Proximal-distal Patterning Research Articles

Human lung branching morphogenesis is orchestrated by the spatiotemporal distribution of ACTA2, SOX2, and SOX9.

Lung morphogenesis relies on a number of important processes, including proximal-distal patterning, cell proliferation, migration and differentiation, as well as epithelial-mesenchymal interactions. In mouse lung development, SOX2+ cells are localized in the proximal epithelium, whereas SOX9+ cells are present in the distal epithelium. We show that, in human lung, expression of these transcription factors differs, in that during the pseudoglandular stage distal epithelial progenitors at the tips coexpress SOX2 and SOX9. This double-positive population was no longer present by the canalicular stages of development. As in mouse, the human proximal epithelial progenitors express solely SOX2 and are surrounded by smooth muscle cells (SMCs) both in the proximal airways and at the epithelial clefts. Upon Ras-related C3 botulinum toxin substrate 1 inhibition, we noted decreased branching, as well as increased SMC differentiation, attenuated peristalsis, and a reduction in the distal double-positive SOX2/SOX9 progenitor cell population. Thus, the presence of SOX2/SOX9 double-positive progenitor cells in the distal epithelium during the pseudoglandular stage of human lung development appears to be critical to proximal-distal patterning and lung branching. Moreover, SMCs promote a SOX2 proximal phenotype and seem to suppress the SOX9+ population.

Smoking-Dependent Distal-to-Proximal Repatterning of the Adult Human Small Airway Epithelium.

Small airways are the primary site of pathologic changes in chronic obstructive pulmonary disease (COPD), the major smoking-induced lung disorder. On the basis of the concept of proximal-distal patterning that determines regional specialization of the airway epithelium during lung development, we hypothesized that a similar program operates in the adult human lung being altered by smoking, leading to decreased regional identity of the small airway epithelium (SAE). The proximal and distal airway signatures were identified by comparing the transcriptomes of large and small airway epithelium samples obtained by bronchoscopy from healthy nonsmokers. The expression of these signatures was evaluated in the SAE of healthy smokers and smokers with COPD compared with that of healthy nonsmokers. The capacity of airway basal stem cells (BCs) to maintain region-associated phenotypes was evaluated using the air-liquid interface model. The distal and proximal airway signatures, containing 134 and 233 genes, respectively, were identified. These signatures included known developmental regulators of airway patterning, as well as novel regulators such as epidermal growth factor receptor, which was associated with the proximal airway phenotype. In the SAE of smokers with COPD, there was a dramatic smoking-dependent loss of the regional transcriptome identity with concomitant proximalization. This repatterning phenotype was reproduced by stimulating SAE BCs with epidermal growth factor, which was up-regulated in the SAE of smokers, during differentiation of SAE BCs in vitro. Smoking-induced global distal-to-proximal reprogramming of the SAE represents a novel pathologic feature of COPD and is mediated by exaggerated epidermal growth factor/epidermal growth factor receptor signaling in SAE BCs.

Retinoic acid regulates avian lung branching through a molecular network.

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rarα, and rarβ) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgfβ2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rarβ, meis2, hoxb5, tgfβ2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgfβ2, and id2 spatial distribution; to increase rarβ, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal-distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.

Gene expression reveals evidence for EGFR-dependent proximal-distal limb patterning in a myriapod.

Evolution of segmented limbs is one of the key innovations of Arthropoda, allowing development of functionally specific specialized head and trunk appendages, a major factor behind their unmatched evolutionary success. Proximodistal limb patterning is controlled by two regulatory networks in the vinegar fly Drosophila melanogaster, and other insects. The first is represented by the function of the morphogens Wingless (Wg) and Decapentaplegic (Dpp); the second by the EGFR-signaling cascade. While the role of Wg and Dpp has been studied in a wide range of arthropods representing all main branches, that is, Pancrustacea (= Hexapoda + Crustacea), Myriapoda and Chelicerata, investigation of the potential role of EGFR-signaling is restricted to insects (Hexapoda). Gene expression analysis of Egfr, its potential ligands, and putative downstream factors in the pill millipede Glomeris marginata (Myriapoda: Diplopoda), reveals that-in at least mandibulate arthropods-EGFR-signaling is likely a conserved regulatory mechanism in proximodistal limb patterning.

