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Quantitative assessment of Hox complex expression in the indirect development of the polychaete annelid Chaetopterus sp.

A prediction from the set-aside theory of bilaterian origins is that pattern formation processes such as those controlled by the Hox cluster genes are required specifically for adult body plan formation. This prediction can be tested in animals that use maximal indirect development, in which the embryonic formation of the larva and the postembryonic formation of the adult body plan are temporally and spatially distinct. To this end, we quantitatively measured the amount of transcripts for five Hox genes in embryos of a lophotrochozoan, the polychaete annelid Chaetopterus sp. The polychaete Hox complex is shown not to be expressed during embryogenesis, but transcripts of all measured Hox complex genes are detected at significant levels during the initial stages of adult body plan formation. Temporal colinearity in the sequence of their activation is observed, so that activation follows the 3'-5' arrangement of the genes. Moreover, Hox gene expression is spatially localized to the region of teloblastic set-aside cells of the later-stage embryos. This study shows that an indirectly developing lophotrochozoan shares with an indirectly developing deuterostome, the sea urchin, a common mode of Hox complex utilization: construction of the larva, whether a trochophore or dipleurula, does not involve Hox cluster expression, but in both forms the complex is expressed in the set-aside cells from which the adult body plan derives.

Animals↗

Spatial transcriptomics of Ciona adult brains reveals functional zonalization and insights into neural gland function.

The ascidian Ciona is a pivotal chordate model for illuminating the evolutionary origins of the vertebrate brain. Here, spatial transcriptomics of the adult Ciona neural complex, combined with image-based computational super-resolution mapping, resolved distinct tissue domains including the cerebral ganglion, neural gland, ciliated funnel, neural gland duct/dorsal strand, and body wall muscle. Within the cerebral ganglion, high-resolution mapping revealed clear molecular zonalization separating the cortex and medulla, alongside regional specialization within the cortex itself. The neural gland exhibited localized enrichment of genes associated with extracellular matrix and cell-cell interactions. These spatial features suggest that the neural gland functions as a homeostatic and signaling interface, reminiscent of primitive vertebrate meninges or choroid plexus. Overall, this spatially defined gene expression map provides a foundational framework for understanding functional regionalization in the tunicate brain and its evolutionary relationship to vertebrate nervous systems.

Ciona↗

Two muscle-specific LIM proteins in Drosophila.

The LIM domain defines a zinc-binding motif found in a growing number of eukaryotic proteins that regulate cell growth and differentiation during development. Members of the cysteine-rich protein (CRP) family of LIM proteins have been implicated in muscle differentiation in vertebrates. Here we report the identification and characterization of cDNA clones encoding two members of the CRP family in Drosophila, referred to as muscle LIM proteins (Mlp). Mlp60A encodes a protein with a single LIM domain linked to a glycine-rich region. Mlp84B encodes a protein with five tandem LIM-glycine modules. In the embryo, Mlp gene expression is spatially restricted to somatic, visceral, and pharyngeal muscles. Within the somatic musculature, Mlp84B transcripts are enriched at the terminal ends of muscle fibers, whereas Mlp60A transcripts are found throughout the muscle fibers. The distributions of the Mlp60A and Mlp84B proteins mirror their respective mRNA localizations, with Mlp84B enrichment occurring at sites of muscle attachment. Northern blot analysis revealed that Mlp gene expression is developmentally regulated, showing a biphasic pattern over the course of the Drosophila life cycle. Peaks of expression occur late in embryogenesis and during metamorphosis, when the musculature is differentiating. Drosophila Mlp60A and Mlp84B, like vertebrate members of the CRP family, have the ability to associate with the actin cytoskeleton when expressed in rat fibroblast cells. The temporal expression and spatial distribution of muscle LIM proteins in Drosophila are consistent with a role for Mlps in myogenesis, late in the differentiation pathway.

Amino Acid Sequence↗

Regulatory analysis of the mouse Hoxb3 gene: multiple elements work in concert to direct temporal and spatial patterns of expression.

