Human nucleoli comprise multiple constrained territories, tethered to individual chromosomes.

It is unknown how ribosomal gene (rDNA) arrays from multiple chromosomal nucleolar organizers (NORs) partition within human nucleoli. Exploration of this paradigm for chromosomal organization is complicated by the shared DNA sequence composition of five NOR-bearing acrocentric chromosome p-arms. Here, we devise a methodology for genetic manipulation of individual NORs. Efficient "scarless" genome editing of rDNA repeats is achieved on "poised" human NORs held within monochromosomal cell hybrids. Subsequent transfer to human cells introduces "active" NORs yielding readily discernible functional customized ribosomes. We reveal that ribosome biogenesis occurs entirely within constrained territories, tethered to individual NORs inside a larger nucleolus.

The p-Arms of Human Acrocentric Chromosomes Play by a Different Set of Rules.

The p-arms of the five human acrocentric chromosomes bear nucleolar organizer regions (NORs) comprising ribosomal gene (rDNA) repeats that are organized in a homogeneous tandem array and transcribed in a telomere-to-centromere direction. Precursor ribosomal RNA transcripts are processed and assembled into ribosomal subunits, the nucleolus being the physical manifestation of this process. I review current understanding of nucleolar chromosome biology and describe current exploration into a role for the NOR chromosomal context. Full DNA sequences for acrocentric p-arms are now emerging, aided by the current revolution in long-read sequencing and genome assembly. Acrocentric p-arms vary from 10.1 to 16.7 Mb, accounting for ∼2.2% of the genome. Bordering rDNA arrays, distal junctions, and proximal junctions are shared among the p-arms, with distal junctions showing evidence of functionality. The remaining p-arm sequences comprise multiple satellite DNA classes and segmental duplications that facilitate recombination between heterologous chromosomes, which is likely also involved in Robertsonian translocations.

A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage.

DNA double-strand breaks (DSBs) are repaired by two main pathways: nonhomologous end-joining and homologous recombination (HR). Repair pathway choice is thought to be determined by cell cycle timing and chromatin context. Nucleoli, prominent nuclear subdomains and sites of ribosome biogenesis, form around nucleolar organizer regions (NORs) that contain rDNA arrays located on human acrocentric chromosome p-arms. Actively transcribed rDNA repeats are positioned within the interior of the nucleolus, whereas sequences proximal and distal to NORs are packaged as heterochromatin located at the nucleolar periphery. NORs provide an opportunity to investigate the DSB response at highly transcribed, repetitive, and essential loci. Targeted introduction of DSBs into rDNA, but not abutting sequences, results in ATM-dependent inhibition of their transcription by RNA polymerase I. This is coupled with movement of rDNA from the nucleolar interior to anchoring points at the periphery. Reorganization renders rDNA accessible to repair factors normally excluded from nucleoli. Importantly, DSBs within rDNA recruit the HR machinery throughout the cell cycle. Additionally, unscheduled DNA synthesis, consistent with HR at damaged NORs, can be observed in G1 cells. These results suggest that HR can be templated in cis and suggest a role for chromosomal context in the maintenance of NOR genomic stability.

Driving nucleolar assembly.

In this issue of Genes & Development, Grob and colleagues (pp. 220-230) identify the minimal molecular requirements to assemble a fully functional nucleolus in human cells and demonstrate the importance of the nucleolar transcription factor upstream binding factor (UBF) as a mitotic bookmark at the ribosomal DNA (rDNA).

Construction of synthetic nucleoli in human cells reveals how a major functional nuclear domain is formed and propagated through cell division.

Human cell nuclei are functionally organized into structurally stable yet dynamic bodies whose cell cycle inheritance is poorly understood. Here, we investigate the biogenesis and propagation of nucleoli, sites of ribosome biogenesis and key regulators of cellular growth. Nucleolar and cell cycles are intimately connected. Nucleoli disappear during mitosis, reforming around prominent uncharacterized chromosomal features, nucleolar organizer regions (NORs). By examining the effects of UBF depletion on both endogenous NORs and synthetic pseudo-NORs, we reveal its essential role in maintaining competency and establishing a bookmark on mitotic NORs. Furthermore, we demonstrate that neo-NORs, UBF-binding site arrays coupled with rDNA transcription units, direct the de novo biogenesis of functional compartmentalized neonucleoli irrespective of their site of chromosomal integration. For the first time, we establish the sequence requirements for nucleolar biogenesis and provide proof that this is a staged process where UBF-dependent mitotic bookmarking precedes function-dependent nucleolar assembly.

