Blastema Cells Research Articles

Expression patterns of dnmt3aa, dnmt3ab, and dnmt4 during development and fin regeneration in zebrafish

Epigenetic modifications such as DNA methylation and chromatin modifications are critical for regulation of spatiotemporal gene expression during development. In mammals, the de novo-type DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. In addition to developmental processes, we recently showed that DNA methylation levels are dynamically changed during zebrafish fin regeneration, suggesting that the de novo-type Dnmts might play roles in the regulation of gene expression during regeneration processes. Here, we showed the detailed expression profiles of three zebrafish dnmt genes (dnmt3aa, dnmt3ab, and dnmt4), which were identified as the orthologues of mammalian dnmt3a and dnmt3b, during embryonic and larval development, as well as fin regeneration processes. dnmt3aa and dnmt3ab are expressed in the brain, pharyngeal arches, pectoral fin buds, intestine, and swim bladder; the specific expression of dnmt3aa is observed in the pronephric duct during larval development. dnmt4 expression is observed in the zona limitans intrathalamica, midbrain–hindbrain boundary, ciliary marginal zone, pharyngeal arches, auditory capsule, pectoral fin buds, intestine, pancreas, liver, and hematopoietic cells in the aorta–gonad–mesonephros and caudal hematopoietic tissue from 48 to 72h post-fertilization. Furthermore, during fin regeneration, strong dnmt3aa expression, and faint dnmt3ab and dnmt4 expression are detected in blastema cells at 72h post-amputation. Taken together, our results suggest that zebrafish Dnmt3aa, Dnmt3ab, and Dnmt4 may play roles in the formation of various organs, such as the brain, kidney, digestive organs, and/or hematopoietic cells, as well as in the differentiation of blastema cells.

Position-specific induction of ectopic limbs in non-regenerating blastemas on axolotl forelimbs.

Ectopic retinoic acid (RA) has been hypothesized to reprogram the positional identity of cells in developing and regenerating limbs to a single positional value corresponding to the posterior‐ventral‐proximal (PVPr) position on the limb. We tested this hypothesis by using RA to reprogram the information of blastema cells that were induced to form at different positions around the limb circumference. We observed that RA treatment of blastemas in anterior and dorsal locations, but not posterior and ventral locations, resulted in the induction of complete ectopic limbs. These position‐specific differences in limb induction are probably due to differences in the positional disparity between the RA‐reprogrammed blastema cells and the cells at the periphery of the wound. These observations are consistent with the hypothesis that RA treatment reprograms the information in blastema cells to the PVPr position on the limb, since anterior and dorsal positions have the largest disparity and posterior and ventral have the smallest disparity from the PVPr identity.

Experimentally induced metamorphosis in axolotls reduces regenerative rate and fidelity.

While most tetrapods are unable to regenerate severed body parts, amphibians display a remarkable ability to regenerate an array of structures. Frogs can regenerate appendages as larva, but they lose this ability around metamorphosis. In contrast, salamanders regenerate appendages as larva, juveniles, and adults. However, the extent to which fundamental traits (e.g., metamorphosis, body size, aging, etc.) restrict regenerative ability remains contentious. Here we utilize the ability of normally paedomorphic adult axolotls (Ambystoma mexicanum) to undergo induced metamorphosis by thyroxine exposure to test how metamorphosis and body size affects regeneration in age‐matched paedomorphic and metamorphic individuals. We show that body size does not affect regeneration in adult axolotls, but metamorphosis causes a twofold reduction in regeneration rate, and lead to carpal and digit malformations. Furthermore, we find evidence that metamorphic blastemal cells may take longer to traverse the cell cycle and display a lower proliferative rate. This study identifies the axolotl as a powerful system to study how metamorphosis restricts regeneration independently of developmental stage, body size, and age; and more broadly how metamorphosis affects tissue‐specific changes.

