A new developmental mechanism for the separation of the mammalian middle ear ossicles from the jaw

Multiple mammalian lineages independently evolved a definitive mammalian middle ear (DMME) through breakdown of Meckel's cartilage (MC). However, the cellular and molecular drivers of this evolutionary transition remain unknown for most mammal groups. Here, we identify such drivers in the living marsupial opossum Monodelphis domestica, whose MC transformation during development anatomically mirrors the evolutionary transformation observed in fossils. Specifically, we link increases in cellular apoptosis and TGF-BR2 signalling to MC breakdown in opossums. We demonstrate that a simple change in TGF-β signalling is sufficient to inhibit MC breakdown during opossum development, indicating that changes in TGF-β signalling might be key during mammalian evolution. Furthermore, the apoptosis that we observe during opossum MC breakdown does not seemingly occur in mouse, consistent with homoplastic DMME evolution in the marsupial and placental lineages.

3 an anti-digoxigenin antibody is conjugated to a fluorescent reporter. Protocol: fix slides in 1% PFA in 1xPBS (938ul of 16% PFA + 15mL PBS), 2x 5 min rinses in 1xPBS, post fix in precooled 2:1 mix of EtOH:Acetic acid at -20 degrees C, 2x 5 min rinses in 1xPBS, gently dry excess liquid, apply 75ul of Equilibration Buffer per slide for 10 sec, remove excess liquid, apply 55ul of Working Strength TdT Enzyme per slide (77ul Reaction Buffer + 33ul TdT Enzyme), coverslip and place in humidified chamber for 1 hour at 37 degrees C, 10 min in Working Strength Stop/Wash Buffer (1mL stop/wash + 34mL ddH 2 O), 3x 1 min washes in 1xPBS, apply 65ul Anti-Digoxigenin Conjugate per slide (62ul anti-Digo + 68ul Blocking Solution), coverslip and place in humidified chamber for 30 min at room temperature (keep in dark), 4x 2 min washes in 1xPBS, 2-3 drops of Vectashield Mounting Medium with DAPI (4',6-diamidino-2phenylindole) per slide, coverslip and dry for several hours in the dark at room temperature, store at -20 degrees C. DAPI fluoresces when bound to DNA and is used as a nuclear counterstain.
All IF slides were imaged using a Leica Microsystems DMI4000 B automated inverted fluorescence microscope with a Hamamatsu ORCA-ER high-resolution digital camera, and using Image-Pro Plus 7.0 software. The Hamamatsu is a black and white camera so all images where taken three times, using blue, green, and red fluorescence. Afterwards, the separate color channels were merged into one, using FIJI/ImageJ software (NIH) (36).

