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Series GSE235779 Query DataSets for GSE235779
Status Public on Mar 11, 2024
Title Compensatory growth and recovery of tissue cytoarchitecture after transient cartilage-specific cell death in foetal mouse limbs
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Summary A major question in developmental and regenerative biology is how organ size is controlled by progenitor cells. For example, while limb bones exhibit catch-up growth (recovery of a normal growth trajectory after transient developmental perturbation), it is unclear how this emerges from the behaviour of chondroprogenitors, the cells sustaining the cartilage anlagen that are progressively replaced by bone. Here we show that transient sparse cell death in the mouse foetal cartilage was repaired postnatally, via a two-step process. During injury, progression of chondroprogenitors towards more differentiated states was delayed, leading to altered cartilage cytoarchitecture and impaired bone growth. Then, once cell death was over, chondroprogenitor differentiation was accelerated and cartilage structure recovered, including partial rescue of bone growth. At the molecular level, ectopic activation of mTORC1 correlated with, and was necessary for, part of the recovery, revealing a specific candidate to be explored during normal growth and in future therapies.
Overall design The use of animals in this study was approved by the Monash Animal Research Platform animal ethics committee at Monash University. The Pitx2-ASE-Cre (aka Pitx2-Cre) mouse line was crossed with the Col2a1-rtTA mouse line and then inter-crossed to generate Pitx2-Cre/Cre; Col2a1-rtTA mice. Experimental and control animals were generated by crossing Pitx2-Cre/Cre; Col2a1-rtTA/+ females with TigreDragon-DTA/DTA males (i.e. homozygous for the conditional misexpression allele). The separation of control and experimental animals was based on the rtTA genotype. Pregnant females were administered doxycycline hyclate (Sigma, 1mg/ml in drinking water, with 0.5% sucrose for palatability) from E12.25 to E13.75. The day of vaginal plug detection was designated as E0.5, and E19.5 was referred to as P0. Mouse embryos were euthanized using hypothermia in cold PBS, while mouse pups were euthanized by decapitation. Control and experimental samples (2-3 biological replicates each) were collected for 4 different stages: E15.5, E17.5, P0 and P3. Left and right cartilage samples (each containing proximal and distal tibia and femur) were kept separated for each sample, and flash-frozen upon dissection. Total RNA was extracted using the Monarch Total RNA Miniprep Kit (New England Biolabs), following manufacturer instructions. From this, RNA-Seq of messenger RNAs (mRNAs) was performed using a custom in-house multiplex method, which allowed us to sequence up to 24 different samples in the same sequencing lane. Briefly, samples were given a unique i7 index (together with UMI) during individual polyA-priming and first-strand synthesis, which also added a template-switch sequence to the 5’ end. Samples were then pooled and amplified using P7 and an oligo which binds the template-switch sequence. Final library construction was completed by tagmentation and addition of P5 (with i5 index) by PCR. Sequencing was performed on an Illumina NSQ2k run with up to 101nt SR (cDNA). An 18-nt i7 read contains the 8-nt index and 10-nt UMI and, where required, an 8-nt i5 index read is also generated. We obtained a read depth of at least 15 million reads per library.
Contributor(s) H’ng CH, Amarasinghe SL, Zhang B, Chang H, Qu X, Powell DR, Rosello-Diez A
Citation(s) 38580631
Submission date Jun 26, 2023
Last update date Apr 24, 2024
Contact name Alberto Rosello-Diez
Organization name Monash University
Department ARMI
Street address Level 1. 15 Innovation walk
City Clayton
State/province VIC
ZIP/Postal code 3800
Country Australia
Platforms (1)
GPL30172 NextSeq 2000 (Mus musculus)
Samples (68)
GSM7509338 Left Cartilage 478 Exp Nov_2021 [478L_S1_L001]
GSM7509339 Right Cartilage 478 Exp Nov_2021 [478R_S2_L001]
GSM7509340 Left Cartilage 473 Ctrl Nov_2021 [473L_S3_L001]
BioProject PRJNA987611

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