The expression pattern of the mouse Hoxb3 gene is exceptionally complex and dynamic compared with that of other members of the Hoxb cluster. There are multiple types of transcripts for Hoxb3 gene, and the anterior boundaries of its expression vary at different stages of development. Two enhancers flanking Hoxb3 on the 3' and 5' sides regulate Hoxb2 and Hoxb4, respectively, and these control regions define the two ends of a 28-kb interval in and around the Hoxb3 locus. To assay the regulatory potential of DNA fragments in this interval we have used transgenic analysis with a lacZ reporter gene to locate cis-elements for directing the dynamic patterns of Hoxb3 expression. Our detailed analysis has identified four new and widely spaced cis-acting regulatory regions that can together account for major aspects of the Hoxb3 expression pattern. Elements Ib, IIIa, and IVb control gene expression in neural and mesodermal tissues; element Va controls mesoderm-specific gene expression. The most anterior neural expression domain of Hoxb3 is controlled by an r5 enhancer (element IVa); element IIIa directs reporter expression in the anterior spinal cord and hindbrain up to r6, and the region A enhancer (in element I) mediates posterior neural expression. Hence, the regulation of segmental expression of Hoxb3 in the hindbrain is different from that of Hoxa3, as two separate enhancer elements contribute to expression in r5 and r6. The mesoderm-specific element (Va) directs reporter expression to prevertebra C1 at 12.5 dpc, which is the anterior limit of paraxial mesoderm expression for Hoxb3. When tested in combinations, these cis-elements appear to work as modules in an additive manner to recapitulate the major endogenous expression patterns of Hoxb3 during embryogenesis. Together our study shows that multiple control elements direct reporter gene expression in diverse tissue-, temporal-, and spatially restricted subset of the endogenous Hoxb3 expression domains and work in concert to control the neural and mesodermal patterns of expression.

Animals↗

Temporal and spatial relationships between segmentation and homeotic gene expression in Drosophila embryos: distributions of the fushi tarazu, engrailed, Sex combs reduced, Antennapedia, and Ultrabithorax proteins.

The specification of segment number and identity in the Drosophila embryo requires the activity of several classes of genes that may be grouped according to the array of pattern elements that they control. Double-label immunofluorescence was used to simultaneously localize the products of genes representative of the pair-rule segmentation class (fushi tarazu), the segment polarity class (engrailed), and the homeotic class (Sex combs reduced, Antennapedia, and Ultrabithorax) of pattern-regulating genes. The temporal order of appearance of each class of proteins and the precise spatial relationships between the products of the different genes are described with single-cell resolution. Boundaries of gene expression, particularly the parasegmental boundaries, are established by early-acting genes such as fushi tarazu and subsequently respected by the expression patterns of later appearing gene products such as engrailed and Ultrabithorax, suggesting regulatory relationships between certain pairs of genes. In addition, the dynamic transitions observed in spatial relationship among the Sex combs reduced, Antennapedia, and Ultrabithorax homeotic protein patterns during the early period of embryogenesis may reflect cross-regulatory interactions among these genes. Finally, some cells contain a single homeotic product, whereas other cells simultaneously contain several, suggesting that certain cells may be determined by the combinatorial action of homeotic genes.

Animals↗

Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena.

The spatially patterned differentiation of heterocysts in the filamentous cyanobacterium Anabaena requires a functional hetR gene. Transcriptional fusions to luxAB show that hetR is transcribed at a low level throughout the filament when Anabaena is grown with combined nitrogen, and that induction of the gene begins within 2 h following nitrogen deprivation. By 3.5 h, induction is localized to spaced foci. By 6 h, there is an overall induction of at least threefold in whole cultures, reflecting at least a 20-fold increase within spatially separated cells. The induction requires the presence of a functional hetR gene, indicating that hetR is autoregulatory. Full induction of a heterocyst structural gene, hepA, also requires a functional hetR locus.

ATP-Binding Cassette Transporters↗

Whorl-specific expression of the SUPERMAN gene of Arabidopsis is mediated by cis elements in the transcribed region.

The SUPERMAN (SUP) gene of Arabidopsis is involved in controlling cell proliferation in stamen and carpel primordia and in ovules during flower development. The SUP gene encodes a transcription factor with a C2H2-type zinc finger motif, a serine/proline-rich domain, a basic domain, and a leucine-zipper-like domain and is expressed in a very limited region in stamen primordia and in the developing ovary during flower development. The SUP gene is susceptible to methylation, resulting in epigenetic gene silencing. To understand how the SUP gene is expressed spatially and temporally in its restricted domain, and why methylation of the transcribed region affects early-stage SUP expression, we have identified the SUP cis regulatory elements by characterizing SUP gene fusions. These studies show that the SUP gene has discrete upstream promoter elements required for expression in stamen primordia in early stages and in the ovary in later stages. The promoter activity for stamen primordia is modulated by several positive and negative elements located in the transcribed and translated regions. Several regulatory elements in the transcribed region correlate with the areas of the gene that are heavily methylated in epigenetic alleles; these data provide a possible explanation of how methylation of the transcribed region represses transcription.

