Live imaging-assisted domain-specific CRISPR genome editing at single cell resolution in plants

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has been widely used for genome engineering in a wide range of organisms, but much of the development of CRISPR-based genome editing has been aimed toward improving its efficiency and accuracy, so as to obtain genetic materials carrying known and stably heritable genome modifications. Precise spatiotemporal control over genome editing technology at cell type resolution is a key challenge for gene function studies. Some tissue-specific CRISPR genome editing methods relying on phenotypic characterization and fluorescent immune-staining techniques have been developed for biomedical research and gene therapy, they function by spatially controlling expression of Cas9. Recent work establishes the presence and location of mutational events at a single cell level in Arabidopsis roots and stomata. Here we present an efficient domain-specific CRISPR-Cas9 system combined with a high resolution live-imaging based screening strategy, applied in the shoot apical meristem of Arabidopsis thaliana. Using the system we investigate PIN-FORMED1 (PIN1) protein functions in tissue morphogenesis and PIN1 mechanical stress response in a cell layer-specific fashion. We find that reported failure to generate new primordia in epidermal PIN1 knockout SAMs is due to a reduction in mechanical stress differences in the sub-epidermal layer. The methods described are applicable to spatial-temporal gene manipulation in plants

Live imaging-assisted domain-specific CRISPR genome editing at single cell resolution in plants

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has been widely used for genome engineering in a wide range of organisms, but much of the development of CRISPR-based genome editing has been aimed toward improving its efficiency and accuracy, so as to obtain genetic materials carrying known and stably heritable genome modifications. Precise spatiotemporal control over genome editing technology at cell type resolution is a key challenge for gene function studies. Some tissue-specific CRISPR genome editing methods relying on phenotypic characterization and fluorescent immune-staining techniques have been developed for biomedical research and gene therapy, they function by spatially controlling expression of Cas9. Recent work establishes the presence and location of mutational events at a single cell level in Arabidopsis roots and stomata. Here we present an efficient domain-specific CRISPR-Cas9 system combined with a high resolution live-imaging based screening strategy, applied in the shoot apical meristem of Arabidopsis thaliana. Using the system we investigate PIN-FORMED1 (PIN1) protein functions in tissue morphogenesis and PIN1 mechanical stress response in a cell layer-specific fashion. We find that reported failure to generate new primordia in epidermal PIN1 knockout SAMs is due to a reduction in mechanical stress differences in the sub-epidermal layer. The methods described are applicable to spatial-temporal gene manipulation in plants

Overlapping and Distinct Roles of HAM Family Genes in Arabidopsis Shoot Meristems

In Arabidopsis shoot apical meristems (SAMs), a well-characterized regulatory loop between WUSCHEL (WUS) and CLAVATA3 (CLV3) maintains stem cell homeostasis by regulating the balance between cell proliferation and cell differentiation. WUS proteins, translated in deep cell layers, move into the overlaying stem cells to activate CLV3. The secreted peptide CLV3 then regulates WUS levels through a ligand-receptor mediated signaling cascade. CLV3 is specifically expressed in the stem cells and repressed in the deep cell layers despite presence of the WUS activator, forming an apical-basal polarity along the axis of the SAM. Previously, we proposed and validated a hypothesis that the HAIRY MERISTEM (HAM) family genes regulate this polarity, keeping the expression of CLV3 off in interior cells of the SAM. However, the specific role of each individual member of the HAM family in this process remains to be elucidated. Combining live imaging and molecular genetics, we have dissected the conserved and distinct functions of different HAM family members in control of CLV3 patterning in the SAMs and in the de novo shoot stem cell niches as well

Control of plant stem cell function by conserved interacting transcriptional regulators

Plant stem cells in the shoot apical meristem (SAM) and root apical meristem are necessary for postembryonic development of aboveground tissues and roots, respectively, while secondary vascular stem cells sustain vascular development. WUSCHEL (WUS), a homeodomain transcription factor expressed in the rib meristem of the Arabidopsis SAM, is a key regulatory factor controlling SAM stem cell populations, and is thought to establish the shoot stem cell niche through a feedback circuit involving the CLAVATA3 (CLV3) peptide signalling pathway. WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which is specifically expressed in the root quiescent centre, defines quiescent centre identity and functions interchangeably with WUS in the control of shoot and root stem cell niches. WOX4, expressed in Arabidopsis procambial cells, defines the vascular stem cell niche. WUS/WOX family proteins are evolutionarily and functionally conserved throughout the plant kingdom and emerge as key actors in the specification and maintenance of stem cells within all meristems. However, the nature of the genetic regime in stem cell niches that centre on WOX gene function has been elusive, and molecular links underlying conserved WUS/WOX function in stem cell niches remain unknown. Here we demonstrate that the Arabidopsis HAIRY MERISTEM (HAM) family of transcription regulators act as conserved interacting cofactors with WUS/WOX proteins. HAM and WUS share common targets in vivo and their physical interaction is important in driving downstream transcriptional programs and in promoting shoot stem cell proliferation. Differences in the overlapping expression patterns of WOX and HAM family members underlie the formation of diverse stem cell niche locations, and the HAM family is essential for all of these stem cell niches. These findings establish a new framework for the control of stem cell production during plant development

Calcium signals are necessary to establish auxin transporter polarity in a plant stem cell niche

