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1.
Genetics ; 2024 May 13.
Article in English | MEDLINE | ID: mdl-38739761

ABSTRACT

In C. elegans, expanded families of divergent Hedgehog-related and Patched-related proteins promote numerous processes ranging from epithelial and sense organ development to pathogen responses to cuticle shedding during the molt cycle. The molecular functions of these proteins have been mysterious since nematodes lack a canonical Hedgehog signaling pathway. Here we show that Hedgehog-related proteins are components of the cuticle and pre-cuticle apical extracellular matrices that coat, shape, and protect external epithelia. Of four Hedgehog-related proteins imaged, two (GRL-2 and GRL-18) stably associated with the cuticles of specific tubes and two (GRL-7 and WRT-10) labelled pre-cuticle substructures such as furrows or alae. We found that wrt-10 mutations disrupt cuticle alae ridges, consistent with a structural role in matrix organization. We hypothesize that most nematode Hedgehog-related proteins are apical extracellular matrix components, a model that could explain many of the reported functions for this family. These results highlight ancient connections between Hedgehog proteins and the extracellular matrix and suggest that any signaling roles of C. elegans Hedgehog-related proteins will be intimately related to their matrix association.

2.
bioRxiv ; 2023 Dec 26.
Article in English | MEDLINE | ID: mdl-38234847

ABSTRACT

In C. elegans, divergent Hedgehog-related (Hh-r) and Patched-related (PTR) proteins promote numerous processes ranging from epithelial and sense organ development to pathogen responses to cuticle shedding during the molt cycle. Here we show that Hh-r proteins are actual components of the cuticle and pre-cuticle apical extracellular matrices (aECMs) that coat, shape, and protect external epithelia. Different Hh-r proteins stably associate with the aECMs of specific tissues and with specific substructures such as furrows and alae. Hh-r mutations can disrupt matrix structure. These results provide a unifying model for the function of nematode Hh-r proteins and highlight ancient connections between Hh proteins and the extracellular matrix.

3.
G3 (Bethesda) ; 12(4)2022 04 04.
Article in English | MEDLINE | ID: mdl-35143646

ABSTRACT

Necrosis was once described as a chaotic unregulated response to cellular insult. We now know that necrosis is controlled by multiple pathways in response to many different cellular conditions. In our pnc-1 NAD+ salvage deficient Caenorhabditis elegans model excess nicotinamide induces excitotoxic death in uterine-vulval uv1 cells and OLQ mechanosensory neurons. We sought to characterize necrosis in our pnc-1 model in the context of well-characterized necrosis, apoptosis, and autophagy pathways in C. elegans. We confirmed that calpain and aspartic proteases were required for uv1 necrosis, but changes in intracellular calcium levels and autophagy were not, suggesting that uv1 necrosis occurs by a pathway that diverges from mec-4d-induced touch cell necrosis downstream of effector aspartic proteases. OLQ necrosis does not require changes in intracellular calcium, the function of calpain or aspartic proteases, or autophagy. Instead, OLQ survival requires the function of calreticulin and calnexin, pro-apoptotic ced-4 (Apaf1), and genes involved in both autophagy and axon guidance. In addition, the partially OLQ-dependent gentle nose touch response decreased significantly in pnc-1 animals on poor quality food, further suggesting that uv1 and OLQ necrosis differ downstream of their common trigger. Together these results show that, although phenotypically very similar, uv1, OLQ, and touch cell necrosis are very different at the molecular level.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , NAD/metabolism , Necrosis/metabolism , Neurons/metabolism
4.
Genetics ; 219(3)2021 11 05.
Article in English | MEDLINE | ID: mdl-34740248

ABSTRACT

The Patched-related superfamily of transmembrane proteins can transport lipids or other hydrophobic molecules across cell membranes. While the Hedgehog receptor Patched has been intensively studied, much less is known about the biological roles of other Patched-related family members. Caenorhabditis elegans has a large number of Patched-related proteins, despite lacking a canonical Hedgehog pathway. Here, we show that PTR-4 promotes the assembly of the precuticle apical extracellular matrix, a transient and molecularly distinct matrix that precedes and patterns the later collagenous cuticle or exoskeleton. ptr-4 mutants share many phenotypes with precuticle mutants, including defects in eggshell dissolution, tube shaping, alae (cuticle ridge) structure, molting, and cuticle barrier function. PTR-4 localizes to the apical side of a subset of outward-facing epithelia, in a cyclical manner that peaks when precuticle matrix is present. Finally, PTR-4 is required to limit the accumulation of the lipocalin LPR-3 and to properly localize the Zona Pellucida domain protein LET-653 within the precuticle. We propose that PTR-4 transports lipids or other hydrophobic components that help to organize the precuticle and that the cuticle and molting defects seen in ptr-4 mutants result at least in part from earlier disorganization of the precuticle.