Wnt/β-Catenin Signaling Defines Organizing Centers that Orchestrate Growth and Differentiation of the Regenerating Zebrafish Caudal Fin

Zebrafish regenerate their fins via the formation of a population of progenitor cells, the blastema. Wnt/β-catenin signaling is essential for blastemal cell proliferation and patterning of the overlying epidermis. Yet, we find that β-catenin signaling is neither active in the epidermis nor the majority of the proliferative blastemal cells. Rather, tissue-specific pathway interference indicates that Wnt signaling in the nonproliferative distal blastema is required for cell proliferation in the proximal blastema, and signaling in cells lining the osteoblasts directs osteoblast differentiation. Thus, Wnt signaling regulates epidermal patterning, blastemal cell proliferation, and osteoblast maturation indirectly via secondary signals. Gene expression profiling, chromatin immunoprecipitation, and functional rescue experiments suggest that Wnt/β-catenin signaling acts through Fgf and Bmp signaling to control epidermal patterning, whereas retinoic acid and Hedgehog signals mediate its effects on blastemal cell proliferation. We propose that Wnt signaling orchestrates fin regeneration by defining organizing centers that instruct cellular behaviors of adjacent tissues.

Progressive Specification Rather than Intercalation of Segments During Limb Regeneration

An amputated salamander limb regenerates the correct number of segments. Models explaining limb regeneration were largely distinct from those for limb development, despite the presence of common patterning molecules. Intercalation has been an important concept to explain salamander limb regeneration, but clear evidence supporting or refuting this model was lacking. In the intercalation model, the first blastema cells acquire fingertip identity, creating a gap in positional identity that triggers regeneration of the intervening region from the stump. We used HOXA protein analysis and transplantation assays to show that axolotl limb blastema cells acquire positional identity in a proximal-to-distal sequence. Therefore, intercalation is not the primary mechanism for segment formation during limb regeneration in this animal. Patterning in development and regeneration uses similar mechanisms.

Positional Information Is Reprogrammed in Blastema Cells of the Regenerating Limb of the Axolotl (Ambystoma mexicanum)

The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP), to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.

SDF-1α/CXCR4 signaling mediates digit tip regeneration promoted by BMP-2

Previously we demonstrated that BMP signaling is required for endogenous digit tip regeneration, and that treatment with BMP-2 or -7 induces a regenerative response following amputation at regeneration-incompetent levels (Yu et al., 2010, 2012). Both endogenous regeneration and BMP-induced regeneration are associated with the transient formation of a blastema, however the formation of a regeneration blastema in mammals is poorly understood. In this study, we focus on how blastema cells respond to BMP signaling during neonatal digit regeneration in mice. First, we show that blastema cells retain regenerative properties after expansion in vitro, and when re-introduced into the amputated digit, these cells display directed migration in response to BMP-2. However, in vitro studies demonstrate that BMP-2 alone does not influence blastema cell migration, suggesting a requirement of another pivotal downstream factor for cell recruitment. We show that blastema cell migration is stimulated by the cytokine, SDF-1α, and that SDF-1α is expressed by the wound epidermis as well as endothelial cells of the blastema. Blastema cells express both SDF-1α receptors, CXCR4 and CXCR7, although the migration response is inhibited by the CXCR4-specific antagonist, AMD3100. Mice treated with AMD3100 display a partial inhibition of skeletal regrowth associated with the regeneration response. We provide evidence that BMP-2 regulates Sdf-1α expression in endothelial cells but not cells of the wound epidermis. Finally, we show that SDF-1α-expressing COS1 cells engrafted into a regeneration-incompetent digit amputation wound resulted in a locally enhanced population of CXCR4 positive cells, and induced a partial regenerative response. Taken together, this study provides evidence that one downstream mechanism of BMP signaling during mammalian digit regeneration involves activation of SDF-1α/CXCR4 signaling by endothelial cells to recruit blastema cells.