Arabidopsis↗

The role of the sporulation gene spoIIIE in the regulation of prespore-specific gene expression in Bacillus subtilis.

The spoIIIG gene encodes a sigma factor that determines prespore-specific gene expression during sporulation in Bacillus subtilis. Correct spatial and temporal expression of the spoIIIG gene depends on a number of other sporulation (spo) genes, but only one of these genes, spoIIIE, has a specific effect on spoIIIG expression and not on gene expression in the other differentiating cell, the mother cell. However, the spoIIIE gene is expressed predominantly before differentiation begins. Thus, its product must play an important role in sensing or determining the spatial localization of prespore-specific gene expression in this system.

Amino Acid Sequence↗

Neoadjuvant Immunotherapy Promotes the Formation of Mature Tertiary Lymphoid Structures in a Remodeled Pancreatic Tumor Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a rapidly progressing cancer that responds poorly to immunotherapies. Intratumoral tertiary lymphoid structures (TLS) have been associated with rare long-term PDAC survivors, but the role of TLS in PDAC and their spatial relationships within the context of the broader tumor microenvironment remain unknown. In this study, we report the generation of a spatial multiomic atlas of PDAC tumors and tumor-adjacent lymph nodes from patients treated with combination neoadjuvant immunotherapies. Using machine learning-enabled hematoxylin and eosin image classification models, imaging mass cytometry, and unsupervised gene expression matrix factorization methods for spatial transcriptomics, we characterized cellular states within and adjacent to TLS spanning distinct spatial niches and pathologic responses. Unsupervised learning identified TLS-specific spatial gene expression signatures that are significantly associated with improved survival in patients with PDAC. We identified spatial features of pathologic immune responses, including intratumoral TLS-associated B-cell maturation colocalizing with IgG dissemination and extracellular matrix remodeling. Our findings offer insights into the cellular and molecular landscape of TLS in PDACs during immunotherapy treatment.

Humans↗

Cartilage-derived morphogenetic proteins induce osteogenic gene expression in the C2C12 mesenchymal cell line.

Cartilage-derived morphogenetic protein-1, -2, and -3 (CDMP-1, -2, and -3) are members of the bone morphogenetic protein (BMP) family and have been shown to exhibit a variety of biological activities. In the present study, effects of these CDMPs on the temporal and spatial expression of genes in the pluripotent mesenchymal cell line C2C12 were examined. Cells cultured in the presence of CDMPs lost the characteristic elongated shape of myoblasts. At the molecular level, CDMP treatment did not change the mRNA expression of MyoD, aggrecan, Six1, and tendin. Scleraxis mRNA level was reduced by CDMP treatment. CDMP-1 and -3, but not CDMP-2, stimulated expression of osteogenic markers, such as alkaline phosphatase (AP), osteocalcin (OC), BSP, and type I collagen, in a dose- and time-dependent manner. With few exceptions, the three CDMPs changed, with different potencies, the expression profile of different members of the BMP family in a similar temporal pattern. Except at the late phase of treatment, CDMP treatment did not change the expression of ActR-IA, BMPR-IA, BMPR-IB, BMPR-II, and ALK-7 mRNAs. Based on the current data, the CDMPs appear to be able to stimulate the C2C12 cells to differentiate into the osteoblast pathway.

Animals↗

Inhibitory patterning of the anterior neural plate in Xenopus by homeodomain factors Dlx3 and Msx1.

Patterning of the embryonic ectoderm is dependent upon the action of negative (antineural) and positive (neurogenic) transcriptional regulators. Msx1 and Dlx3 are two antineural genes for which the anterior epidermal-neural boundaries of expression differ, probably due to differential sensitivity to BMP signaling in the ectoderm. In the extreme anterior neural plate, Dlx3 is strongly expressed while Msx1 is silent. While both of these factors prevent the activation of genes specific to the nascent central nervous system, Msx1 inhibits anterior markers, including Otx2 and cement gland-specific genes. Dlx3 has little, if any, effect on these anterior neural plate genes, instead providing a permissive environment for their expression while repressing more panneural markers, including prepattern genes belonging to the Zic family and BF-1. These properties define a molecular mechanism for translating the organizer-dependent morphogenic gradient of BMP activity into spatially restricted gene expression in the prospective anterior neural plate.