Subject(s)
Extracellular Matrix , Membrane Proteins , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , CRISPR-Cas Systems/genetics , Extracellular Matrix/metabolism , Extracellular Matrix/ultrastructure , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Electron, Transmission , Molting/genetics , Mucins/metabolism , Mutation , Protein Domains/genetics
5.
EMBO J ; 40(9): e106163, 2021 05 03.
Article in English | MEDLINE | ID: mdl-33792936

ABSTRACT

Transcytosis is a form of specialized transport through which an extracellular cargo is endocytosed, shuttled across the cytoplasm in membrane-bound vesicles, and secreted at a different plasma membrane surface. This important process allows membrane-impermeable macromolecules to pass through a cell and become accessible to adjacent cells and tissue compartments. Transcytosis also promotes redistribution of plasma membrane proteins and lipids to different regions of the cell surface. Here we review transcytosis and highlight in vivo studies showing how developing epithelial cells use it to change shape, to migrate, and to relocalize signaling molecules.


Subject(s)
Epithelium/physiology , Membrane Proteins/metabolism , Animals , Cytoplasm/metabolism , Humans , Lipid Metabolism , Morphogenesis , Transcytosis
6.
Biol Reprod ; 103(5): 1132-1143, 2020 10 29.
Article in English | MEDLINE | ID: mdl-32716476

ABSTRACT

Sirolimus, also known as rapamycin, and its closely related rapamycin analog (rapalog) Everolimus inhibit "mammalian target of rapamycin complex 1" (mTORC1), whose activity is required for spermatogenesis. Everolimus is Food and Drug Administration approved for treating human patients to slow growth of aggressive cancers and preventing organ transplant rejection. Here, we test the hypothesis that rapalog inhibition of mTORC1 activity has a negative, but reversible, impact upon spermatogenesis. Juvenile (P20) or adult (P>60) mice received daily injections of sirolimus or Everolimus for 30 days, and tissues were examined at completion of treatment or following a recovery period. Rapalog treatments reduced body and testis weights, testis weight/body weight ratios, cauda epididymal sperm counts, and seminal vesicle weights in animals of both ages. Following rapalog treatment, numbers of differentiating spermatogonia were reduced, with concomitant increases in the ratio of undifferentiated spermatogonia to total number of remaining germ cells. To determine if even low doses of Everolimus can inhibit spermatogenesis, an additional group of adult mice received a dose of Everolimus ∼6-fold lower than a human clinical dose used to treat cancer. In these animals, only testis weights, testis weight/body weight ratios, and tubule diameters were reduced. Return to control values following a recovery period was variable for each of the measured parameters and was duration and dose dependent. Together, these data indicate rapalogs exerted a dose-dependent restriction on overall growth of juvenile and adult mice and negative impact upon spermatogenesis that were largely reversed; following treatment cessation, males from all treatment groups were able to sire offspring.


Subject(s)
Cell Differentiation/drug effects , Everolimus/pharmacology , Fertility/drug effects , Spermatogenesis/drug effects , Spermatogonia/drug effects , Animals , Male , Mice
7.
Development ; 146(12)2019 05 13.
Article in English | MEDLINE | ID: mdl-31023878

ABSTRACT

In the mammalian testis, sustained spermatogenesis relies on spermatogonial stem cells (SSCs); their progeny either remain as stem cells (self-renewal) or proliferate and differentiate to enter meiosis in response to retinoic acid (RA). Here, we sought to uncover elusive mechanisms regulating a key switch fundamental to spermatogonial fate: the capacity of spermatogonia to respond to RA. Using the developing mouse testis as a model, we found that spermatogonia and precursor prospermatogonia exhibit a heterogeneous capacity to respond to RA with at least two underlying causes. First, progenitor spermatogonia are prevented from responding to RA by catabolic activity of cytochrome P450 family 26 enzymes. Second, a smaller subset of undifferentiated spermatogonia enriched for SSCs exhibit catabolism-independent RA insensitivity. Moreover, for the first time, we observed that precursor prospermatogonia are heterogeneous and comprise subpopulations that exhibit the same differential RA responsiveness found in neonatal spermatogonia. We propose a novel model by which mammalian prospermatogonial and spermatogonial fates are regulated by their intrinsic capacity to respond (or not) to the differentiation signal provided by RA before, and concurrent with, the initiation of spermatogenesis.