De novo transcriptome sequencing of axolotl blastema for identification of differentially expressed genes during limb regeneration

BackgroundSalamanders are unique among vertebrates in their ability to completely regenerate amputated limbs through the mediation of blastema cells located at the stump ends. This regeneration is nerve-dependent because blastema formation and regeneration does not occur after limb denervation. To obtain the genomic information of blastema tissues, de novo transcriptomes from both blastema tissues and denervated stump ends of Ambystoma mexicanum (axolotls) 14 days post-amputation were sequenced and compared using Solexa DNA sequencing.ResultsThe sequencing done for this study produced 40,688,892 reads that were assembled into 307,345 transcribed sequences. The N50 of transcribed sequence length was 562 bases. A similarity search with known proteins identified 39,200 different genes to be expressed during limb regeneration with a cut-off E-value exceeding 10-5. We annotated assembled sequences by using gene descriptions, gene ontology, and clusters of orthologous group terms. Targeted searches using these annotations showed that the majority of the genes were in the categories of essential metabolic pathways, transcription factors and conserved signaling pathways, and novel candidate genes for regenerative processes. We discovered and confirmed numerous sequences of the candidate genes by using quantitative polymerase chain reaction and in situ hybridization.ConclusionThe results of this study demonstrate that de novo transcriptome sequencing allows gene expression analysis in a species lacking genome information and provides the most comprehensive mRNA sequence resources for axolotls. The characterization of the axolotl transcriptome can help elucidate the molecular mechanisms underlying blastema formation during limb regeneration.

Nerve independent limb induction in axolotls

Urodele amphibians can regenerate their limbs. During limb regeneration, dermal fibroblasts are transformed into undifferentiated cells called blastema cells. These dermis–blastema cells show multipotency. Such so-called endogenous reprogramming of cell differentiation is one of the main targets of amphibian limb regeneration studies. It is well recognized that nerve presence controls the initiation of limb regeneration. Accordingly, nerve factors have been sought in amphibian limb regeneration. To investigate it, a relatively new study system called the accessory limb model (ALM) was developed. Using ALM, two signaling cascades (Fgf and Gdf5 signaling) came under focus. In the present study, Growth and differentiation factor-5 (Gdf5) application to wounded skin initiated limb regeneration responses and resulted in induction of a blastema-like structure in the absence of a nerve. However, the Gdf5-induced structure showed defects as a regeneration blastema, such as absence of detectable Prrx1 expression by in situ hybridization. The defects could be remedied by additional Fibroblasts growth factor (Fgf) inputs. These two inputs (Gdf5 and Fgfs) were sufficient to substitute for the nerve functions in the induction of limb regeneration. Indeed, Fgf2, Fgf8, and Gdf5 applications with the contralateral skin graft resulted in limb formation without nerve supply. Furthermore, acquisition of cartilage differentiation potential of dermal fibroblasts was tested in an in vivo and in vitro combination assay. Dermal fibroblasts cultured with Gdf5 were difficult to participate in cartilage formation when the cultured cells were grafted into cartilage forming region. In contrast, dermal fibroblasts cultured with Fgf2 and Fgf8 became easier to participate into cartilage formation in the same procedure. These results contribute to our understanding of molecular mechanisms of the early phase of amphibian limb regeneration.

Histological observations of blastema formation during earthworm tail regeneration

The earthworm is a unique and valuable model for investigating the mechanism of regeneration. We studied the developmental change in blastemal cells during earthworm tail regeneration. By histological observation, we have found that the major origin of earthworm blastemal cells is the longitudinal muscle layer of the body wall. The blastema formed beneath the wound dermis is segmented and becomes redifferentiated. The results obtained demonstrate that the blastema formation in earthworm tail regeneration provides a useful epimorphic model of regeneration.

Chemical activation of RARβ induces post-embryonically bilateral limb duplication during Xenopus limb regeneration

The anuran amphibian Xenopus laevis can regenerate its limbs for a limited time during the larval stage, while limbs are still developing. Using this regeneration model, we evaluated the proximo-distal blastema cell identity when endogenous retinoids were increased by CYP26 inhibition or when RAR-specific agonists altered RA signaling. Simultaneous proximo-distal and antero-posterior limb duplications were generated, and the RAR-specific agonist can modify blastema identity after amputation, because chemical activation of RARβ produced bilateral hindlimb duplications that resulted in a drastic duplication phenotype of regenerating limbs.

