Pharmacological Manipulation of Early Zebrafish Skeletal Development Shows an Important Role for Smad9 in Control of Skeletal Progenitor Populations

Biomolecules

2021 Feb 13

McDonald, GLK;Wang, M;Hammond, CL;Bergen, DJM;
PMID: 33668680 | DOI: 10.3390/biom11020277

Osteoporosis and other conditions associated with low bone density or quality are highly prevalent, are increasing as the population ages and with increased glucocorticoid use to treat conditions with elevated inflammation. There is an unmet need for therapeutics which can target skeletal precursors to induce osteoblast differentiation and osteogenesis. Genes associated with high bone mass represent interesting targets for manipulation, as they could offer ways to increase bone density. A damaging mutation in SMAD9 has recently been associated with high bone mass. Here we show that Smad9 labels groups of osteochondral precursor cells, which are not labelled by the other Regulatory Smads: Smad1 or Smad5. We show that Smad9+ cells are proliferative, and that the Smad9+ pocket expands following osteoblast ablation which induced osteoblast regeneration. We further show that treatment with retinoic acid, prednisolone, and dorsomorphin all alter Smad9 expression, consistent with the effects of these drugs on the skeletal system. Taken together these results demonstrate that Smad9+ cells represent an undifferentiated osteochondral precursor population, which can be manipulated by commonly used skeletal drugs. We conclude that Smad9 represents a target for future osteoanabolic therapies.

GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation

Nature neuroscience

2021 Dec 01

Pellegrino, G;Martin, M;Allet, C;Lhomme, T;Geller, S;Franssen, D;Mansuy, V;Manfredi-Lozano, M;Coutteau-Robles, A;Delli, V;Rasika, S;Mazur, D;Loyens, A;Tena-Sempere, M;Siepmann, J;Pralong, FP;Ciofi, P;Corfas, G;Parent, AS;Ojeda, SR;Sharif, A;Prevot, V;
PMID: 34795451 | DOI: 10.1038/s41593-021-00960-z

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation.

Pluripotent stem cell-derived endometrial stromal fibroblasts in a cyclic, hormone-responsive, coculture model of human decidua

Cell reports

2021 May 18

Cheung, VC;Peng, CY;Marinić, M;Sakabe, NJ;Aneas, I;Lynch, VJ;Ober, C;Nobrega, MA;Kessler, JA;
PMID: 34010658 | DOI: 10.1016/j.celrep.2021.109138

Various human diseases and pregnancy-related disorders reflect endometrial dysfunction. However, rodent models do not share fundamental biological processes with the human endometrium, such as spontaneous decidualization, and no existing human cell cultures recapitulate the cyclic interactions between endometrial stromal and epithelial compartments necessary for decidualization and implantation. Here we report a protocol differentiating human pluripotent stem cells into endometrial stromal fibroblasts (PSC-ESFs) that are highly pure and able to decidualize. Coculture of PSC-ESFs with placenta-derived endometrial epithelial cells generated organoids used to examine stromal-epithelial interactions. Cocultures exhibited specific endometrial markers in the appropriate compartments, organization with cell polarity, and hormone responsiveness of both cell types. Furthermore, cocultures recapitulate a central feature of the human decidua by cyclically responding to hormone withdrawal followed by hormone retreatment. This advance enables mechanistic studies of the cyclic responses that characterize the human endometrium.