In plants mechanical signals pattern morphogenesis through the polar transport of the hormone auxin and through regulation of interphase microtubule (MT) orientation. To date, the mechanisms by which such signals induce changes in cell polarity remain unknown. Through a combination of time-lapse imaging, and chemical and mechanical perturbations, we show that mechanical stimulation of the SAM causes transient changes in cytoplasmic calcium ion concentration (Ca^(2+)) and that transient Ca^(2+) response is required for downstream changes in PIN-FORMED 1 (PIN1) polarity. We also find that dynamic changes in Ca^(2+) occur during development of the SAM and this Ca^(2+) response is required for changes in PIN1 polarity, though not sufficient. In contrast, we find that Ca^(2+) is not necessary for the response of MTs to mechanical perturbations revealing that Ca^(2+) specifically acts downstream of mechanics to regulate PIN1 polarity response

HAIRY MERISTEM with WUSCHEL confines CLAVATA3 expression to the outer apical meristem layers

The control of the location and activity of stem cells depends on spatial regulation of gene activities in the stem cell niche. Using computational and experimental approaches, we have tested and found support for a hypothesis for gene interactions that specify the Arabidopsis apical stem cell population. The hypothesis explains how the WUSCHEL gene product, synthesized basally in the meristem, induces CLAVATA3-expressing stem cells in the meristem apex but, paradoxically, not in the basal domain where WUSCHEL itself is expressed. The answer involves the activity of the small family of HAIRY MERISTEM genes, which prevent the activation of CLAVATA3 and which are expressed basally in the shoot meristem

Overlapping and Distinct Roles of HAM Family Genes in Arabidopsis Shoot Meristems

In Arabidopsis shoot apical meristems (SAMs), a well-characterized regulatory loop between WUSCHEL (WUS) and CLAVATA3 (CLV3) maintains stem cell homeostasis by regulating the balance between cell proliferation and cell differentiation. WUS proteins, translated in deep cell layers, move into the overlaying stem cells to activate CLV3. The secreted peptide CLV3 then regulates WUS levels through a ligand-receptor mediated signaling cascade. CLV3 is specifically expressed in the stem cells and repressed in the deep cell layers despite presence of the WUS activator, forming an apical-basal polarity along the axis of the SAM. Previously, we proposed and validated a hypothesis that the HAIRY MERISTEM (HAM) family genes regulate this polarity, keeping the expression of CLV3 off in interior cells of the SAM. However, the specific role of each individual member of the HAM family in this process remains to be elucidated. Combining live imaging and molecular genetics, we have dissected the conserved and distinct functions of different HAM family members in control of CLV3 patterning in the SAMs and in the de novo shoot stem cell niches as well

HAIRY MERISTEM with WUSCHEL confines CLAVATA3 expression to the outer apical meristem layers

The control of the location and activity of stem cells depends on spatial regulation of gene activities in the stem cell niche. Using computational and experimental approaches, we have tested and found support for a hypothesis for gene interactions that specify the Arabidopsis apical stem cell population. The hypothesis explains how the WUSCHEL gene product, synthesized basally in the meristem, induces CLAVATA3-expressing stem cells in the meristem apex but, paradoxically, not in the basal domain where WUSCHEL itself is expressed. The answer involves the activity of the small family of HAIRY MERISTEM genes, which prevent the activation of CLAVATA3 and which are expressed basally in the shoot meristem

Genetic Characterization of Five Hatchery Populations of the Pacific Abalone (Haliotis discus hannai) Using Microsatellite Markers

The Pacific abalone, Haliotis discus hannai, is a popular food in Eastern Asia. Aquacultural production of this species has increased because of recent resource declines, the growing consumption, and ongoing government-operated stock release programs. Therefore, the genetic characterization of hatchery populations is necessary to maintain the genetic diversity of this species and to develop more effective aquaculture practices. We analyzed the genetic structures of five cultured populations in Korea using six microsatellite markers. The number of alleles per locus ranged from 15 to 64, with an average of 23.5. The mean observed and expected heterozygosities were 0.797 and 0.904, respectively. The inbreeding coefficient FIS ranged from 0.054 to 0.184 (mean FIS = 0.121 ± 0.056). The genetic differentiation across all populations was low but significant (overall FST = 0.009, P < 0.01). Pairwise multilocus FST tests, estimates of genetic distance, and phylogenetic and principal component analyses did not show a consistent relationship between geographic and genetic distances. These results could reflect extensive aquaculture, the exchange of breeds and eggs between hatcheries and/or genetic drift due to intensive breeding practices. Thus, for optimal resource management, the genetic variation of hatchery stocks should be monitored and inbreeding controlled within the abalone stocks that are being released every year. This genetic information will be useful for the management of both H. discus hannai fisheries and the aquaculture industry

Apocrine Hidradenocarcinoma of the Scalp: A Classification Conundrum

Introduction The classification of malignant sweat gland lesions is complex. Traditionally, cutaneous sweat gland tumors have been classified by either eccrine or apocrine features. Methods A case report of a 33-year-old Hispanic man with a left scalp mass diagnosed as a malignancy of adnexal origin preoperatively is discussed. After presentation at our multidisciplinary tumor board, excision with ipsilateral neck dissection was undertaken. Results Final pathology revealed an apocrine hidradenocarcinoma. The classification and behavior of this entity are discussed in this report. Conclusion Apocrine hidradenocarcinoma can be viewed as an aggressive malignant lesion of cutaneous sweat glands on a spectrum that involves both eccrine and apoeccrine lesions