Notch signaling coordinates cellular proliferation with differentiation during zebrafish fin regeneration

Zebrafish can completely regenerate amputated fins via formation of a blastema, a proliferative mass of undifferentiated precursor cells. During regenerative growth, blastema proliferation must be tightly coordinated with cellular differentiation, but little is known about how this is achieved. Here, we show that Notch signaling is essential for maintenance of blastema cells in a proliferative undifferentiated state. We found that the Notch pathway is activated in response to fin amputation in the highly proliferative region of the blastema. Chemical interference with Notch signaling resulted in a complete block of regeneration. Notch signaling was not required for the earliest known cellular processes during blastema formation, i.e. dedifferentiation and migration of osteoblasts, but specifically interfered with proliferation of blastema cells. Interestingly, overactivation of the pathway via misexpression of the intracellular domain of the Notch receptor (NICD) likewise inhibited regenerative outgrowth. In NICD-overexpressing fins, overall blastemal cell proliferation was not enhanced, but expanded into proximal regions where cellular differentiation normally occurs. Similarly, blastemal and epidermal gene expression territories invaded proximal regions upon sustained Notch activation. Concomitantly, NICD overexpression suppressed differentiation of osteoblasts and caused an expansion of the undifferentiated blastema. Together, these data suggest that Notch signaling activity maintains blastemal cells in a proliferative state and thus coordinates proliferation with differentiation during regenerative growth.

Specialized progenitors and regeneration

Planarians are flatworms capable of regenerating all body parts. Planarian regeneration requires neoblasts, a population of dividing cells that has been studied for over a century. Neoblast progeny generate new cells of blastemas, which are the regenerative outgrowths at wounds. If the neoblasts comprise a uniform population of cells during regeneration (e.g. they are all uncommitted and pluripotent), then specialization of new cell types should occur in multipotent, non-dividing neoblast progeny cells. By contrast, recent data indicate that some neoblasts express lineage-specific transcription factors during regeneration and in uninjured animals. These observations raise the possibility that an important early step in planarian regeneration is the specialization of neoblasts to produce specified rather than naïve blastema cells.

AR Highlights

Xenopus laevis, commonly known as the African clawed frog, is a useful model in developmental biology. The tail of the Xenopus tadpole completely regenerates by 7–14 days after amputation. This regeneration is similar to tissue renewal in mammals. A ‘‘refractory period’’ occurs during development during which tail regenerative ability is lost in tadpoles. Because of this refractory period, the Xenopus tadpole tail is an ideal model for studying changes in regenerative ability in mammals. Tail regeneration in Xenopus tadpoles requires bioelectrical signaling, which involves slow changes in ion content and resting membrane voltage. For optimal treatment to provide bioelectrical signals for successful regeneration, techniques need to be developed for precise temporal control of resting membrane voltage. In this article, the authors discuss a technique that assists with control of resting membrane voltage in vivo. They demonstrate that this method controls regenerative responses in the tadpole tail. The authors propose a new hypothesis for how ion content is converted into alterations in cell behavior and transcription. The authors hypothesize that the sodium/butyratre transporter SLC5A8 is a mechanism by which some systems may link epigenetic chromatin modifications with bioelectric events (changes in ion content). Determining the mechanisms by which ion flow modulates gene expression will likely provide further understanding on cell biology and evolution of morphogenetic pathways, and contribute to development of treatments that use bioelectricity. Mammalian tissue repair involves a complex, dynamic interplay among cells located at the site of injury and white blood cells recruited to the site. These interactions lead to various processes, including inflammation and tissue differentiation, necessary for repair. In contrast, limb regeneration in urodeles (e.g., salamanders and newts) involves a short period of inflammation, but this is followed by blastema formation that results in a new organ that is fully functional. The authors focus on limb regeneration in Xenopus laevis, which as well as being a useful embryological model, also plays an important role in developmental studies of the immune system and for investigation of regenerative mechanisms. They first review studies on gene expression in amputated Xenopus limbs at various developmental stages involving regeneration and non-regeneration. These studies show that inflammation and regenerative capacity are inversely correlated. The authors also review non-mammalian immune system features involved in the role of inflammation in the balance between regeneration and fibrosis. The authors show results testing the hypothesis that factors controlling the inflammatory response to injury affect regeneration. They compare the effects of antiinflammatory agents and a proinflammatory adjuvant. The data reviewed by the authors suggest that studies on ontogenic immunological changes that occur in Xenopus laevis are useful for determining how inflammation affects blastema formation. An important question is the extent to which differentiating blastema cells resemble mammalian mesenchymal stem cells. Many lizards have the remarkable ability of tail and spinal cord regeneration. Although tail regeneration in lizards has been well studied, further characterization of the anatomy of the regenerated tale is required. The authors examined the anatomy of the original and regenerated tail in the green anole Anolis carolinensis. They chose this species as a model because the process of tail shedding and regeneration is well understood, as well as the fact that the genome has been sequenced, making it a good model for understanding genetic regulation for tail regeneration. The authors investigated the musculature, chondrology, and osteology of the original and regenerated tail in A. carolinensis. The authors found differences in musculature between the original and regenerated tail, suggesting major functional differences between the two types of tails. Notably, the regenerated tail is thought to be less able to perform fine movements. The authors' findings of detailed anatomy of the tail in A. carolinensis could be useful for future genetic and developmental studies on tail regeneration in lizards, and potentially to regeneration in mammals. Ellen C. Jensen*

