Development of Outer Hair Cells in Ames Waltzer Mice: Mutation in Protocadherin 15 Affects Development of Cuticular Plate and Associated Structures

The Ames waltzer (av) mouse mutant harbors a mutation in the protocadherin 15 gene (Pcdh15) and is a model for deafness in Usher syndrome 1F and nonsyndromic deafness DFNB23. Mutation in Pcdh15 affects stereocilia morphogenesis and polarity. Disruptions of apical cellular components in outer hair cells have also been described in av mutants. Organization of stereocilia and cell polarization may be dependent on proper orientation of structural components residing in the apical portion of the cell during development. We used electron and immunofluorescent microscopy to examine structural maturation of outer hair cells in av3J mice with emphasis on the fonticulus, basal body/centriole complex, actin mesh, and the microtubule network during initiation of bundle organization, between embryonic day (E) 16.5 and postnatal day 5 (P5). We found major ultrastructural rearrangements near the hair cell surface in av3J mice. Earliest changes were in kinocilia, basal body, and stereocilia positioning and microtubule arrangement once the kinocilia had lateralized to the side of the cell (between E16.5 and postnatal day [P] 0, before cuticular plate formation and stereocilia elongation). By P0, the developing fonticulus in av mice appeared enlarged, with a normal vesicle density. Stereocilia bundle disorganization increased after P0, with disruptions of the actin mesh within the cuticular plate. These observations support the hypothesis that mutations in Pcdh15 in av3J mice adversely affect coordinated maturation of apical cell components, resulting in disturbed stereocilia bundle polarity in av mice. Anat Rec, 2007. © 2008 Wiley‐Liss, Inc.     

Glomerular Hypertrophy in Offspring of Subtotally Nephrectomized Ewes

We have shown that fetuses whose mothers underwent subtotal nephrectomy (STNx) before pregnancy had high urine flow rates and sodium excretions, but lower hematocrits, plasma chloride, and plasma renin levels compared with controls. To see if these functional differences in utero persist after birth and are the result of altered renal development, we studied 8 lambs born to STNx mothers (STNxL) and 10 controls (ConL) in the second week of life. These lambs were of similar body weights, nose–rump lengths and abdominal girths. Their kidney weights were not different (ConL 36.1 ± 1.9 vs. STNxL 39.8 ± 3.3 g), nor were kidney dimensions or glomerular number (ConL 423,520 ± 22,194 vs. STNxL 429,530 ± 27,471 glomeruli). However, STNxL had 30% larger glomerular volumes (both mean and total, P < 0.01) and there was a positive relationship between total glomerular volume and urinary protein excretion (P < 0.05) in STNxL. Despite this change in glomerular morphology, glomerular filtration rate, tubular function, urine flow, and sodium excretion rates were not different between STNxL and ConL, nor were plasma electrolytes, osmolality, and plasma renin levels. Thus while many of the functional differences seen in late gestation were not present at 1–2 weeks after birth, the alteration in glomerular size and its relationship to protein excretion suggests that exposure to this altered intrauterine environment may predispose offspring of mothers with renal dysfunction to renal disease in adult life. Anat Rec, 291:318–324, 2008. © 2008 Wiley‐Liss, Inc.     

Identification of mutations in TCOF1: use of molecular analysis in the pre- and postnatal diagnosis of Treacher Collins syndrome

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of facial development, which results from mutations in TCOF1. TCS comprises conductive hearing loss, hypoplasia of the mandible and maxilla, downward sloping palpebral fissures and cleft palate. Although, there is usually a reasonable degree of bilateral symmetry, a high degree of both inter- and intrafamilial variability is characteristic of TCS. The wide variation in the clinical presentation of different patients, together with the fact that more than 60% of cases arise de novo, can complicate the diagnosis of mild cases and genetic counselling. In the current study, we describe how molecular techniques have been used to facilitate pre- and postnatal disease diagnoses in 13 TCS families.     

Sperm analysis by FISH in a case of t(17; 22) (q11; q12) balanced translocation: case report

Individual sperm from men with balanced translocations have different chromosomal contents. Thus, an estimation of the overall sperm chromosomal imbalance of such patients could help to give the couple an adapted genetic counselling. We report here the study of a balanced translocation carrier, t(17;22) (q11;q12) whose reproductive history reported four miscarriages. Moreover, he had an abnormal semen analysis with oligoteratozoospermia. The meiotic segregation pattern was examined in 700 sperm, using fluorescence in-situ hybridization (FISH). Nineteen percent of the sperm had balanced translocations or were normal. All other sperm were unbalanced (81%) and their distribution was observed as follows: the frequencies of adjacent 1, adjacent 2 and 3:1 segregations were 12.9, 5.8 and 46.8% respectively. Among the segregations scored, 13.7% were related to second meiotic division abnormalities. Less than 2% of the total sperm scored were not explained. The 3:1 segregation was present at a very high rate, which is very unusual. In cases of balanced translocations, we believe that no general features can be drawn. Thus, the FISH technique may be very helpful for genetic counselling, which remains an important step and must be done with care.     

Tissue fixation effects on immunohistochemical staining of caspase-3 in brain tissue.

Fixation methods for tissue often vary amongst clinical and research laboratories. To evaluate the effects of fixation method on studies of brain tissue, we examined immunohistochemical outcomes amongst 2 fixatives, 4 caspase-3 antibodies, and 2 species (human infants and piglets). Fixatives were 10% neutral buffered formalin (NBF) or 10% NBF and glacial acetic acid (FAA). Antibodies for caspase-3 were commercially obtained and included 2 for active caspase-3, and 2 for procaspase-3 (CASP3 and CPP32). Immunohistochemical staining of caspase-3 varied with fixation method, with the greatest effect of fixation method observed for the active caspase-3 antibodies and this effect was present in both species. In NBF-fixed tissue, active caspase-3 immunoreactivity was only visible microscopically, and was specific to neuronal cell bodies. In FAA-fixed tissue, active caspase-3 immunoreactivity was visible macroscopically, and predominantly present in fiber tracts and fasciculi compared with neuronal bodies. Fixation and species differences were also identified for the procaspase-3 antibodies, CASP3 and CPP32, where FAA-fixed pig tissue showed abundant staining of blood vessels that were not observed in the NBF-fixed pig tissue or in the human tissue. This study characterizes differences in immunohistochemical outcomes using commercially available antibodies for caspase-3, according to tissue fixation method and species.     

The architecture of variant surface glycoprotein gene expression sites in Trypanosoma brucei

Trypanosoma brucei evades the immune system by switching between Variant Surface Glycoprotein (VSG) genes. The active VSG gene is transcribed in one of approximately 20 telomeric expression sites (ESs). It has been postulated that ES polymorphism plays a role in host adaptation. To gain more insight into ES architecture, we have determined the complete sequence of Bacterial Artificial Chromosomes (BACs) containing DNA from three ESs and their flanking regions. There was variation in the order and number of ES-associated genes (ESAGs). ESAGs 6 and 7, encoding transferrin receptor subunits, are the only ESAGs with functional copies in every ES that has been sequenced until now. A BAC clone containing the VO2 ES sequences comprised approximately half of a 330 kb 'intermediate' chromosome. The extensive similarity between this intermediate chromosome and the left telomere of T. brucei 927 chromosome I, suggests that this previously uncharacterised intermediate size class of chromosomes could have arisen from breakage of megabase chromosomes. Unexpected conservation of sequences, including pseudogenes, indicates that the multiple ESs could have arisen through a relatively recent amplification of a single ES.