Single-cell RNA-sequencing analysis of the developing mouse inner ear identifies molecular logic of auditory neuron diversification

Nature communications

2022 Jul 05

Petitpré, C;Faure, L;Uhl, P;Fontanet, P;Filova, I;Pavlinkova, G;Adameyko, I;Hadjab, S;Lallemend, F;
PMID: 35790771 | DOI: 10.1038/s41467-022-31580-1

Different types of spiral ganglion neurons (SGNs) are essential for auditory perception by transmitting complex auditory information from hair cells (HCs) to the brain. Here, we use deep, single cell transcriptomics to study the molecular mechanisms that govern their identity and organization in mice. We identify a core set of temporally patterned genes and gene regulatory networks that may contribute to the diversification of SGNs through sequential binary decisions and demonstrate a role for NEUROD1 in driving specification of a Ic-SGN phenotype. We also find that each trajectory of the decision tree is defined by initial co-expression of alternative subtype molecular controls followed by gradual shifts toward cell fate resolution. Finally, analysis of both developing SGN and HC types reveals cell-cell signaling potentially playing a role in the differentiation of SGNs. Our results indicate that SGN identities are drafted prior to birth and reveal molecular principles that shape their differentiation and will facilitate studies of their development, physiology, and dysfunction.

Single-cell atlas of craniogenesis uncovers SOXC-dependent, highly proliferative, and myofibroblast-like osteodermal progenitors

Cell reports

2022 Jul 12

Angelozzi, M;Pellegrino da Silva, R;Gonzalez, MV;Lefebvre, V;
PMID: 35830813 | DOI: 10.1016/j.celrep.2022.111045

The mammalian skull vault is essential to shape the head and protect the brain, but the cellular and molecular events underlying its development remain incompletely understood. Single-cell transcriptomic profiling from early to late mouse embryonic stages provides a detailed atlas of cranial lineages. It distinguishes various populations of progenitors and reveals a high expression of SOXC genes (encoding the SOX4, SOX11, and SOX12 transcription factors) early in development in actively proliferating and myofibroblast-like osteodermal progenitors. SOXC inactivation in these cells causes severe skull and skin underdevelopment due to the limited expansion of cell populations before and upon lineage commitment. SOXC genes enhance the expression of gene signatures conferring dynamic cellular and molecular properties, including actin cytoskeleton assembly, chromatin remodeling, and signaling pathway induction and responsiveness. These findings shed light onto craniogenic mechanisms and SOXC functions and suggest that similar mechanisms could decisively control many developmental, adult, pathological, and regenerative processes.

Mechanical forces couple bone matrix mineralization with inhibition of angiogenesis to limit adolescent bone growth

Nature communications

2022 Jun 01

Dzamukova, M;Brunner, TM;Miotla-Zarebska, J;Heinrich, F;Brylka, L;Mashreghi, MF;Kusumbe, A;Kühn, R;Schinke, T;Vincent, TL;Löhning, M;
PMID: 35650194 | DOI: 10.1038/s41467-022-30618-8

Bone growth requires a specialised, highly angiogenic blood vessel subtype, so-called type H vessels, which pave the way for osteoblasts surrounding these vessels. At the end of adolescence, type H vessels differentiate into quiescent type L endothelium lacking the capacity to promote bone growth. Until now, the signals that switch off type H vessel identity and thus limit adolescent bone growth have remained ill defined. Here we show that mechanical forces, associated with increased body weight at the end of adolescence, trigger the mechanoreceptor PIEZO1 and thereby mediate enhanced production of the kinase FAM20C in osteoblasts. FAM20C, the major kinase of the secreted phosphoproteome, phosphorylates dentin matrix protein 1, previously identified as a key factor in bone mineralization. Thereupon, dentin matrix protein 1 is secreted from osteoblasts in a burst-like manner. Extracellular dentin matrix protein 1 inhibits vascular endothelial growth factor signalling by preventing phosphorylation of vascular endothelial growth factor receptor 2. Hence, secreted dentin matrix protein 1 transforms type H vessels into type L to limit bone growth activity and enhance bone mineralization. The discovered mechanism may suggest new options for the treatment of diseases characterised by aberrant activity of bone and vessels such as osteoarthritis, osteoporosis and osteosarcoma.