Notch regulates blastema proliferation and prevents differentiation during adult zebrafish fin regeneration

Zebrafish have the capacity to regenerate several organs, including the heart and fins. Fin regeneration is epimorphic, involving the formation at the amputation plane of a mass of undifferentiated, proliferating mesenchymal progenitor-like cells, called blastema. This tissue provides all the cell types that form the fin, so that after damage or amputation the fin pattern and structure are fully restored. How blastema cells remain in this progenitor-like state is poorly understood. Here, we show that the Notch pathway plays an essential role during fin regeneration. Notch signalling is activated during blastema formation and remains active throughout the regeneration process. Chemical inhibition or morpholino-mediated knockdown of Notch signalling impairs fin regeneration via decreased proliferation accompanied by reduced expression of Notch target genes in the blastema. Conversely, overexpression of a constitutively active form of the Notch1 receptor (N1ICD) in the regenerating fin leads to increased proliferation and to the expansion of the blastema cell markers msxe and msxb, as well as increased expression of the proliferation regulator aldh1a2. This blastema expansion prevents regenerative fin outgrowth, as indicated by the reduction in differentiating osteoblasts and the inhibition of bone regeneration. We conclude that Notch signalling maintains blastema cells in a plastic, undifferentiated and proliferative state, an essential requirement for fin regeneration.

Analysis of RNA-Seq data reveals involvement of JAK/STAT signalling during leg regeneration in the cricket Gryllus bimaculatus

In the cricket Gryllus bimaculatus, missing distal parts of the amputated leg are regenerated from the blastema, a population of dedifferentiated proliferating cells that forms at the distal tip of the leg stump. To identify molecules involved in blastema formation, comparative transcriptome analysis was performed between regenerating and normal unamputated legs. Components of JAK/STAT signalling were upregulated more than twofold in regenerating legs. To verify their involvement, Gryllus homologues of the interleukin receptor Domeless (Gb'dome), the Janus kinase Hopscotch (Gb'hop) and the transcription factor STAT (Gb'Stat) were cloned, and RNAi was performed against these genes. Gb'dome(RNAi), Gb'hop(RNAi) and Gb'Stat(RNAi) crickets showed defects in leg regeneration. Blastema expression of Gb'cyclinE was decreased in the Gb'Stat(RNAi) cricket compared with that in the control. Hyperproliferation of blastema cells caused by Gb'fat(RNAi) or Gb'warts(RNAi) was suppressed by RNAi against Gb'Stat. The results suggest that JAK/STAT signalling regulates blastema cell proliferation during leg regeneration.

Expression analysis of XPhyH-like during development and tail regeneration in Xenopus tadpoles: Possible role of XPhyH-like expressing immune cells in impaired tail regenerative ability