Nucleolus structural integrity during the first meiotic prophase in rat spermatocytes.

A typical nucleolus structure is shaped by three components. A meshwork of fine fibers forming the fibrillar center (FC) is surrounded by densely packed fibers forming the dense fibrillar component (DFC). Meanwhile, wrapping the FC and DFC is the granular component (GC). During the mitotic prophase, the nucleolus undergoes disassembling of its components. On the contrary, throughout the first meiotic prophase that occurs in the cells of the germ line, small nucleoli are assembled into one nucleolus by the end of the prophase. These nucleoli are transcriptionally active, suggesting that they are fully functional. Electron microscopy analysis has suggested that these nucleoli display their three main components but a typical organization has not been observed. Here, by immunolabeling and electron microscopy, we show that the nucleolus has its three main components. The GC is interlaced with the DFC and is not as well defined as previously thought during leptotene and zygotene stage.

Structural features of two major nucleolar organizer regions (NORs), Nor-B1 and Nor-B2, and chromosome-specific rRNA gene expression in wheat.

The reference genome sequence of wheat 'Chinese Spring' (CS) is now available (IWGSC RefSeq v1.0), but the core sequences defining the nucleolar organizer regions (NORs) have not been characterized. We estimated that the total copy number of the rDNA units in the wheat genome is 11 160, of which 30.5%, 60.9% and 8.6% are located on Nor-B1 (1B), Nor-B2 (6B) and other NORs, respectively. The total length of the NORs is estimated to be 100 Mb, corresponding to approximately 10% of the unassembled portion of the genome not represented in RefSeq v1.0. Four subtypes (S1-S4) of the rDNA units were identified based on differences within the 3' external transcribed spacer regions in Nor-B1 and Nor-B2, and quantitative PCR indicated locus-specific variation in rDNA subtype contents. Expression analyses of rDNA subtypes revealed that S1 was predominantly expressed and S2 weakly expressed, in contrast to the relative abundance of rDNA subtypes in the wheat genome. These results suggest a regulation mechanism of differential rDNA expression based on sequence differences. S3 expression increased in the ditelosomic lines Dt1BL and Dt6BL, suggesting that S3 is subjected to chromosome-mediated silencing. Structural differences were detected in the regions surrounding the NOR among homoeologous chromosomes of groups 1 and 6. The adjacent regions distal to the major NORs were expanded compared with their homoeologous counterparts, and the gene density of these expanded regions was relatively low. We provide evidence that these regions are likely to be important for autoregulation of the associated major NORs as well as silencing of minor NORs.

Upstream binding factor-dependent and pre-rRNA transcription-independent association of pre-rRNA processing factors with rRNA gene.

The nucleolus is the ribosome biogenesis center. The nucleolar structure is disrupted upon entry into mitosis and is formed in early G1 phase. To understand the molecular mechanisms of nucleolar assembly and disassembly, we have studied the mechanism of association between factors involved in pre-ribosome RNA (rRNA) processing and rRNA gene chromatin (r-chromatin). We found that the pre-rRNA transcription-processing linking factor Nopp140 and pre-rRNA processing factors such as DKC1 and fibrillarin (FBL) associate with r-chromatin during interphase, while Nopp140, DKC1, and FBL were released from r-chromatin in mitosis. The association of these factors with r-chromatin was found to be restored independent of pre-rRNA transcription in early G1 phase, but a mature nucleolar structure was not formed, suggesting that nucleolar assembly can be divided into at least two steps with respect to pre-rRNA transcription. Moreover, we found that the r-chromatin association of Nopp140, DKC1, and FBL was dependent on the transcription factor upstream binding factor (UBF). However, we demonstrated that UBF alone was not sufficient to recruit these pre-rRNA processing factors to r-chromatin. Thus, UBF is necessary but not sufficient for the associations between pre-rRNA processing factors and r-chromatin.

Argentophilic nucleolus organizer region as a proliferation marker in cervical intraepithelial neoplasia grade 1 of the uterine cervix.