CHIP inhibits odontoblast differentiation through promoting DLX3 polyubiquitylation and degradation

Development (Cambridge, England)

2023 May 15

Zheng, H;Zhang, X;Fu, J;Xue, Y;Chen, Z;Yang, G;Chen, Y;Chen, D;Yuan, G;
PMID: 37213079 | DOI: 10.1242/dev.200848

Dentin is the major hard tissue of teeth formed by differentiated odontoblasts. How odontoblast differentiation is regulated remains enigmatic. Here, we report that the E3 ubiquitin ligase CHIP is highly expressed in undifferentiated dental mesenchymal cells and downregulated after differentiation of odontoblasts. Ectopic expression of CHIP inhibits odontoblastic differentiation of mouse dental papilla cells, whereas knockdown of endogenous CHIP has opposite effects. Chip (Stub1) knockout mice display increased formation of dentin and enhanced expression of odontoblast differentiation markers. Mechanistically, CHIP interacts with and induces K63 polyubiquitylation of the transcription factor DLX3, leading to its proteasomal degradation. Knockdown of DLX3 reverses the enhanced odontoblastic differentiation caused by knockdown of CHIP. These results suggest that CHIP inhibits odontoblast differentiation by targeting its tooth-specific substrate DLX3. Furthermore, our results indicate that CHIP competes with another E3 ubiquitin ligase, MDM2, that promotes odontoblast differentiation by monoubiquitylating DLX3. Our findings suggest that the two E3 ubiquitin ligases CHIP and MDM2 reciprocally regulate DLX3 activity by catalyzing distinct types of ubiquitylation, and reveal an important mechanism by which differentiation of odontoblasts is delicately regulated by divergent post-translational modifications.

The developmental basis of fingerprint pattern formation and variation

Cell

2023 Mar 02

Glover, JD;Sudderick, ZR;Shih, BB;Batho-Samblas, C;Charlton, L;Krause, AL;Anderson, C;Riddell, J;Balic, A;Li, J;Klika, V;Woolley, TE;Gaffney, EA;Corsinotti, A;Anderson, RA;Johnston, LJ;Brown, SJ;Wang, S;Chen, Y;Crichton, ML;Headon, DJ;
PMID: 36764291 | DOI: 10.1016/j.cell.2023.01.015

Fingerprints are complex and individually unique patterns in the skin. Established prenatally, the molecular and cellular mechanisms that guide fingerprint ridge formation and their intricate arrangements are unknown. Here we show that fingerprint ridges are epithelial structures that undergo a truncated hair follicle developmental program and fail to recruit a mesenchymal condensate. Their spatial pattern is established by a Turing reaction-diffusion system, based on signaling between EDAR, WNT, and antagonistic BMP pathways. These signals resolve epithelial growth into bands of focalized proliferation under a precociously differentiated suprabasal layer. Ridge formation occurs as a set of waves spreading from variable initiation sites defined by the local signaling environments and anatomical intricacies of the digit, with the propagation and meeting of these waves determining the type of pattern that forms. Relying on a dynamic patterning system triggered at spatially distinct sites generates the characteristic types and unending variation of human fingerprint patterns.

Single-cell transcriptomic profiling redefines the origin and specification of early adrenogonadal progenitors

Cell reports

2023 Feb 28

Neirijnck, Y;Sararols, P;Kühne, F;Mayère, C;Weerasinghe Arachchige, LC;Regard, V;Nef, S;Schedl, A;
PMID: 36862551 | DOI: 10.1016/j.celrep.2023.112191

Adrenal cortex and gonads represent the two major steroidogenic organs in mammals. Both tissues are considered to share a common developmental origin characterized by the expression of Nr5a1/Sf1. The precise origin of adrenogonadal progenitors and the processes driving differentiation toward the adrenal or gonadal fate remain, however, elusive. Here, we provide a comprehensive single-cell transcriptomic atlas of early mouse adrenogonadal development including 52 cell types belonging to twelve major cell lineages. Trajectory reconstruction reveals that adrenogonadal cells emerge from the lateral plate rather than the intermediate mesoderm. Surprisingly, we find that gonadal and adrenal fates have already diverged prior to Nr5a1 expression. Finally, lineage separation into gonadal and adrenal fates involves canonical versus non-canonical Wnt signaling and differential expression of Hox patterning genes. Thus, our study provides important insights into the molecular programs of adrenal and gonadal fate choice and will be a valuable resource for further research into adrenogonadal ontogenesis.