Animals↗

Dynamic distribution of Spatial during mouse spermatogenesis and its interaction with the kinesin KIF17b.

The Spatial gene is expressed in highly polarized cell types, such as epithelial cells in the thymus, neurons in the brain and germ cells in the testis. In this study, we report the characterization and distribution of Spatial proteins during mouse spermatogenesis. Besides Spatial-epsilon and -delta, we show that the newly described short isoform Spatial-beta is expressed specifically in round spermatids. Using indirect immunofluorescence, we detected Spatial in the cytosol of the early round spermatid. By the end stages of round spermatids, Spatial is concentrated at the opposite face of the acrosome near the nascent flagellum and in the manchette during the elongation process. Finally in mature sperm, Spatial persists in the principal piece of the tail. Moreover, we found that Spatial colocalizes with KIF17b, a testis-specific isoform of the brain kinesin-2 motor KIF17. This colocalization is restricted to the manchette and the principal piece of the sperm tail. Further, coimmunoprecipitation experiments of native proteins from testis lysates confirmed Spatial-KIF17b association through the long Spatial-epsilon isoform. Together, these findings imply a function of Spatial in spermatid differentiation as a new cargo of kinesin KIF17b, in a microtubule-dependent mechanism specific to the manchette and the principal piece of the sperm tail.

Animals↗

A homeobox gene, PRESSED FLOWER, regulates lateral axis-dependent development of Arabidopsis flowers.

It is postulated that the symmetric organization of plant lateral organs is based on two crossed axes, the abaxial-adaxial and the lateral axes. The PRESSED FLOWER (PRS) gene, the expression and function of which are dependent on the lateral axis, is reported in this study. In the prs mutant, growth of the lateral sepals is repressed, and although the size and shape of the abaxial and adaxial sepals are normal, the cell files at the lateral margins are missing. Double-mutant analyses showed that the PRS gene functions independently of the determinations of both floral organ identity and floral meristem size. The PRS gene, encoding a putative transcriptional factor with a homeodomain, was shown to be required for cell proliferation. PRS gene expression is spatially and temporally unique and is expressed in a restricted number of L1 cells at the lateral regions of flower primordia, floral organ primordia, and young leaf primordia. Our study strongly suggests that the PRS gene is involved in the molecular mechanism of lateral axis-dependent development of lateral organs in Arabidopsis.

Alleles↗

Hox genes, homology and axis formation--the application of morphological concepts to evolutionary developmental biology.

This article focuses on the interphyletic comparison of gene expression patterns. By means of the hypothesis of the inversion of the dorsoventral axis during the evolution of the Bilateria, it is demonstrated, that evolutionary developmental biologists use similarities in spatial and temporal gene expression patterns as evidence for the formulation of hypotheses of homology concerning either developing structures or body regions. The molecular genetic and morphogenetic evidence used is discussed within the framework of a cladistic-phylogenetic analysis based on the phylogenetic tree of the Bilateria. I argue that similarity of spatial and temporal gene expression patterns is not a sufficient criterion for homology inference. Therefore, gene expression patterns should be coded as characters. Their homology should be tested in concert with other characters. Furthermore, it is demonstrated, that spatial and temporal similar gene expression patterns, indicating similar molecular genetic mechanisms, were interpreted as an analytical criterion of homology, offering the possibility to identify similar structures. In contrast to this, the evolutionary developmental biologists have not developed a causal-analytically extended concept of shape, from which a causal-analytical concept of homology could be deduced. Instead, the homology concept from evolutionary morphology is used.

Animals↗

Differential transcription and translation of immediate early genes in the gerbil hippocampus after transient global ischemia.

Excitotoxic activation of glutamate receptors is thought to be a key event for the molecular pathogenesis of postischemic delayed neuronal death of CA-1 neurons in the gerbil hippocampus. Glutamate receptor stimulation also causes induction of transcription factors that belong to the class of immediate early genes. We examined the expression of six different immediate early genes in the gerbil hippocampus after transient global ischemia. Comparative analysis of c-fos and Krox-24 expression was carried out in the same animals at the transcriptional and translational level by in situ hybridization and immunocytochemistry. Postischemic synthesis of four additional immediate early gene (IEG)-encoded proteins (FOS-B, c-JUN, JUN-B, and JUN-D) was investigated by immunocytochemistry at recirculation intervals between 1 and 48 h. After 5 min of ischemia, transcription of c-fos and Krox-24 mRNA was induced in all hippocampal subpopulations with peak expression at 1 h after recirculation. In vulnerable CA-1 neurons, increased transcription of c-fos and Krox-24 was not followed by translation into protein. Induction of immediate early gene-encoded proteins was restricted to neuronal populations less vulnerable to brief ischemia and identified neurons that are targets of glutamate receptor-mediated neurotoxicity but that are destined to survive. Our data indicate an asynchronous synthesis and persistence of individual IEG-encoded proteins in these neurons. The staggered induction implies that combinatorial changes of transcription factors allow a differential postischemic regulation of target gene expression both spatially and over time.