AIM: p16INK4a and argentophilic nucleolus organizer region (AgNOR) can be used as markers for progression of cervical intraepithelial neoplasia grade 1 (CIN1) of the uterine cervix. Our objective was to study the predictive value of the AgNOR technique as a progression marker of CIN1 and its correlation with p16INK4A. MATERIAL AND METHODS: One uterine cervix biopsy from each of 75 patients with diagnosis of CIN1 was selected. All of these patients underwent a second biopsy, and these were also used for the study. RESULTS: The second biopsies showed: regression (20 patients), persistent CIN1 (38 patients), progression to CIN2 (10 patients) and progression to CIN3 (seven patients). p16INK4A showed reactivity in 67 of the 75 first CIN1 biopsies: 12 of the 20 cases that cleared the lesions and the 55 cases with persistent or progressive lesions were positive for p16INK4a (specificity: 40%; sensitivity: 100%; positive predictive value [PPV]: 82%; negative predictive value [NPV]: 100%). Samples with AgNOR areas less than 3.0 µ(2) returned in all cases, but patients whose lesions persisted or progressed to CIN2/CIN3, showed AgNOR areas greater than 3.0 µ(2) in 50/55 cases (specificity: 100%; sensitivity: 91%; PPV: 100%; NPV: 80%). CONCLUSIONS: p16INK4a is expressed in a high percentage of returning lesions. AgNOR might be a better marker of proliferation of CIN1 than p16INK4a (PPV = 100%), which means that a value greater than 3.0 µ(2) indicates the persistence or progression of the lesion. As its NPV is 80%, a value of AgNOR area less than 3.0 µ(2) in CIN1 leaves a margin of doubt about the future behavior of the lesion.

Computer-Assisted Morphometric Comparative Analysis of Argyrophilic Nucleolar Organizer Regions (AgNORs) in Leukoplakia With Dysplasia and Oral Squamous Cell Carcinoma.

Background Oral squamous cell carcinoma (OSCC) and oral leukoplakia (OL) with dysplasia are closely linked conditions in the oral cavity, with the latter often indicating precancerous changes, underscoring the urgency of early detection and intervention. Histopathological confirmation is crucial for accurate diagnosis. The nucleolar organizer region (NOR), specifically analyzed through silver-staining (argyrophilic NORs), provides insights into nuclear changes associated with the lesion. Computer-assisted morphometric analysis enhances precision and objectivity in evaluating AgNOR-related parameters. Aim To conduct a computer-assisted morphometric comparison of AgNORs using various NOR-related parameters in cases of OSCC and leukoplakia with dysplasia and to evaluate their diagnostic significance. Materials and methods A computer-assisted morphometric analysis was conducted using various NOR-related parameters, such as nuclear profile area, single AgNOR profile area per nucleus, total AgNOR profile area per nucleus, and number of AgNOR profiles per nucleus on a total sample of 90 specimens, which includes leukoplakia with dysplasia (30), OSCC (30), and a control group, including 30 samples of normal oral mucosa. A comparison was conducted on the morphometric values between the groups under investigation. Tukey's multiple comparison tests and ANOVA were used to analyze the data and determine the differences between the groups. Results The present investigation revealed a significant difference in all four AgNOR-related parameters between leukoplakia and OSCC in comparison to the control group (normal oral mucosa). Comparing OL (41.78 ± 0.46) and OSCC (62.78 ± 0.47) to the control group (35.93 ± 0.99), the mean value of nuclear profile area (A Nuc) was significantly greater. In comparison to the control group (3.40 ± 0.09), the mean value of a single AgNOR profile area per nucleus (A NOR) was found to be relatively lower in both research groups, OL (2.00 ± 0.02) and OSCC (1.39 ± 0.01). The total AgNOR profile area per nucleus (TA NOR) had a mean value of 10.61 ± 0.69 in OL and 12.05 ± 0.28 in OSCC, respectively, compared to 7.82 ± 0.38 in the control group. The study found that there was more number of profiles of AgNORs per nucleus (n NOR) in the study groups of OL (5.30 ± 0.29) and OSCC (8.69 ± 0.19) than in the control group (2.32 ± 0.11). Conclusion The parameters linked to the NOR are biologically informative and easy to check regularly in a pathology lab. Additionally, AgNORs give us important information that enables us to study the range of nuclear changes in malignant and potentially malignant lesions.

Uniqueness of the Gossypium mustelinum genome revealed by GISH and 45S rDNA FISH.

