Morpho-Histology, Endogenous Hormone Dynamics and Transcriptome Profiling in Dacrydium Pectinatum during Male Cone Development

Dacrydium pectinatum de Laubenfels is a perennial gymnosperm species dominant in tropical montane rain forests. Due to severe damages by excessive deforestation, typhoons, and other external forces, the population of the species has been significantly reduced. Furthermore, its natural regeneration is poor. To better understand the male cone development in D. pectinatum, we examined the morphological and anatomical changes, analyzed the endogenous hormone dynamics, and profiled gene expression. The morpho-histological observations suggest that the development of D. pectinatum male cone can be largely divided into four stages: microspore primordium formation (April to May), microspore sac and pollen mother cell formation (July to November), pollen mother cell division (January), and pollen grain formation (February). The levels of gibberellins (GA), auxin (IAA), abscisic Acid (ABA), cytokinin (CTK), and jasmonic acid (JA) fluctuated during the process of male cone development. The first transcriptome database for a Dacrydium species was generated, revealing >70,000 unigene sequences. Differential expression analyses revealed several floral and hormone biosynthesis and signal transduction genes that could be critical for male cone development. Our study provides new insights on the cone development in D. pectinatum and the foundation for male cone induction with hormones and studies of factors contributing to the species’ low rate of seed germination.

ATF6 is essential for human cone photoreceptor development

Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying ATF6 disease variants and from gene-edited ATF6 null hESCs. We found that achromatopsia patient and ATF6 null retinal organoids failed to form cone structures concomitant with loss of cone phototransduction gene expression, while rod photoreceptors developed normally. Adaptive optics retinal imaging of achromatopsia patients carrying ATF6 variants also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the defect in cone formation observed in our retinal organoids. These results establish that ATF6 is essential for human cone development. Interestingly, we find that a selective small molecule ATF6 signaling agonist restores the transcriptional activity of some ATF6 disease-causing variants and stimulates cone growth and gene expression in patient retinal organoids carrying these variants. These findings support that pharmacologic targeting of the ATF6 pathway can promote human cone development and should be further explored for blinding retinal diseases.

Cone-Bearing Branches of Pinus koraiensis Are Not Carbon Autonomous during Cone Development

Cone development in conifer species is crucial to ensure sexual regeneration. A better understanding of carbon (C) source-sink relations at the branch level can guide strategies for improving resource allocation to reproduction. In particular, the evaluation of C relations between vegetative and reproductive branches is helpful to test whether tree branches are carbon autonomous. With this aim, we integrated girdling and defoliation treatments with 13C pulse labeling in situ to evaluate C autonomy in cone-bearing branches of P. koraiensis during the growing season. Girdling significantly reduced branch volumetric development, branch biomass, and non-structural carbohydrates across foliar, twig, and cone tissues; it also arrested cone development. Defoliation effects on these variables were minor, although they tended to increase with defoliation intensity. In addition, 13C increased by 4.5% and 45.4% after 4 h and 24 h of 13C labeling in unlabeled cone-bearing branches, respectively, indicating the C translocation from labeled vegetative branches. These results indicate that the cone-bearing branches are not C autonomous and that the development of female cones relies to a great extent on C import from neighboring branches. However, the amount of C translocated was largely dependent on manipulative alterations of the source-sink balance, thereby denoting extensive plasticity in the degree of branch C autonomy. These results shed light on the reproductive physiology of P. koraiensis.

Cis-regulatory dissection of cone development reveals a broad role for Otx2 and Oc transcription factors

ABSTRACT The vertebrate retina is generated by retinal progenitor cells (RPCs), which produce >100 cell types. Although some RPCs produce many cell types, other RPCs produce restricted types of daughter cells, such as a cone photoreceptor and a horizontal cell (HC). We used genome-wide assays of chromatin structure to compare the profiles of a restricted cone/HC RPC and those of other RPCs in chicks. These data nominated regions of regulatory activity, which were tested in tissue, leading to the identification of many cis-regulatory modules (CRMs) active in cone/HC RPCs and developing cones. Two transcription factors, Otx2 and Oc1, were found to bind to many of these CRMs, including those near genes important for cone development and function, and their binding sites were required for activity. We also found that Otx2 has a predicted autoregulatory CRM. These results suggest that Otx2, Oc1 and possibly other Onecut proteins have a broad role in coordinating cone development and function. The many newly discovered CRMs for cones are potentially useful reagents for gene therapy of cone diseases.

Axonal Organelles as Molecular Platforms for Axon Growth and Regeneration after Injury

Investigating the molecular mechanisms governing developmental axon growth has been a useful approach for identifying new strategies for boosting axon regeneration after injury, with the goal of treating debilitating conditions such as spinal cord injury and vision loss. The picture emerging is that various axonal organelles are important centers for organizing the molecular mechanisms and machinery required for growth cone development and axon extension, and these have recently been targeted to stimulate robust regeneration in the injured adult central nervous system (CNS). This review summarizes recent literature highlighting a central role for organelles such as recycling endosomes, the endoplasmic reticulum, mitochondria, lysosomes, autophagosomes and the proteasome in developmental axon growth, and describes how these organelles can be targeted to promote axon regeneration after injury to the adult CNS. This review also examines the connections between these organelles in developing and regenerating axons, and finally discusses the molecular mechanisms within the axon that are required for successful axon growth.