The apical ectodermal ridge of the mouse model of ectrodactyly Dlx5;Dlx6-/- shows altered stratification and cell polarity, which are restored by exogenous Wnt5a ligand.

The congenital malformation split hand/foot (SHFM) is characterized by missing central fingers and dysmorphology or fusion of the remaining ones. Type-1 SHFM is linked to deletions/rearrangements of the DLX5–DLX6 locus and point mutations in the DLX5 gene. The ectrodactyly phenotype is reproduced in mice by the double knockout (DKO) of Dlx5 and Dlx6. During limb development, the apical ectodermal ridge (AER) is a key-signaling center responsible for early proximal–distal growth and patterning. In Dlx5;6 DKO hindlimbs, the central wedge of the AER loses multilayered organization and shows down-regulation of FGF8 and Dlx2. In search for the mechanism, we examined the non-canonical Wnt signaling, considering that Dwnt-5 is a target of distalless in Drosophila and the knockout of Wnt5, Ryk, Ror2 and Vangl2 in the mouse causes severe limb malformations. We found that in Dlx5;6 DKO limbs, the AER expresses lower levels of Wnt5a, shows scattered β-catenin responsive cells and altered basolateral and planar cell polarity (PCP). The addition of Wnt5a to cultured embryonic limbs restored the expression of AER markers and its stratification. Conversely, the inhibition of the PCP molecule c-jun N-terminal kinase caused a loss of AER marker expression. In vitro, the addition of Wnt5a on mixed primary cultures of embryonic ectoderm and mesenchyme was able to confer re-polarization. We conclude that the Dlx-related ectrodactyly defect is associated with the loss of basoapical and PCP, due to reduced Wnt5a expression and that the restoration of the Wnt5a level is sufficient to partially reverts AER misorganization and dysmorphology.

Gremlin1 induces anterior–posterior limb bifurcations in developing Xenopus limbs but does not enhance limb regeneration

Gremlin1 (grem1) has been previously identified as being significantly up-regulated during regeneration of Xenopus laevis limbs. Grem1 is an antagonist of bone morphogenetic proteins (BMPs) with a known role in limb development in amniotes. It forms part of a self-regulating feedback loop linking epithelial (FGF) and mesenchymal (shh) signalling centres, thereby controlling outgrowth, anterior posterior and proximal distal patterning. Spatiotemporal regulation of the same genes in developing and regenerating Xenopus limb buds supports conservation of this mechanism. Using a heat shock inducible grem1 (G) transgene to created temperature regulated stable lines, we have shown that despite being upregulated in regeneration, grem1 overexpression does not enhance regeneration of tadpole hindlimbs. However, both the regenerating and contralateral, developing limb of G transgenics developed skeletal defects, suggesting that overexpressing grem1 negatively affects limb patterning. When grem1 expression was targeted earlier in limb bud development, we saw dramatic bifurcations of the limbs resulting in duplication of anterior posterior (AP) pattern, forming a phenotypic continuum ranging from duplications arising at the level of the femoral head to digit bifurcations, but never involving the pelvis. Intriguingly, the original limbs have AP pattern inversion due to de-restricted Shh signalling. We discuss a possible role for Grem1 regulation of limb BMPs in regulation of branching pattern in the limbs.

Hox5 Genes Regulate the Wnt2/2b-Bmp4-Signaling Axis during Lung Development.

Hox genes are required for proper anteroposterior axial patterning and the development of several organ systems. Here, we show that all three Hox5 paralogous genes play redundant roles in the developing lung. Hoxa5;Hoxb5;Hoxc5 triple-mutant embryos develop severely hypoplastic lungs with reduced branching and proximal-distal patterning defects. Hox5 genes are exclusively expressed in the lung mesoderm; however, defects are observed in both lung mesenchyme and endodermally derived epithelium, demonstrating that Hox5 genes act to regulate mesodermal-epithelial crosstalk during development. We show that Hox5 loss of function leads to loss of Wnt2/2b expression in the distal lung mesenchyme and the downregulation of previously identified downstream targets of Wnt2/2b signaling, including Lef1, Axin2, and Bmp4. Wnt2/2b-enriched media rescue proper Sox2/Sox9 patterning and restore Bmp4 expression in Hox5 triple-mutant lung explants. Taken together, these data show that Hox5 genes are key upstream mesenchymal regulators of the Wnt2/2b-Bmp4-signaling axis critical for proper lung patterning.