Gossypium mustelinum ((AD)4 ) is one of five disomic species in Gossypium. Three 45S ribosomal DNA (rDNA) loci were detected in (AD)4 with 45S rDNA as probe, and three pairs of brighter signals were detected with genomic DNA (gDNA) of Gossypium D genome species as probes. The size and the location of these brighter signals were the same as those detected with 45S rDNA as probe, and were named GISH-NOR. One of them was super-major, which accounted for the fact that about one-half of its chromosome at metaphase was located at chromosome 3, and other two were minor and located at chromosomes 5 and 9, respectively. All GISH-NORs were located in A sub-genome chromosomes, separate from the other four allopolyploid cotton species. GISH-NOR were detected with D genome species as probe, but not A. The greatly abnormal sizes and sites of (AD)4 NORs or GISH-NORs indicate a possible mechanism for 45S rDNA diversification following (AD)4 speciation. Comparisons of GISH intensities and GISH-NOR production with gDNA probes between A and D genomes show that the better relationship of (AD)4 is with A genome. The shortest two chromosomes of A sub-genome of G. mustelinum were shorter than the longest chromosome of D sub-genome chromosomes. Therefore, the longest 13 chromosomes of tetraploid cotton being classified as A sub-genome, while the shorter 13 chromosomes being classified as D sub-genome in traditional cytogenetic and karyotype analyses may not be entirely correct.

A Comparative Study of Morphometric Analysis of Nucleolar Organizer Regions in Oral Leukoplakia and Oral Squamous Cell Carcinoma and Significance of AgNOR as a Diagnostic Tool.

Background Silver stainable nucleolar organizer regions (AgNORs) have proven to exhibit utmost importance due to their higher occurrence in the nucleus especially in malignant cells than in normal. Thus, they assist in the examination of nucleolar structures and variations in nucleolar activity. Aim Quantitative and qualitative analysis in relation to the number and area of AgNOR in tissue sections of the normal oral mucosa (NOM), oral leukoplakia (OL), and oral squamous cell carcinoma (OSCC) was the main aim of the study. Materials & method A total of 50 cases comprising 20 OL with dysplasia, 20 OSCC cases, and 10 samples of normal oral mucosa were taken. Silver nitrate (Sol A) & gelatin (Sol B) solutions were freshly prepared for staining the lesional slides. Results The mean value of nuclear profile area (A Nuc) was comparatively higher in oral leukoplakia i.e. 41.97 and in oral squamous cell carcinoma i.e. 62.36 in comparison to the control group where it was 36.19. The mean value of a single AgNOR profile area per nucleus (A NOR) was found to be comparatively lower in both study groups i.e. oral leukoplakia (2.76) and oral squamous cell carcinoma (1.61) in comparison to the control group (3.45) . The mean value of total AgNOR profile area per nucleus (TA NOR) and the number of profiles of AgNORs per nucleus (n NOR) were found higher in both study groups (oral leukoplakia and oral squamous cell carcinoma) as compared to normal oral mucosa of the control group. However, the findings of all four parameters of morphometric analysis were found to be significantly associated with disorder of oral mucosa i.e. cases of oral leukoplakia and oral squamous cell carcinoma (P value <0.01). Conclusion It can thus be suggested that the mean AgNOR count displayed a higher value in OSCC. Hence, the number of AgNORs in nuclei increases as epithelial cells undergo malignant transformation which is designated that mean AgNOR count may contribute to establishing the prognosis of a lesion.

On the Origin of Neo-Sex Chromosomes in the Neotropical Dragonflies Rhionaeschna bonariensis and R. planaltica (Aeshnidae, Odonata).

Odonata have holokinetic chromosomes. About 95% of species have an XX/X0 sex chromosome system, with heterogametic males. There are species with neo-XX/neo-XY sex chromosomes resulting from an X chromosome/autosome fusion. The genus Rhionaeschna includes 42 species found in the Americas. We analyzed the distribution of the nucleolar organizer region (NOR) using FISH with rDNA probes in Rhionaeschna bonariensis (n = 12 + neo-XY), R. planaltica (n = 7 + neo-XY), and Aeshna cyanea (n = 13 + X0). In R. bonariensis and A. cyanea, the NOR is located on a large pair of autosomes, which have a secondary constriction in the latter species. In R. planaltica, the NOR is located on the ancestral part of the neo-X chromosome. Meiotic analysis and FISH results in R. planaltica led to the conclusion that the neo-XY system arose by insertion of the ancestral X chromosome into an autosome. Genomic in situ hybridization, performed for the first time in Odonata, highlighted the entire neo-Y chromosome in meiosis of R. bonariensis, suggesting that it consists mainly of repetitive DNA. This feature and the terminal chiasma localization suggest an ancient origin of the neo-XY system. Our study provides new information on the origin and evolution of neo-sex chromosomes in Odonata, including new types of chromosomal rearrangements, NOR transposition, and heterochromatin accumulation.