The Transcriptome of Cunninghamia lanceolata male/female cone reveal the association between MIKC MADS-box genes and reproductive organs development

Background Cunninghamia lanceolata (Chinese fir), a member of the conifer family Cupressaceae, is one of the most popular cultivated trees for wood production in China. Continuous research is being performed to improve C. lanceolata breeding values. Given the high rate of seed abortion (one of the reasons being the failure of ovule and pollen development) in C. lanceolata, the proper formation of female/male cones could theoretically increase the number of offspring in future generations. MIKC MADS-box genes are well-known for their roles in the flower/cone development and comprise the typical/atypical floral development model for both angiosperms and gymnosperms. Results We performed a transcriptomic analysis to find genes differentially expressed between female and male cones at a single, carefully determined developmental stage, focusing on the MIKC MADS-box genes. We finally obtained 47 unique MIKC MADS-box genes from C. lanceolata and divided these genes into separate branches. 27 out of the 47 MIKC MADS-box genes showed differential expression between female and male cones, and most of them were not expressed in leaves. Out of these 27 genes, most B-class genes (AP3/PI) were up-regulated in the male cone, while TM8 genes were up-regulated in the female cone. Then, with no obvious overall preference for AG (class C + D) genes in female/male cones, it seems likely that these genes are involved in the development of both cones. Finally, a small number of genes such as GGM7, SVP, AGL15, that were specifically expressed in female/male cones, making them candidate genes for sex-specific cone development. Conclusions Our study identified a number of MIKC MADS-box genes showing differential expression between female and male cones in C. lanceolata, illustrating a potential link of these genes with C. lanceolata cone development. On the basis of this, we postulated a possible cone development model for C. lanceolata. The gene expression library showing differential expression between female and male cones shown here, can be used to discover unknown regulatory networks related to sex-specific cone development in the future.

Sexual Dimorphism in the Chemical Composition of Male and Female in the Dioecious Tree, Juniperus communis L., Growing under Different Nutritional Conditions

We hypothesized that female and male individuals of the dioecious tree species, Juniperus communis, exhibit different strategies of resource allocation when growing under stress conditions. To test this hypothesis, we performed a two-year pot experiment on plants exposed to different levels of nutrient availability. Analysis of the plants revealed a higher concentration of carbohydrates, carbon, and phenolic compounds in needles of female plants, indicating that females allocate more resources to storage and defense than males. This difference was independent of nutrient availability. Differences in carbohydrates levels between the sexes were most often significant in June, during the most intensive phase of vegetative growth in both sexes, but could also be attributed to female resources investment in cone development. A higher level of nitrogen and other macroelements was observed in males than in females, which may have been connected to the accumulation of resources (nitrogen) for pollen grain production in males or greater allocation of these elements to seeds and cones in females. The interaction between sex and soil fertilization for the C:N ratio may also indicate sex-specific patterns of resource allocation and utilization, which is impacted by their availability during specific periods of J. communis annual life cycle.

Temporal and Spatial Characteristics Endogenous Hormone Regulation During Male Cone Development of Dacrydium pectinatum

Background: Dacrydiumpectinatum de Laubenfels is a perennial gymnosperm dominant in tropical montane rain forests. Due to severe damages by excessive deforestation, typhoons, and other external forces, the population of the species has been significantly reduced. Furthermore, natural regeneration is poor. In order to better understand the reproductive process in D. pectinatum, we examined the morphological and anatomical changes during the development of male cone and analyzed the endogenous hormone dynamics.Results: Our study indicates that D. pectinatum male buds become distinguishable in April in tropical montane rain forests, while microspore sac forms in September and pollen mother cell forms and divides in December. Pollen grains mature and disperse in the following February. A mature male cone averages 8.5 mm in length. Level of GA, IAA, ABA and JA and their ratios fluctuated during late August to late November when sporogenous tissues were actively differentiated.Conclusions: The differentiation of sporogenous tissues is accompanied by variations in levels of endogenous hormones (GA, ABA, IAA, and JA) and their balances. The new insights about the cone development in D. pectinatum lay the foundation for future cone induction with hormones and study of factors contributing to the species’ low rate of seed germination.

Otx2 and Oc1 directly regulate the transcriptional program of cone photoreceptor development

The vertebrate retina is a highly organized structure of approximately 110 cell types. Retinal progenitor cells (RPCs) produce these cell types in a temporal order that is highly conserved. While some RPCs produce many cell types, some terminally dividing RPCs produce restricted types of daughter cells, such as a cone photoreceptor and a horizontal cell (HC). Here, we compared the transcriptomes and chromatin profiles of such a restricted cone/HC RPC with those of other RPCs. We identified many cis-regulatory modules (CRMs) active in cone/HC RPCs and developing cones. We then showed that Otx2 and Oc1 directly regulate the activity of multiple CRMs genome-wide, including near genes important for cone development, such as Rxrg and Neurod1. In addition, we found that Otx2 regulates itself. These results suggest that Otx2 and Oc1 have a broader role than previously appreciated, and deepen our understanding of retinal development, which may benefit therapies for retinal diseases.