The complex of ASYMMETRIC LEAVES (AS) proteins plays a central role in antagonistic interactions of genes for leaf polarity specification in Arabidopsis

Leaf primordia are born around meristem‐containing stem cells at shoot apices, grow along three axes (proximal–distal, adaxial–abaxial, medial–lateral), and develop into flat symmetric leaves with adaxial–abaxial polarity. Axis development and polarity specification of Arabidopsis leaves require a network of genes for transcription factor‐like proteins and small RNAs. Here, we summarize present understandings of adaxial‐specific genes, ASYMMETRIC LEAVES1 (AS1) and AS2. Their complex (AS1–AS2) functions in the regulation of the proximal–distal leaf length by directly repressing class 1 KNOX homeobox genes (BP, KNAT2) that are expressed in the meristem periphery below leaf primordia. Adaxial–abaxial polarity specification involves antagonistic interaction of adaxial and abaxial genes including AS1 and AS2 for the development of two respective domains. AS1–AS2 directly represses the abaxial gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3) and indirectly represses ETT/ARF3 and ARF4 through tasiR‐ARF. Modifier mutations have been identified that abolish adaxialization and enhance the defect in the proximal–distal patterning in as1 and as2. AS1–AS2 and its modifiers synergistically repress both ARFs and class 1 KNOXs. Repression of ARFs is critical for establishing adaxial–abaxial polarity. On the other hand, abaxial factors KANADI1 (KAN1) and KAN2 directly repress AS2 expression. These data delineate a molecular framework for antagonistic gene interactions among adaxial factors, AS1, AS2, and their modifiers, and the abaxial factors ARFs as key regulators in the establishment of adaxial–abaxial polarity. Possible AS1–AS2 epigenetic repression and activities downstream of ARFs are discussed. WIREs Dev Biol 2015, 4:655–671. doi: 10.1002/wdev.196For further resources related to this article, please visit the WIREs website.

The barley Uniculme4 gene encodes a BLADE-ON-PETIOLE-like protein that controls tillering and leaf patterning.

Tillers are vegetative branches that develop from axillary buds located in the leaf axils at the base of many grasses. Genetic manipulation of tillering is a major objective in breeding for improved cereal yields and competition with weeds. Despite this, very little is known about the molecular genetic bases of tiller development in important Triticeae crops such as barley (Hordeum vulgare) and wheat (Triticum aestivum). Recessive mutations at the barley Uniculme4 (Cul4) locus cause reduced tillering, deregulation of the number of axillary buds in an axil, and alterations in leaf proximal-distal patterning. We isolated the Cul4 gene by positional cloning and showed that it encodes a BROAD-COMPLEX, TRAMTRACK, BRIC-À-BRAC-ankyrin protein closely related to Arabidopsis (Arabidopsis thaliana) BLADE-ON-PETIOLE1 (BOP1) and BOP2. Morphological, histological, and in situ RNA expression analyses indicate that Cul4 acts at axil and leaf boundary regions to control axillary bud differentiation as well as the development of the ligule, which separates the distal blade and proximal sheath of the leaf. As, to our knowledge, the first functionally characterized BOP gene in monocots, Cul4 suggests the partial conservation of BOP gene function between dicots and monocots, while phylogenetic analyses highlight distinct evolutionary patterns in the two lineages.

RING1 proteins contribute to early proximal-distal specification of the forelimb bud by restricting Meis2 expression.