RNA-sequencing.
Additional samples were cryosectioned for the specific purpose of collecting tissue for RNA-Sequencing. N=3 specimens were collected for each stage (16, 18, and 20 day) and immediately snap-frozen, without fixation, then cryosectioned on Arcturus PEN membrane glass slides (Applied Biosystems), five slides per specimen. An Arcturus Veritas Microdissection Instrument was used for laser capture microdissection (LCM) of Meckel's cartilage and malleus (with minimal surrounding perichondrium) focusing on their connection area. An UV cutting laser was used to excise the tissue of interest. Afterward, an IR capture laser was fired through Arcturus Capsure HS LCM Caps, melting an attached transfer film, which would then bond with 4 the tissue. The cells attached to the caps were removed with an Arcturus PicoPure RNA Isolation Kit. An ExtracSure Extraction Device was placed on each cap and 10ul of Extraction Buffer (XB) was added to each. A 0.5mL microcentrifuge tube was placed on top, then covered with an incubation block preheated to 42 degrees C, and left to incubate for 30 min. Afterward, they were centrifuged for 2 min at 800x g, the extracted RNA was pooled with the other slides for each specimen, and the cell extract was stored at -80 degrees C. We also took slide scrapes for each set (used for baseline comparison) by placing RNA extraction buffer directly on the slides.
The remainder of the RNA isolation was completed following the PicoPure RNA Isolation Kit guidelines. RNA integrity was checked by an Agilent 2100 Bioanalyzer, revealing RIN values of 7.8 -8.9. As the quantity of RNA in cartilage is commonly very low, we used the Clontech SMARTer Ultra Low Input RNA Kit for amplification. We began the Clontech amplification with 9ul of pooled RNA from each specimen. The first portion of this protocol, first-strand cDNA synthesis, was completed inside of a PCR clean hood workstation. The final portion, cDNA purification, was completed using Agencourt Ampure XP beads with a magnetic block for separation. All steps were conducted following standard Clontech Kit guidelines, with the final supernatant containing purified cDNA from the original tissue samples stored at -20 degrees C.
Resultant cDNA samples were run through the Bioanalyzer again, as well as a Qubit Fluorometer, to determine accurate concentrations. Next, we built libraries for sequencing using a Nextera XT DNA Sample Preparation Kit, along with a Nextera XT Index Kit and TruSeq Dual Index Sequencing Primers. All samples started with 5ul of input DNA at a concentration of 0.2ng/ul. The standard protocol was followed for the Nextera XT DNA Library Preparation Guide. DNA was tagged and fragmented, amplified via PCR, and cleaned up with AMPure XP beads to purify the library. Afterwards, the library was again validated on the Agilent 2100 Bioanalyzer, followed by library normalization and pooling for HiSeq sequencing. High-throughput sequencing was conducted on an Illumina HiSeq 2500, at the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois (37).
Initial RNA-Seq analysis was conducted on the UIUC web-based Galaxy (38) platform (galaxy.illinois.edu), using the Tuxedo protocol. Sequence files were uploaded, along with an opossum reference genome (monDom5) (39) from ensemble.org. The basic sequence for analyses was as follows: Edit Sequences was used to trim ends, TopHat was used to align reads to the genome, Cufflinks was used to assemble the reads into transcripts, Cuffmerge was used to blend multiple samples from the same stage, and Cuffdiff was used to report genes and transcripts that are differentially expressed between samples (40). We also used the Database for Mounting Medium with DAPI per slide, glass coverslip, store at 4 degrees C until imaging.
Functional Assays -After identifying TGFbr2 as having a potential role in Meckel's cartilage separation, we knocked down TGFb signaling to investigate the impact on phenotype. A TGFb1,2,3 antibody (MAB1835) from R&D Systems was used to neutralize the biological activity of TGFb signaling (43). The antibody was reconstituted at 0.5mg/mL in sterile PBS, and 38ul intraperitoneal (IP) injections (based upon estimated 1.9g weight of pups at 20 days) were administered to neonatal opossum for six consecutive days beginning on postnatal day 16.
Control pups were injected with an equal dosage of 1xPBS. Mothers were anesthetized with isoflurane during injections. This precluded the necessity of physically removing the pups from the mothers, as the pups are continually attached to the mother's nipples at this developmental stage. We utilized a precision vaporizer isoflurane anesthesia machine with the oxygen flowmeter set to 1 litter per minute (LPM) and the vaporizer dial set to 2½. Mothers were placed in an induction chamber until they lost consciousness, at which point they were switched to a nose cone for the remaining duration. Injections took less than five minutes and the mothers fully recovered within a few minutes after returning to their cages. Pups were euthanized on postnatal day 22. MC morphology was visualized using micro-CT scanning (as described above) and 7 clearing and staining for at least 3 control and 3 treatment pups. For clearing and staining, following 4% PFA fixation and dehydration into ethanol, P22 samples were placed in acetone, then stained with 0.3% Alcian Blue 8GX (A5268 Sigma) and 0.1% Alizarin Red S (A5533) at 37° C for 5 days. Samples were then cleared in a 1% KOH solution (changed daily) for ~2 weeks. Once cleared, specimens were imaged using a Leica M205 C stereo microscope with a Leica DFC425 digital camera, utilizing the Leica Application Suite (LAS) version 3.8. At least 3 treatment and 3 control pups were also sectioned and IF used to test for apoptotic cells (as described above) and anti-p-Smad2 (44) (at a working concentration of 1:100; otherwise as described above for the Phosphohistone H3 antibody) (Cell Signaling Technology).    and the metatherian Kokopelia (56), it is not parsimonious to interpret them differently. Thus we consider the latter scenario to be less likely. All evo-devo hypotheses (Fig. 3 vs. Fig. S5) are contingent on the interpretation of the presence/absence of the MC and its osteological correlates on the dentary in stem eutherians and metatherians, and their immediate zatherian outgroups. But current evidence is more favorable for the scenario presented in Fig 3 than in Fig S5. Our preferred hypothesis is that parallel up regulations of TGF-β signaling occurred in multiple early mammal lineages, facilitating the independent acquisitions (Fig 3). However, if the scenario in  Table S1. Genes that are differentially expressed in the opossum Meckel's cartilage and its perichondrium by a log-fold change of ³2 at and before P20 (Sample1 = P16 or P18, and Sample2 = P20), as identified by RNA-seq.