Expression studies have implicated FLRT2 in cranial neural crest cell migration and prechondrogenic cell condensation during craniofacial skeletogenesis. We aimed to determine whether FLRT2 was involved in mediating cell-matrix interactions in the ATDC5 chondroprogenitor cell line. Immunolocalization experiments of ATDC5 cells revealed that FLRT2 was present on the cell membrane as well as extracellularly, where it colocalized with Fibronectin (Fn). After cell extraction of the matrix, FLRT2 was identified in the ATDC5-derived extracellular matrix (ECM) and was further found to be associated with Fn-coated beads in cell cultures. Blockage of Fn fibril formation via a blocking peptide resulted in a concomitant decrease in extracellular FLRT2 accumulation. Over a 7-day period following the replenishment of the Fn blocking peptide to the cultures, there was a partial rebound in Fn fibril formation that was accompanied by a concomitant reappearance of FLRT2 co-expression. Co-immunoprecipitation confirmed that FLRT2 and Fn interacted, either directly or indirectly. Immunoprecipitation and Western blot analyses with antibodies recognizing epitopes located on the extra- and intracellular domains of FLRT2 further revealed the presence of different sized bands, suggesting that FLRT2 may exist in both membrane-bound and shed forms. Our data therefore provide evidence that FLRT2 and/or its cleavage products may be cooperating with Fn and other ECM proteins to regulate critical cellular events. Further studies will be necessary in delineate more precisely the roles of FLRT2 in mediating cell- and cell-matrix interactions during normal development.

Craniosynostosis is a relatively common birth defect characterized by the premature fusion of one or more cranial sutures. Examples of craniosynostosis syndromes include Crouzon (CS), Pfeiffer (PS) and Apert (AS) syndrome, with clinical characteristics such as midface hypoplasia, hypertelorism and in some cases, limb defects. Mutations in Fibroblast Growth Factor Receptor-2 comprise the majority of known mutations in syndromic forms of craniosynostosis. A number of clinical reports of FGFR-associated craniosynostosis patients and mouse mutants have been linked to gastrointestinal tract (GIT) disorders, leading to the hypothesis of a direct link between FGFR-associated craniosynostosis syndromes and GIT malformations. We conducted an investigation to determine GIT symptoms in a sample of FGFR-associated craniosynostosis syndrome patients and a mouse model of CS containing a mutation (W290R) in Fgfr2. We found that, compared to the general population, the incidence of intestinal/bowel malrotation (IM) was present at a higher level in our sample population of patients with FGFR-associated craniosynostosis syndromes. We also showed that the mouse model of CS had an increased incidence of cecal displacement, suggestive of IM. These findings suggest a direct relationship between FGFR-related craniosynostosis syndromes and GIT malformations. Our study may shed further light on the potential widespread impact FGFR mutations on different developmental systems. Based on reports of GIT malformations in children with craniosynostosis syndromes and substantiation with our animal model, GIT malformations should be considered in any child with an FGFR2-associated craniosynostosis syndrome.

To understand the roles of thymic stromal cells in T-lymphocyte development, we semiquantitatively analysed rat thymi recovering from irradiation (6 Gy), using a transmission electron microscope. The most striking findings were that the percentage of subcapsular epithelial cells significantly increased in the cortex on day 3 after irradiation compared with the control; the percentage of intermediate epithelial cells significantly increased in the cortex on days 3 and 5 after irradiation and in the medulla on days 5 and 7 compared with the control; the interdigitating cells disappeared from the medulla by day 7 after irradiation and reappeared on day 9. The present data thus reveal that during recovery after irradiation (6 Gy), marked changes occur in the relative proportions of different epithelial cell subtypes in the cortex and medulla of the rat thymus. In addition, the percentages of macrophages and interdigitating cells also changed during the recovery. These changes, which may be associated with the abrupt proliferation of thymocytes after irradiation, should shed light on the significance of stromal cells in the T cell development.

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To understand the roles of cortical thymic epithelial cells (CTECs) in T-lymphocyte development, we analyzed rat thymi recovering from irradiation (6Gy), at the ultrastructural level. The morphological alterations in the CTECs were most prominent during the third to fifth day of recovery, when proliferating thymocytes were observed in the vicinity of the CTECs. The most striking finding among the alterations in the CTECs after irradiation was a cytoplasmic vacuolization with an increased amount of granular and membranous content. The granular content was observed as loosely aggregated structures or finely dispersed granules and dense bodies. The membranous content appeared in various forms including vesicular, tubular, and irregular membranous structures and myelin figures. The above features are characteristic of the hyperfunctional state of CTECs with increased secretion activities, which suggests their important roles in the repopulation and maturation of the cortical thymocytes during recovery after irradiation.