Single-Nucleus RNA Sequencing of Developing and Mature Superior Colliculus Identifies Neuronal Diversity and Candidate Mediators of Circuit Assembly

bioRxiv : the preprint server for biology

2023 Feb 07

Ayupe, AC;Choi, JS;Beckedorff, F;Mccartan, R;Levay, K;Park, KK;
PMID: 36778361 | DOI: 10.1101/2023.02.01.526254

The superior colliculus (SC) is a sensorimotor structure in the midbrain that integrates input from multiple sensory modalities to initiate motor commands. It undergoes well-characterized steps of circuit assembly during development, rendering the mouse SC a popular model to study establishment and refinement of neural connectivity. Here we performed single nucleus RNA-sequencing analysis of the mouse SC isolated at various developmental time points. Our study provides a transcriptomic landscape of the cell types that comprise the SC across murine development with particular emphasis on neuronal heterogeneity. We used these data to identify Pax7 as a marker for an anatomically homogeneous population of GABAergic neurons. Lastly, we report a repertoire of genes differentially expressed across the different postnatal ages, many of which are known to regulate axon guidance and synapse formation. Our data provide a valuable resource for interrogating the mechanisms of circuit development, and identifying markers for manipulating specific SC neuronal populations and circuits.

Human theca arises from ovarian stroma and is comprised of three discrete subtypes

Communications biology

2023 Jan 04

Guahmich, NL;Man, L;Wang, J;Arazi, L;Kallinos, E;Topper-Kroog, A;Grullon, G;Zhang, K;Stewart, J;Schatz-Siemers, N;Jones, SH;Bodine, R;Zaninovic, N;Schattman, G;Rosenwaks, Z;James, D;
PMID: 36599970 | DOI: 10.1038/s42003-022-04384-8

Theca cells serve multiple essential functions during the growth and maturation of ovarian follicles, providing structural, metabolic, and steroidogenic support. While the function of theca during folliculogenesis is well established, their cellular origins and the differentiation hierarchy that generates distinct theca sub-types, remain unknown. Here, we performed single cell multi-omics analysis of primary cell populations purified from human antral stage follicles (1-3 mm) to define the differentiation trajectory of theca/stroma cells. We then corroborated the temporal emergence and growth kinetics of defined theca/stroma subpopulations using human ovarian tissue samples and xenografts of cryopreserved/thawed ovarian cortex, respectively. We identified three lineage specific derivatives termed structural, androgenic, and perifollicular theca cells, as well as their putative lineage-negative progenitor. These findings provide a framework for understanding the differentiation process that occurs in each primordial follicle and identifies specific cellular/molecular phenotypes that may be relevant to either diagnosis or treatment of ovarian pathologies.

Single-cell transcriptomic and spatial landscapes of the developing human pancreas

Cell metabolism

2022 Dec 06

Olaniru, OE;Kadolsky, U;Kannambath, S;Vaikkinen, H;Fung, K;Dhami, P;Persaud, SJ;
PMID: 36513063 | DOI: 10.1016/j.cmet.2022.11.009

Current differentiation protocols have not been successful in reproducibly generating fully functional human beta cells in vitro, partly due to incomplete understanding of human pancreas development. Here, we present detailed transcriptomic analysis of the various cell types of the developing human pancreas, including their spatial gene patterns. We integrated single-cell RNA sequencing with spatial transcriptomics at multiple developmental time points and revealed distinct temporal-spatial gene cascades. Cell trajectory inference identified endocrine progenitor populations and branch-specific genes as the progenitors differentiate toward alpha or beta cells. Spatial differentiation trajectories indicated that Schwann cells are spatially co-located with endocrine progenitors, and cell-cell connectivity analysis predicted that they may interact via L1CAM-EPHB2 signaling. Our integrated approach enabled us to identify heterogeneity and multiple lineage dynamics within the mesenchyme, showing that it contributed to the exocrine acinar cell state. Finally, we have generated an interactive web resource for investigating human pancreas development for the research community.