Polycomb group (PcG) proteins play a pivotal role in silencing developmental genes and help to maintain various stem and precursor cells and regulate their differentiation. PcG factors also regulate dynamic and complex regional specification, particularly in mammals, but this activity is mechanistically not well understood. In this study, we focused on proximal-distal (PD) patterning of the mouse forelimb bud to elucidate how PcG factors contribute to a regional specification process that depends on developmental signals. Depletion of the RING1 proteins RING1A (RING1) and RING1B (RNF2), which are essential components of Polycomb repressive complex 1 (PRC1), led to severe defects in forelimb formation along the PD axis. We show that preferential defects in early distal specification in Ring1A/B-deficient forelimb buds accompany failures in the repression of proximal signal circuitry bound by RING1B, including Meis1/2, and the activation of distal signal circuitry in the prospective distal region. Additional deletion of Meis2 induced partial restoration of the distal gene expression and limb formation seen in the Ring1A/B-deficient mice, suggesting a crucial role for RING1-dependent repression of Meis2 and likely also Meis1 for distal specification. We suggest that the RING1-MEIS1/2 axis is regulated by early PD signals and contributes to the initiation or maintenance of the distal signal circuitry.

Sp6 and Sp8 transcription factors control AER formation and dorsal-ventral patterning in limb development.

The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. The induction of the AER is a complex process that relies on integrated interactions among the Fgf, Wnt, and Bmp signaling pathways that operate within the ectoderm and between the ectoderm and the mesoderm of the early limb bud. The transcription factors Sp6 and Sp8 are expressed in the limb ectoderm and AER during limb development. Sp6 mutant mice display a mild syndactyly phenotype while Sp8 mutants exhibit severe limb truncations. Both mutants show defects in AER maturation and in dorsal-ventral patterning. To gain further insights into the role Sp6 and Sp8 play in limb development, we have produced mice lacking both Sp6 and Sp8 activity in the limb ectoderm. Remarkably, the elimination or significant reduction in Sp6;Sp8 gene dosage leads to tetra-amelia; initial budding occurs, but neither Fgf8 nor En1 are activated. Mutants bearing a single functional allele of Sp8 (Sp6−/−;Sp8+/−) exhibit a split-hand/foot malformation phenotype with double dorsal digit tips probably due to an irregular and immature AER that is not maintained in the center of the bud and on the abnormal expansion of Wnt7a expression to the ventral ectoderm. Our data are compatible with Sp6 and Sp8 working together and in a dose-dependent manner as indispensable mediators of Wnt/βcatenin and Bmp signaling in the limb ectoderm. We suggest that the function of these factors links proximal-distal and dorsal-ventral patterning.

Cdon acts as a Hedgehog decoy receptor during proximal-distal patterning of the optic vesicle.

Patterning of the vertebrate optic vesicle into proximal/optic stalk and distal/neural retina involves midline-derived Hedgehog (Hh) signalling, which promotes stalk specification. In the absence of Hh signalling, the stalks are not specified, causing cyclopia. Recent studies showed that the cell adhesion molecule Cdon forms a heteromeric complex with the Hh receptor Patched 1 (Ptc1). This receptor complex binds Hh and enhances signalling activation, indicating that Cdon positively regulates the pathway. Here we show that in the developing zebrafish and chick optic vesicle, in which cdon and ptc1 are expressed with a complementary pattern, Cdon acts as a negative Hh signalling regulator. Cdon predominantly localizes to the basolateral side of neuroepithelial cells, promotes the enlargement of the neuroepithelial basal end-foot and traps Hh protein, thereby limiting its dispersion. This Ptc-independent function protects the retinal primordium from Hh activity, defines the stalk/retina boundary and thus the correct proximo-distal patterning of the eye.

Distinct requirements for beta-catenin in pancreatic epithelial growth and patterning

Pancreatic exocrine and endocrine lineages arise from multipotent pancreatic progenitor cells (MPCs). Exploiting the mechanisms that govern expansion and differentiation of these cells could enhance efforts to generate β-cells from stem cells. Although our prior work indicates that the canonical Wnt signaling component β-catenin is required qualitatively for exocrine acinar but not endocrine development, precisely how this requirement plays out at the level of MPCs and their lineage-restricted progeny is unknown. In addition, the contribution of β-catenin function to β-cell development remains controversial. To resolve the potential roles of β-catenin in development of MPCs and β-cells, we generated pancreas- and pre-endocrine-specific β-catenin knockout mice. Pancreas-specific loss of β-catenin produced not only a dramatic reduction in acinar cell numbers, but also a significant reduction in β-cell mass. The loss of β-cells is due not to a defect in the differentiation of endocrine precursors, but instead correlates with an early and specific loss of MPCs. In turn, this reflects a novel role for β-catenin in maintaining proximal–distal patterning of the early epithelium, such that distal MPCs resort to a proximal, endocrine-competent “trunk” fate when β-catenin is deleted. Moreover, β-catenin maintains proximal–distal patterning, in part, by inhibiting Notch signaling. Subsequently, β-catenin is required for proliferation of both distal and proximal cells, driving overall organ growth. In distinguishing two distinct roles for β-catenin along the route of β-cell development, we suggest that temporally appropriate positive and negative manipulation of this molecule could enhance expansion and differentiation of stem cell-derived MPCs.