Establishment and Maintenance of Open Ribosomal RNA Gene Chromatin States in Eukaryotes.

In growing eukaryotic cells, nuclear ribosomal (r)RNA synthesis by RNA polymerase (RNAP) I accounts for the vast majority of cellular transcription. This high output is achieved by the presence of multiple copies of rRNA genes in eukaryotic genomes transcribed at a high rate. In contrast to most of the other transcribed genomic loci, actively transcribed rRNA genes are largely devoid of nucleosomes adapting a characteristic "open" chromatin state, whereas a significant fraction of rRNA genes resides in a transcriptionally inactive nucleosomal "closed" chromatin state. Here, we review our current knowledge about the nature of open rRNA gene chromatin and discuss how this state may be established.

Insights into the Karyotype Evolution of Charinidae, the Early-Diverging Clade of Whip Spiders (Arachnida: Amblypygi).

Whip spiders (Amblypygi) represent an ancient order of tetrapulmonate arachnids with a low diversity. Their cytogenetic data are confined to only a few reports. Here, we analyzed the family Charinidae, a lineage almost at the base of the amblypygids, providing an insight into the ancestral traits and basic trajectories of amblypygid karyotype evolution. We performed Giemsa staining, selected banding techniques, and detected 18S ribosomal DNA and telomeric repeats by fluorescence in situ hybridization in four Charinus and five Sarax species. Both genera exhibit a wide range of diploid chromosome numbers (2n = 42-76 and 22-74 for Charinus and Sarax, respectively). The 2n reduction was accompanied by an increase of proportion of biarmed elements. We further revealed a single NOR site (probably an ancestral condition for charinids), the presence of a (TTAGG)n telomeric motif localized mostly at the chromosome ends, and an absence of heteromorphic sex chromosomes. Our data collectively suggest a high pace of karyotype repatterning in amblypygids, with probably a high ancestral 2n and its subsequent gradual reduction by fusions, and the action of pericentric inversions, similarly to what has been proposed for neoamblypygids. The possible contribution of fissions to charinid karyotype repatterning, however, cannot be fully ruled out.

Quantitative assessment of tumor-associated tissue eosinophilia and nuclear organizing region activity to validate the significance of the pattern of invasion in oral squamous cell carcinoma: A retrospective study.

INTRODUCTION: Pattern of invasion (POI) in scoring system of oral squamous cell carcinoma (OSCC) can predict local recurrence and overall survival rate. Argyrophilic nucleolar organizer region (AGNOR) counts are considered to reflect the biosynthetic and nucleolar activity of a cell and thus serve as an indicator of the rapidity of the cell cycle thereby indicating the proliferative index of the tumor. It is implied that higher tumor associated tissue eosinophilia (TATE) showed lesser venous invasion, lymph node metastasis and clinical recurrence. The aim of the study was to assess and evaluate the following criteria's: POI-1 to POI-4 as defined by Bryne et al. in OSCC, proliferative index by AgNOR stain and TATE with carbol chromotrope stain in OSCC, validity of POI by correlating the AgNOR proliferative index and TATE. MATERIALS AND METHODS: Forty samples of formalin fixed paraffin embedded tissue blocks diagnosed of OSCC were taken for the study. Three sections were taken from a single block and then the tissues were stained differently with H & E Stain, AgNOR stain and Carbol chromotrope stain. First section stained with H & E was observed for POI and grading was done according to Bryne's criteria. The second and third sections were stained with AgNOR stain and Carbol chromotrope stain for proliferative index and TATE. One way analysis of variance was used to test the significance. RESULTS: Mean AgNORs count increases gradually from type 1 to type 4, depicting the increase in the nucleolar proliferative index of the cells and was statistically significant. In the case of the mean eosinophilic count, type 1 shows the highest mean eosinophilic count and the count shows drastic decrease till type 3 and from type 3 to type 4 the decrease is more gradual and was statistically significant. CONCLUSION: The study validated that POI is a good predictor for prognosis and also can be included in grading OSCC along with routine histopathological criteria.

Argyrophilic nucleolar organizer regions staining for cytology smears in dogs and cats.