Subject(s)
Gene Expression Regulation , Spermatogenesis , Spermatogonia/cytology , Stem Cells/cytology , Testis/growth & development , Tretinoin/metabolism , Animals , Cell Differentiation , Cell Lineage , Cytochrome P450 Family 26/metabolism , Genomics , Green Fluorescent Proteins/metabolism , Male , Meiosis , Mice , Sertoli Cells/cytology , Signal Transduction , Testis/embryology
8.
Development ; 145(15)2018 08 13.
Article in English | MEDLINE | ID: mdl-29980567

ABSTRACT

Throughout the male reproductive lifespan, spermatogonial stem cells (SSCs) produce committed progenitors that proliferate and then remain physically connected in growing clones via short cylindrical intercellular bridges (ICBs). These ICBs, which enlarge in meiotic spermatocytes, have been demonstrated to provide a conduit for postmeiotic haploid spermatids to share sex chromosome-derived gene products. In addition to ICBs, spermatogonia exhibit multiple thin cytoplasmic projections. Here, we have explored the nature of these projections in mice and find that they are dynamic, span considerable distances from their cell body (≥25 µm), either terminate or physically connect multiple adjacent spermatogonia, and allow for sharing of macromolecules. Our results extend the current model that subsets of spermatogonia exist as isolated cells or clones, and support a model in which spermatogonia of similar developmental fates are functionally connected through a shared dynamic cytoplasm mediated by thin cytoplasmic projections.


Subject(s)
Cytoplasm/metabolism , Mammals/metabolism , Spermatogonia/metabolism , Animals , Cell Differentiation , Cytoplasm/ultrastructure , Diffusion , Green Fluorescent Proteins/metabolism , Intracellular Space/metabolism , Macromolecular Substances/metabolism , Male , Meiosis , Mice, Transgenic , Papio , Rats , Spermatocytes/cytology , Spermatocytes/metabolism , Spermatogonia/cytology , Spermatogonia/ultrastructure
9.
Biol Reprod ; 96(4): 816-828, 2017 Apr 01.
Article in English | MEDLINE | ID: mdl-28379293

ABSTRACT

Spermatogonial stem cells must balance self-renewal with production of transit-amplifying progenitors that differentiate in response to retinoic acid (RA) before entering meiosis. This self-renewal vs. differentiation fate decision is critical for maintaining tissue homeostasis, as imbalances cause defects that can lead to human testicular cancer or infertility. Little is currently known about the program of differentiation initiated by RA, and the pathways and proteins involved are poorly defined. We recently found that RA stimulation of the Phosphatidylinositol 3-kinase (PI3K)/AKT/Mammalian target of rapamycin (mTOR) kinase signaling pathway is required for differentiation, and that short-term inhibition of mTOR complex 1 (mTORC1) by rapamycin blocked spermatogonial differentiation in vivo and prevented RA-induced translational activation. Since this phenotype resulted from global inhibition of mTORC1, we created conditional germ cell knockout mice to investigate the germ cell-autonomous role of MTOR in spermatogonial differentiation. MTOR germ cell KO mice were viable and healthy, but testes from neonatal (postnatal day (P)8), juvenile (P18), and adult (P > 60) KO mice were smaller than littermate controls, and no sperm were produced in adult testes. Histological and immunostaining analyses revealed that spermatogonial differentiation was blocked, and no spermatocytes were formed at any of the ages examined. Although spermatogonial proliferation was reduced in the neonatal testis, it was blocked altogether in the juvenile and adult testis. Importantly, a small population of self-renewing undifferentiated spermatogonia remained in adult testes. Taken together, these results reveal that MTOR is dispensable for the maintenance of undifferentiated spermatogonia, but is cell autonomously required for their proliferation and differentiation.


Subject(s)
Cell Differentiation/physiology , Cell Proliferation/physiology , Spermatogonia/physiology , TOR Serine-Threonine Kinases/metabolism , Animals , Animals, Newborn , Male , Mice , Mice, Knockout , Spermatogenesis , TOR Serine-Threonine Kinases/genetics , Testis/physiology
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