Animals↗

Nod factor induction of reactive oxygen species production is correlated with expression of the early nodulin gene rip1 in Medicago truncatula.

Plant genes that are specifically activated by the rhizobial lipochitooligosaccharide signal molecule (Nod factor) in legume hosts are collectively referred to as nodulins. Although nodulin gene expression is both spatially and temporally correlated with symbiosis, the function of these genes and the molecular events underlying their expression remain unknown. Sequence analysis of rip1, an early nodulin gene encoding a putative peroxidase protein, revealed the existence of sequence motifs with homology to reactive oxygen species (ROS) responsive cis elements. Here we report that recognition of compatible Nod factor rapidly stimulates a spatially localized production of reactive oxygen species in legume roots. Sinorhizobium meliloti mutants that produce an altered Nod factor structure and a nonnodulating plant mutant, dmi1-1, that is implicated in Nod factor signal transduction are equally impaired in the ability to elicit ROS production and rip1 expression. Interestingly, both rip1 transcription and ROS production exhibit the same tissue-specific pattern of localization. Moreover, exogenous hydrogen peroxide is sufficient to activate rip1 transcription. Taken together, these results suggest that ROS production is a consequence of specific Nod factor perception and implicate H2O2 produced during this response as a mediator of Nod factor-induced rip1 expression.

Gene Expression Regulation, Developmental↗

Microarray analysis of gene expression on herbal glycoside recipes improving deficient ability of spatial learning memory in ischemic mice.

In order to reveal the mechanism of herbal glycoside recipes retrieving deficient ability of spatial learning memory in mice suffering from cerebral ischemia/reperfusion, a microarray system was used to analyze gene expression in those groups with increasing ability of spatial learning memory who were different from ischemic mice. In this work, we reported a comprehensive characterization of gene expression profiles of mouse hippocampus by the use of cDNA microarray system containing 1176 known genes in middle cerebral artery occlusion (MCAO) ischemic mice after treating with different dosage recipes of glycoside herbs (30, 90, and 270 mg/kg). The ability of spatial learning memory in ischemic mice was found to be decreased. The pathological process in ischemic mouse brain showed that a complex related to 100 genes' expression yielded 1.8-fold. Dose-dependent effects showed an improvement in the deficient ability and reduction in infarct volume when treated with glycoside recipes. Many genes (38-46) in expression were found greater than 1.8-fold in those effective recipes groups, including genes in cell cycle regulation, signal transduction, nerve system transcription factors, DNA binding protein, etc. Nine genes related to retrieving deficient ability of spatial learning memory treated with glycoside recipes were also found in this study. These results suggest that microarray analysis of gene expression might be useful for elucidating the mechanisms of pharmacological function of recipes.

14-3-3 Proteins↗

Gibberellin induces alpha-amylase gene in seed coat of Ipomoea nil immature seeds.

Two full-length cDNAs encoding gibberellin 3-oxidases, InGA3ox1 and InGA3ox2, were cloned from developing seeds of morning glory (Ipomoea nil (Pharbitis nil) Choisy cv. Violet) with degenerate-PCR and RACEs. The RNA-blot analysis for these clones revealed that the InGA3ox2 gene was organ-specifically expressed in the developing seeds at 6-18 days after anthesis. In situ hybridization showed the signals of InGA3ox2 mRNA in the seed coat, suggesting that active gibberellins (GAs) were synthesized in the tissue, although no active GA was detected there by immunohistochemistry. In situ hybridization analysis for InAmy1 (former PnAmy1) mRNA showed that InAmy1 was also synthesized in the seed coat. Both InGA3ox2 and InAmy1 genes were expressed spatially overlapped without a clear time lag, suggesting that both active GAs and InAmy1 were synthesized almost simultaneously in seed coat and secreted to the integument. These observations support the idea that GAs play an important role in seed development by inducing alpha-amylase.

Amino Acid Sequence↗