Multiple roles of the gene zinc finger homeodomain-2 in the development of the Drosophila wing

The gene zfh2 and its human homolog Atbf1 encode huge molecules with several homeo- and zinc finger domains. It has been reported that they play important roles in neural differentiation and promotion of apoptosis in several tissues of both humans and flies. In the Drosophila wing imaginal disc, Zfh2 is expressed in a dynamic pattern and previous results suggest that it is involved is proximal–distal patterning. In this report we go further in the analysis of the function of this gene in wing development, performing ectopic expression experiments and studying its effects in genes involved in wing development. Our results suggest that Zfh2 plays an important role controlling the expression of several wing genes and in the specification of those cellular properties that define the differences in cell proliferation between proximal and distal domains of the wing disc.

Orbital and solar forcing of shifts in Mid- to Late Holocene flood intensity from varved sediments of pre-alpine Lake Ammersee (southern Germany)

Microfacies analyses and X-ray fluorescence scanning (μ-XRF) at sub-mm resolution were conducted on the varved Mid- to Late Holocene interval of two sediment profiles from pre-alpine Lake Ammersee (southern Germany). The coring sites are located in a proximal (AS10prox) and distal (AS10dist) position towards the main tributary River Ammer, in 1.8 km distance from each other. To shed light on sediment distribution within the lake, particular emphasis was (1) the detection of intercalated detrital layers and their micro-sedimentological features, and (2) intra-basin correlation of these event deposits. Detrital layers were dated by microscopic varve counting, verified by accelerator mass spectrometry 14C dating of terrestrial plant macrofossils.Since ∼5500 varve years (vyr) BP, in total 1573 detrital layers were detected in either one or both of the investigated sediment profiles. Based on their microfacies, geochemistry, and proximal-distal deposition pattern, detrital layers were interpreted as River Ammer flood deposits. Earlier studies on flood layer seasonality have proven that flood layer deposition occurs predominantly during spring and summer, the flood season at Lake Ammersee. Most prominent features of the record are the onset of regular flood layer deposition at ∼5500 vyr BP in AS10prox and ∼2800 vyr BP in AS10dist as well as three major increases in mean flood layer thickness at ∼5500, 2800, and 400 vyr BP. Integrating information from both sediment profiles allowed to interpret these changes in terms of shifts towards higher mean flood intensity. Proposed triggering mechanisms are gradual reduction in Northern Hemisphere orbital summer forcing and superimposed centennial-scale solar activity minima. Likely responses to this forcing are enhanced equator-to-pole temperature gradients and changes in synoptic-scale atmospheric circulation. The consequences for the Ammersee region are more intense cyclones leading to extremer rainfall and flood events in spring and summer.

The Liguleless narrow mutation affects proximal-distal signaling and leaf growth

How cells acquire competence to differentiate according to position is an essential question in developmental biology. Maize leaves provide a unique opportunity to study positional information. In the developing leaf primordium, a line is drawn across a field of seemingly identical cells. Above the line, the cells become blade, below the line the cells become sheath and at the line, the cells differentiate into the specialized tissues of ligule and auricle. We identified a new mutation, Liguleless narrow (Lgn), that affects this patterning and shows striking defects in lateral growth as well, thus linking proximal-distal patterning to medial-lateral growth. In characterizing the defect we discovered that both the auxin transport protein ZmPIN1a and the squamosa promoter-binding protein LIGULELESS1 are expressed precisely at this positionally cued line and are disrupted by Lgn. Positional cloning and a transposon-derived allele demonstrate that LGN is a kinase. These results suggest that LGN participates in setting up positional information through a signaling cascade. Interestingly, LGN has a paralog that is upregulated in the mutant, suggesting an important feedback mechanism involved in setting the positional boundary.

