Dataset Information

Morphometric analysis of testis cord formation in Sox9-EGFP mice.

ABSTRACT: Sox9-EGFP knockin mice were generated to label Sertoli cells and visualize testis cord formation during development. Confocal microscopy and morphometric analysis of developing cords were performed. Serial histological sections were used for three-dimensional cord reconstruction. Initially, gonad length decreased from embryonic day (E) 11.5 to E13.5, but increased thereafter, while gonad width doubled every 12 hours from E11.5 through E14.5. At E12.5, the average number of cords was 12.5, whereas this decreased to 10.4 at E13.5 and E14.5. Cord number at a given time point varied between gonads and influenced dimensions. The initial cords that formed were complex and branches were common. Time-lapse imaging revealed an intricate behavior of the Sertoli-germ cell mass and cellular exchange between connected neighboring cords. These results suggest that cord formation is a highly dynamic process that subsequently becomes refined to establish the final number of seminiferous tubule precursors.

SUBMITTER: Nel-Themaat L

PROVIDER: S-EPMC2838451 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Morphometric analysis of testis cord formation in Sox9-EGFP mice.

Nel-Themaat Liesl L Vadakkan Tegy J TJ Wang Ying Y Dickinson Mary E ME Akiyama Haruhiko H Behringer Richard R RR

Developmental dynamics : an official publication of the American Association of Anatomists 20090501 5

Sox9-EGFP knockin mice were generated to label Sertoli cells and visualize testis cord formation during development. Confocal microscopy and morphometric analysis of developing cords were performed. Serial histological sections were used for three-dimensional cord reconstruction. Initially, gonad length decreased from embryonic day (E) 11.5 to E13.5, but increased thereafter, while gonad width doubled every 12 hours from E11.5 through E14.5. At E12.5, the average number of cords was 12.5, wherea ...[more]

PMID: 19384968

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Project description:Purpose: SOX9Y440X is the most commonly reported SOX9 mutation identified in human patients with Campomelic dysplasia, a dominant semi-lethal skeletal disease characterized by congenital shortening and angulation of long bones (campomelia). To understand how the SOX9Y440X mutant cause the abnormal limb development and compare with previously heterozygous Sox9 null mutant, a mouse model with Sox9Y440X mutation has been generated. The goal of this study is to carry out transcriptome analyses (RNA-seq) of E13.5 limbs collected from Sox9+/IRES-EGFP and Sox9+/Y440X-IRES-EGFP mice. GFP+ and GFP- cells were collected from each genotype by FACS. Comparing transcriptome profiles of cells from these two genotypes aims to reveal how the SOX9Y440X mutant causes abnormal limb development. Method: Forelimbs (FL) and hindlimbs (HL) mRNA profiles of E13.5 Sox9+/IRES-EGFP (WT) and Sox9+/Y440X-IRES-EGFP (CD) mice were generated by sequencing. 4-6 pairs of FL and HL buds were collected separately for each genotype. Limb tissues were then digested into single cells, then EGFP positive and negative cells were collected by FACS for each sample. Total RNA from each sample was prepared using Ambion RNA isolation kit (Cat. AM1580). 100ng of total RNA was used from each sample for RNA-sequencing. cDNA libraries were prepared by TruSeq Stranded mRNA Sample Prep Kit (Illumina). Pair-End sequencing of 101bp was done using HiSeq 1500 Sequencer Model. Data processing: Reads was pre-processed and aligned to the mouse GRCm38 (mm10) reference genome and expression levels were quantified using the nfcore/rnaseq workflow. The count matrix from featureCounts was used in the downstream analyses. The Nextflow pipeline for downstream analysis is available at https://github.com/fuxialexander/sox9. With R library edgeR 3.36.0, read counts were normalized using the ‘TMM’ method. We identified differentially expressed genes between the WT and CD groups in GFP+ or GFP- samples with the ‘glmQLFTest’ function and a Benjamini & Hochberg adjusted P-value cutoff of 0.05. Conclusions: Our study represents the first detailed analysis of mouse foetal limbs transcriptomes between Sox9Y440X mutant and wild type. Our work contributes to gain insight into the molecular signalling pathways affected by SOX9Y440X in vivo.

Dataset Information

Morphometric analysis of testis cord formation in Sox9-EGFP mice.

Publications

Morphometric analysis of testis cord formation in Sox9-EGFP mice.

Similar Datasets

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