Nodal signaling establishes a competency window for stochastic cell fate switching

Developmental cell

2022 Dec 05

Economou, AD;Guglielmi, L;East, P;Hill, CS;
PMID: 36473458 | DOI: 10.1016/j.devcel.2022.11.008

Specification of the germ layers by Nodal signaling has long been regarded as an archetype of how graded morphogens induce different cell fates. However, this deterministic model cannot explain why only a subset of cells at the early zebrafish embryo margin adopt the endodermal fate, whereas their immediate neighbours, experiencing a similar signaling environment, become mesoderm. Combining pharmacology, quantitative imaging and single cell transcriptomics, we demonstrate that sustained Nodal signaling establishes a bipotential progenitor state from which cells can switch to an endodermal fate or differentiate into mesoderm. Switching is a random event, the likelihood of which is modulated by Fgf signaling. This inherently imprecise mechanism nevertheless leads to robust endoderm formation because of buffering at later stages. Thus, in contrast to previous deterministic models of morphogen action, Nodal signaling establishes a temporal window when cells are competent to undergo a stochastic cell fate switch, rather than determining fate itself.

Nail-associated mesenchymal cells contribute to and are essential for dorsal digit tip regeneration

Cell reports

2022 Dec 20

Mahmud, N;Eisner, C;Purushothaman, S;Storer, MA;Kaplan, DR;Miller, FD;
PMID: 36543145 | DOI: 10.1016/j.celrep.2022.111853

Here, we ask why the nail base is essential for mammalian digit tip regeneration, focusing on the inductive nail mesenchyme. We identify a transcriptional signature for these cells that includes Lmx1b and show that the Lmx1b-expressing nail mesenchyme is essential for blastema formation. We use a combination of Lmx1bCreERT2-based lineage-tracing and single-cell transcriptional analyses to show that the nail mesenchyme contributes cells for two pro-regenerative mechanisms. One group of cells maintains their identity and regenerates the new nail mesenchyme. A second group contributes specifically to the dorsal blastema, loses their nail mesenchyme phenotype, acquires a blastema transcriptional state that is highly similar to blastema cells of other origins, and ultimately contributes to regeneration of the dorsal but not ventral dermis and bone. Thus, the regenerative necessity for an intact nail base is explained, at least in part, by a requirement for the inductive nail mesenchyme.

Spatiotemporal single-cell regulatory atlas reveals neural crest lineage diversification and cellular function during tooth morphogenesis

Nature communications

2022 Aug 16

Jing, J;Feng, J;Yuan, Y;Guo, T;Lei, J;Pei, F;Ho, TV;Chai, Y;
PMID: 35974052 | DOI: 10.1038/s41467-022-32490-y

Cranial neural crest cells are an evolutionary innovation of vertebrates for craniofacial development and function, yet the mechanisms that govern the cell fate decisions of postmigratory cranial neural crest cells remain largely unknown. Using the mouse molar as a model, we perform single-cell transcriptome profiling to interrogate the cell fate diversification of postmigratory cranial neural crest cells. We reveal the landscape of transcriptional heterogeneity and define the specific cellular domains during the progression of cranial neural crest cell-derived dental lineage diversification, and find that each domain makes a specific contribution to distinct molar mesenchymal tissues. Furthermore, IGF signaling-mediated cell-cell interaction between the cellular domains highlights the pivotal role of autonomous regulation of the dental mesenchyme. Importantly, we reveal cell-type-specific gene regulatory networks in the dental mesenchyme and show that Foxp4 is indispensable for the differentiation of periodontal ligament. Our single-cell atlas provides comprehensive mechanistic insight into the cell fate diversification process of the cranial neural crest cell-derived odontogenic populations.

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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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