Xenopus tadpoles have high regenerative ability of amputated tails except during the ‘refractory period’, when the ability is transiently lost. We previously demonstrated that distinct immune responses occur in tail stumps between the refractory and pre/post-refractory regeneration periods. Furthermore, treatment with an immunosuppressant, FK506, restores the tail regenerative ability during the refractory period. Based on these findings, we previously proposed that autoreactive immune cells infiltrate the tail stumps to attack blastema cells as ‘non-self’ during the refractory period, resulting in the impaired regenerative ability. The immune cells that attack the blastema cells, however, remained unclear. Here we screened for genes whose expression in the tail stumps was altered by FK506 treatment during the refractory period and identified a Xenopus homolog of phytanoyl-CoA dioxygenase (PhyH)-like. XPhyH-like expression transiently increased in tail stumps after amputation during the refractory period, and was reduced by FK506 treatment. XPhyH-like expression in the whole tadpole body specifically increased during the refractory period and was enriched in the blood cell fraction. These findings suggest that XPhyH-like is expressed in autoreactive immune cells that are distributed in the whole body during the refractory period and transiently infiltrate the tail stumps to attack the blastema cells as ‘non-self’.

MiRNA Profiles as a Predictor of Chemoresponsiveness in Wilms’ Tumor Blastema

The current SIOP treatment protocol for Wilms’ tumor involves pre-operative chemotherapy followed by nephrectomy. Not all patients benefit equally from such chemotherapy. The aim of this study was to generate a miRNA profile of chemo resistant blastemal cells in high risk Wilms’ tumors which might serve as predictive markers of therapeutic response at the pre-treatment biopsy stage. We have shown here that unsupervised hierarchical clustering of genome-wide miRNA expression profiles can clearly separate intermediate risk tumors from high risk tumors. A total of 29 miRNAs were significantly differentially expressed between post-treatment intermediate risk and high risk groups, including miRNAs that have been previously linked to chemo resistance in other cancer types. Furthermore, 7 of these 29 miRNAs were already at the pre-treatment biopsy stage differentially expressed between cases ultimately deemed intermediate risk compared to high risk. These miRNA alterations include down-regulation in high risk cases of miR-193a.5p, miR-27a and the up-regulation of miR-483.5p, miR-628.5p, miR-590.5p, miR-302a and miR-367. The demonstration of such miRNA markers at the pre-treatment biopsy stage could permit stratification of patients to more tailored treatment regimens.

Connective tissue cells, but not muscle cells, are involved in establishing the proximo-distal outcome of limb regeneration in the axolotl

During salamander limb regeneration, only the structures distal to the amputation plane are regenerated, a property known as the rule of distal transformation. Multiple cell types are involved in limb regeneration; therefore, determining which cell types participate in distal transformation is important for understanding how the proximo-distal outcome of regeneration is achieved. We show that connective tissue-derived blastema cells obey the rule of distal transformation. They also have nuclear MEIS, which can act as an upper arm identity regulator, only upon upper arm amputation. By contrast, myogenic cells do not obey the rule of distal transformation and display nuclear MEIS upon amputation at any proximo-distal level. These results indicate that connective tissue cells, but not myogenic cells, are involved in establishing the proximo-distal outcome of regeneration and are likely to guide muscle patterning. Moreover, we show that, similarly to limb development, muscle patterning in regeneration is influenced by β-catenin signalling.

Pleuropulmonary blastoma in an adult: A case report

Introduction: Pleuropulmonary blastoma (PPB) is a dysontogenetic neoplasm of childhood that appears as a pulmonary and/or pleural­based mass. PPB is of three types: cystic (type I), mixed (type II), or solid (type III). It is rarely observed in adults with only a few cases reported in literature. Case Report: We present a case of a 30­year­old male with complaints of chest pain, dyspnea and low grade fever. His computed tompgraphy scan of chest showed three multicystic lesions in right hemithorax. Cytomorphological examination of imprint smears of biopsies taken from the masses suggested malignant lesion and on histological examination, diagnosis of pleuropulmonary blastoma type II was made. Conclusion: Pleuropulmonary blastoma is a rare and aggressive malignant tumor of childhood. Familial disposition and association of PPB tumor with other childhood malignancies has been suggested in literature. Pathologically, the tumor tissue is composed of primitive blastemal cells with focal areas of malignant mesenchyme. Therapy should include surgical tumor resection and subsequent chemotherapy and radiotherapy. PPB is very rare tumor in adults and presentation as multiple lesions in one sided hemithorax has not been reported. PPB should be considered in the clinical and radiographic differential diagnosis of multicystic lesions, even when the patient is a young adult.