Egfr signaling regulates distal as well as medial fate in the embryonic leg of Tribolium castaneum

The evolution of a mechanism to generate a proximal–distal axis perpendicular to the anterior–posterior body axis was a key event in arthropod evolution and facilitated the formation of multi-functional limbs. The study of proximodistal limb patterning in extant arthropods can provide insight into the origin and evolution of the proximal–distal axis. In Drosophila melanogaster, proximal–distal patterning is mainly organized by Wg/Dpp signaling. Egfr signaling is also involved, but is restricted to late stages and distal leg parts (tarsus and pretarsus). Here we study the role of Epidermal growth factor receptor (Egfr), spitz (spi), and pointed (pnt) in leg development in the beetle Tribolium castaneum. We show that Egfr signaling has a more complex role in T. castaneum than in D. melanogaster and is not only required in the distal leg, but is also involved in formation of the medial leg (including femur and tibia). Egfr and spi are required for the regulation of clawless (cll), Distal-less (Dll) and dachshund (dac), and RNAi leads to thickened and fused leg segments. Intriguingly, regulation of Dll and dac by Egfr signaling appears functionally separate from its role in cll regulation, because it is not mediated by the transcription factor Pnt. This suggests that Egfr signaling has a dual role with separate mediators in proximodistal axis patterning. While the regulation of distal factors like cll is evolutionarily conserved, regulation of Dll and dac appears to function in parallel or redundant with Wg/Dpp signaling, thus providing a possible explanation why this role is less evolutionarily conserved in the insects.

Numb Expression and Asymmetric versus Symmetric Cell Division in Distal Embryonic Lung Epithelium

Proper balance between self-renewal and differentiation of lung-specific progenitors is absolutely required for normal lung morphogenesis/regeneration. Therefore, understanding the behavior of lung epithelial stem/progenitor cells could identify innovative solutions for restoring normal lung morphogenesis and/or regeneration. The Notch inhibitor Numb is a key determinant of asymmetric or symmetric cell division and hence cell fate. Yet Numb proximal-distal expression pattern and symmetric versus asymmetric division are uncharacterized during lung epithelial development. Herein, the authors find that the cell fate determinant Numb is highly expressed and asymmetrically distributed at the apical side of distal epithelial progenitors and segregated to one daughter cell in most mitotic cells. Knocking down Numb in MLE15 epithelial cells significantly increased the number of cells expressing the progenitor cell markers Sox9/Id2. Furthermore, cadherin hole analysis revealed that most distal epithelial stem/progenitor cells in embryonic lungs divide asymmetrically; with their cleavage, planes are predicted to bypass the cadherin hole, resulting in asymmetric distribution of the cadherin hole to the daughter cells. These novel findings provide evidence for asymmetric cell division in distal epithelial stem/progenitor cells of embryonic lungs and a framework for future translationally oriented studies in this area.

Proximal–distal patterning of the vertebrate limb is initiated by altered exposure to secreted signals

Proximal–distal patterning of the vertebrate limb is initiated by altered exposure to secreted signals

Expression patterns of two Arabidopsis endo-β-1,4-glucanase genes (At3g43860, At4g39000) in reproductive development

Endo-beta-1,4-D-glucanases (EGases) are a widespread and vital group of glycosyl hydrolases that generally break the beta-1,4-glucosyl linkages. Studies of plant EGases have mainly been concentrated on vegetative growth, while little is currently known about their role in reproductive processes. Using the GUS reporter aided analysis of promoter activities, we identified the expression patterns of two putative Arabidopsis EGases genes (At3g43860 and At4g39000) whose promoters conferred specific localization of the GUS activity in reproductive organs. We found that At3g43860, which is similar to KOR in its protein structural organization, is expressed in mature pollen and the pollen tube, implying that it may have a role in pollen and pollen tube growth. At4g39000 was found to be activated in the developing ovules and seeds, especially at the micropylar end of the inner integuments and nucellus in a proximal-distal pattern. Our results suggested that the two EGases play specific roles in Arabidopsis sexual reproduction.