understanding of BMP function and its contributions to human disease.