The argyrophilic nucleolar organizer regions (AgNORs) are cellular proliferation markers, crucial for predicting the clinical course and aggressiveness of tumors. The purpose of this study was to establish an easy and practical AgNOR staining method in the cytology of dogs and cats. Air-dried cytological slides were prepared from dogs (n=14) and cats (n=12). Acetone, formalin, ethanol and methanol were tested as fixatives for AgNOR staining. Subsequently, various methods of Romanowsky-based counterstains were tested before and after AgNOR staining. Clear and strong AgNOR spots were observed with all fixatives, and post-May-Grünwald staining was the best counterstaining method. The established method showed clear AgNOR spots even in the long-term storage samples and Romanowsky-stained ones.

Comparative Study using Papanicolaou Stain and Silver-stained Nucleolar Organizer Region Counts in Exfoliative Smear of Oral Mucosa in Bidi Smokers and Nonsmokers.

AIM: The aim of this study is to compare the proliferative activity of exfoliated cells in bidi smoker's and nonsmoker's oral mucosa. MATERIALS AND METHODS: The oral mucosal exfoliate smears were prepared from 40 individuals (20 nonsmokers and 20 smokers) with the age group ranging from 25 to 70 years, in and around Akola (Maharashtra). The Papanicolaou (PAP) stain and silver-stained nucleolar organizer region (AgNOR) were used to prepare cytogenic smear to evaluate the presence of cytological alterations, suggestive of inflammation, dysplasia, keratinization, and proliferative activity of epithelial cells. The present study involves PAP Class I and Class II smears. The obtained data were tabulated and statistically analyzed using statistical software IBM SPSS IBM Corp., Statistics for Windows, Version 20.0. Armonk, NY, USA: IBM Corp., and using t-test. RESULTS: There was a significant difference in mean number of AgNORs/nucleus between nonsmokers (0.947 ± 0.2533) and smokers (3.021 ± 0.2256). There were 90% inflammatory changes observed in smokers whereas nonsmokers showed only 75% changes. PAP Class II changes, i.e., significant proliferative activity, were found between smokers and nonsmokers mucosa. CONCLUSION: A significant difference of AgNORs/nucleus was found between nonsmokers and smokers.

Analysis of silver binding nucleolar organizer regions in exfoliative cytology smears of potentially malignant and malignant oral lesions.

Nucleolar organizer regions are nucleolar components that contain proteins that are stained selectively by silver methods; they can be identified as black dots throughout the nucleolus and are known as silver binding nucleolar organizer regions (AgNOR). The number of AgNOR is related to the cell cycle and the proliferative activity of the cells. We investigated AgNOR using exfoliative cytology smears of potentially malignant oral lesions. Eighty individuals were divided into four equal groups: healthy controls, oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. The mean number of AgNOR in each study group gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. The proliferative index was increased in the oral premalignant and malignant patients compared to normal subjects. The mean AgNOR size gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. Spherical shaped AgNOR were most common in controls, whereas large, clustered and kidney shapes were most common in oral squamous cell carcinoma. Multiparameter analysis of AgNOR in oral exfoliative smears is a simple, sensitive and cost-effective method for differentiating premalignant from malignant lesions and can be used in conjunction with routine cytomorphological evaluation.

Integrating the genomic architecture of human nucleolar organizer regions with the biophysical properties of nucleoli.

Nucleoli are the sites of ribosome biogenesis and the largest membraneless subnuclear structures. They are intimately linked with growth and proliferation control and function as sensors of cellular stress. Nucleoli form around arrays of ribosomal gene (rDNA) repeats also called nucleolar organizer regions (NORs). In humans, NORs are located on the short arms of all five human acrocentric chromosomes. Multiple NORs contribute to the formation of large heterochromatin-surrounded nucleoli observed in most human cells. Here we will review recent findings about their genomic architecture. The dynamic nature of nucleoli began to be appreciated with the advent of photodynamic experiments using fluorescent protein fusions. We review more recent data on nucleoli in Xenopus germinal vesicles (GVs) which has revealed a liquid droplet-like behavior that facilitates nucleolar fusion. Further analysis in both XenopusGVs and Drosophila embryos indicates that the internal organization of nucleoli is generated by a combination of liquid-liquid phase separation and active processes involving rDNA. We will attempt to integrate these recent findings with the genomic architecture of human NORs to advance our understanding of how nucleoli form and respond to stress in human cells.