Mouse Dspp frameshift model of human dentinogenesis imperfecta

Scientific reports

2021 Oct 19

Liang, T;Hu, Y;Zhang, H;Xu, Q;Smith, CE;Zhang, C;Kim, JW;Wang, SK;Saunders, TL;Lu, Y;Hu, JC;Simmer, JP;
PMID: 34667213 | DOI: 10.1038/s41598-021-00219-4

Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5' mutations affecting an N-terminal targeting sequence and 3' mutations that shift translation into the - 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp-1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp-1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp-1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5' and 3' Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

Characterisation of lamina I anterolateral system neurons that express Cre in a Phox2a-Cre mouse line

Scientific reports

2021 Sep 09

Alsulaiman, WAA;Quillet, R;Bell, AM;Dickie, AC;Polgár, E;Boyle, KA;Watanabe, M;Roome, RB;Kania, A;Todd, AJ;Gutierrez-Mecinas, M;
PMID: 34504158 | DOI: 10.1038/s41598-021-97105-w

A recently developed Phox2a::Cre mouse line has been shown to capture anterolateral system (ALS) projection neurons. Here, we used this line to test whether Phox2a-positive cells represent a distinct subpopulation among lamina I ALS neurons. We show that virtually all lamina I Phox2a cells can be retrogradely labelled from injections targeted on the lateral parabrachial area (LPb), and that most of those in the cervical cord also belong to the spinothalamic tract. Phox2a cells accounted for ~ 50-60% of the lamina I cells retrogradely labelled from LPb or thalamus. Phox2a was preferentially associated with smaller ALS neurons, and with those showing relatively weak neurokinin 1 receptor expression. The Phox2a cells were also less likely to project to the ipsilateral LPb. Although most Phox2a cells phosphorylated extracellular signal-regulated kinases following noxious heat stimulation, ~ 20% did not, and these were significantly smaller than the activated cells. This suggests that those ALS neurons that respond selectively to skin cooling, which have small cell bodies, may be included among the Phox2a population. Previous studies have defined neurochemical populations among the ALS cells, based on expression of Tac1 or Gpr83. However, we found that the proportions of Phox2a cells that expressed these genes were similar to the proportions reported for all lamina I ALS neurons, suggesting that Phox2a is not differentially expressed among cells belonging to these populations. Finally, we used a mouse line that resulted in membrane labelling of the Phox2a cells and showed that they all possess dendritic spines, although at a relatively low density. However, the distribution of the postsynaptic protein Homer revealed that dendritic spines accounted for a minority of the excitatory synapses on these cells. Our results confirm that Phox2a-positive cells in lamina I are ALS neurons, but show that the Phox2a::Cre line preferentially captures specific types of ALS cells.

Genetically Distinct Parallel Pathways in the Entopeduncular Nucleus for Limbic and Sensorimotor Output of the Basal Ganglia

Neuron

2017 Apr 05

Wallace ML, Saunders A, Huang KW, Philson AC, Goldman M, Macosko EZ, McCarroll SA, Sabatini BL.
PMID: 28384468 | DOI: 10.1016/j.neuron.2017.03.017

The basal ganglia (BG) integrate inputs from diverse sensorimotor, limbic, and associative regions to guide action-selection and goal-directed behaviors. The entopeduncular nucleus (EP) is a major BG output nucleus and has been suggested to channel signals from distinct BG nuclei to target regions involved in diverse functions. Here we use single-cell transcriptional and molecular analyses to demonstrate that the EP contains at least three classes of projection neurons-glutamate/GABA co-releasing somatostatin neurons, glutamatergic parvalbumin neurons, and GABAergic parvalbumin neurons. These classes comprise functionally and anatomically distinct output pathways that differentially affect EP target regions, such as the lateral habenula (LHb) and thalamus. Furthermore, LHb- and thalamic-projecting EP neurons are differentially innervated by subclasses of striatal and pallidal neurons. Therefore, we identify previously unknown subdivisions within the EP and reveal the existence of cascading, molecularly distinct projections through striatum and globus pallidus to EP targets within epithalamus and thalamus.

Hair Cell Mechanotransduction Regulates Spontaneous Activity and Spiral Ganglion Subtype Specification in the Auditory System

Cell

2018 Aug 02

Sun S, Babola T, Pregernig G, So KS, Nguyen M, Su SSM, Palermo AT, Bergles DE, Burns JC, Müller U.
PMID: - | DOI: 10.1016/j.cell.2018.07.008

Type I spiral ganglion neurons (SGNs) transmit sound information from cochlear hair cells to the CNS. Using transcriptome analysis of thousands of single neurons, we demonstrate that murine type I SGNs consist of subclasses that are defined by the expression of subsets of transcription factors, cell adhesion molecules, ion channels, and neurotransmitter receptors. Subtype specification is initiated prior to the onset of hearing during the time period when auditory circuits mature. Gene mutations linked to deafness that disrupt hair cell mechanotransduction or glutamatergic signaling perturb the firing behavior of SGNs prior to hearing onset and disrupt SGN subtype specification. We thus conclude that an intact hair cell mechanotransduction machinery is critical during the pre-hearing period to regulate the firing behavior of SGNs and their segregation into subtypes. Because deafness is frequently caused by defects in hair cells, our findings have significant ramifications for the etiology of hearing loss and its treatment.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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