Secondary Wall Formation Research Articles

Temporal water deficit and wood formation in Cryptomeria japonica.

Cell behavior in the cambium and developing xylem of 3-year-old Japanese cedar (Cryptomeria japonica D. Don.) trees, during and after an 11-day suspension of irrigation, was analyzed. Leaf xylem pressure potential and tangential strain of the stem surface were monitored throughout the experiment. Anatomical features and numbers of developing tracheids and cambial cells were observed in four trees, sampled on Days 0, 4, 8 and 11 after irrigation was suspended. Daytime xylem pressure potential decreased to -1.9 MPa on Day 7 and remained the same until irrigation was resumed on Day 11. The transverse dimensions of the tracheids, which began to form secondary walls, began to decrease on Day 4. The number of cells in the cambial zone and cell expansion zone decreased abruptly on Day 8. Tangentially aligned developing tracheids with collapsed cell walls were observed in samples harvested on Days 8 and 11. Secondary wall formation was recognized in these tracheids. After the resumption of irrigation, xylem pressure potential recovered rapidly to the same value as before the suspension of irrigation. Tangential strain increased within 30 min after the resumption of irrigation, and continued to increase until the onset of light the next day. Eighteen days after the resumption of irrigation, anatomical features of cells in the cambium and cell-expansion zone were similar to those observed before suspension of irrigation.

Growth and Differentiation of Root Endodermis in Primula acaulis Jacq.

Adventitious roots of Primula acaulis Jacq. are characterized by broad cortex and narrow stele during the primary development. Secondary thickening of roots occurs through limited cambial growth together with secondary dilatation growth of the persisting cortex. Close to the root tip, at a distance of ca. 4 mm from the apex, Casparian bands (state I of endodermal development) within endodermal cells develop synchronously. During late, asynchronous deposition of suberin lamellae (state II of endodermal development), a positional effect is clearly expressed - suberization starts in the cells opposite to the phloem sectors of the vascular cylinder at a distance of 30 - 40 mm from the root tip. The formation of secondary walls in endodermis (state III of endodermal development) correlates with the beginning of secondary growth of the root at a distance of ca. 60 mm. Endodermis is the only cortical layer of primrose, where not only cell enlargement but also renewed cell division participate in the secondary dilatation growth. The original endodermal cells additionally divide anticlinally only once. Newly-formed radial walls acquire a typical endodermal character by forming Casparian bands and deposition of suberin lamellae. A network of endodermal Casparian bands of equal density develops during the root thickening by the tangential expansion of cells and by the formation of new radial walls with characteristic wall modifications. These data are important since little attention has been paid up till now to the density of endodermal network as a generally significant structural and functional trait of the root.

Development of gelatinous (reaction) fibers in stems of Gnetum gnemon (Gnetales)

In extraxylary tissues of the stem Gnetum gnemon produces gelatinous fibers that can also function as reaction or tension fibers. These gelatinous fibers occur in all axes in the outer cortex and in displaced axes progressively in the middle and inner cortex and finally in the secondary phloem. Early cell differentiation in the cortex produces initials of laticifers that are unique in gymnosperms. Subsequently narrow fibers differentiate from cells that undergo both extensive passive elongation, as a result of internodal elongation, together with their active apical intrusive growth. Outer fibers always complete secondary wall development and become an important mechanical component of stems. Differentiation of fiber initials continues in the middle and inner cortex, but secondary wall formation can only be determined by a gravimorphic stimulus that produces eccentric development of fibers. Further eccentric development of fibers then continues in the outer secondary phloem from dedifferentiated phloem parenchyma cells that initially undergo extensive intrusive growth. All such cells have characteristic features of tension fibers of angiosperms. They exhibit a pronounced purely cellulosic innermost layer of the secondary wall (Sg layer). In addition, fiber initials are coenocytic, including up to eight nuclei that become distributed uniformly throughout the length of the cell. Mature macerated fibers are markedly brittle, making accurate length measurements difficult. Although cytologically uniform, these fibers thus originate from two kinds of initial (primary and secondary). They also differ in their response to a gravimorphic stimulus determined by their times of inception and their eccentric location. These cells show a suite of positional and gravimorphic responses that illustrate the complexity of plant cell differentiation.

Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis.

Cellulose synthase catalytic subunits (CesAs) have been implicated in catalyzing the biosynthesis of cellulose, the major component of plant cell walls. Interactions between CesA subunits are thought to be required for normal cellulose synthesis, which suggests that incorporation of defective CesA subunits into cellulose synthase complex could potentially cause a dominant effect on cellulose synthesis. However, all CesA mutants so far reported have been shown to be recessive in terms of cellulose synthesis. In the course of studying the molecular mechanisms regulating secondary wall formation in fibers, we have found that a mutant allele of AtCesA7 gene in the fra5 (fragile fiber 5) mutant causes a semidominant phenotype in the reduction of fiber cell wall thickness and cellulose content. The fra5 missense mutation occurred in a conserved amino acid located in the second cytoplasmic domain of AtCesA7. Overexpression of the fra5 mutant cDNA in wild-type plants not only reduced secondary wall thickness and cellulose content but also decreased primary wall thickness and cell elongation. In contrast, overexpression of the fra6 mutant form of AtCesA8 did not cause any reduction in cell wall thickness and cellulose content. These results suggest that the fra5 mutant protein may interfere with the function of endogenous wild-type CesA proteins, thus resulting in a dominant negative effect on cellulose biosynthesis.

168 Cell Wall Proteomics of the Green Alga Haematococcus Pluvialis (Chlorophyceae)

The green microalga, Haematococcus pluvialis, which can synthesize and accumulate large amounts of the ketocarotenoid astaxanthin, undergoes profound changes in cell wall composition and architecture during the cell cycle and in response to environmental stress. In this study, cell wall proteins (CWPs) of H. pluvialis were systematically analyzed by SDS‐PAGE coupled with peptide mass fingerprinting (PMF) and sequence‐database searching. In total, 163 protein bands were analyzed, which resulted in positive identification of 106 protein orthologues. The highly complex and dynamic composition of CWPs are manifested by the fact that the majority of identified CWPs are differentially expressed during specific stages of the cell cycle along with a number of common wall‐associated ‘housekeeping’ proteins. The detection of cellulose synthase orthologue in the vegetative cells suggested that the biosynthesis of cellulose occurred during primary wall formation. A transient accumulation of a putative cytokinin oxidase at the early stage of encystment pointed to a possible role in cytokinin degradation while facilitating secondary wall formation. This work represents the first attempt to use a proteomic approach to investigate CWPs of microalgae. The reference protein map constructed and specific protein markers obtained from this study provides a framework for future characterization of the expression and physiological functions of the putative proteins involved in the biogenesis and modifications of the cell wall of Haematococcus and other related organisms.

Cambial variants in the roots of Glinus lotoides L. and G. oppositifolius (L.) A. DC. (Molluginaceae)

Structure and development of successive cambia and their derivatives are studied histologically in the root of two species of Glinus (Molluginaceae). In both species first ring of cambium functions for a short time followed by the development of second ring from the cortical parenchyma. The subsequent cambial rings formed followed the similar pattern of development. The mature roots are composed of three to five successive rings of xylem alternating with phloem. Absence of ray initials in the cambium leads to the development of rayless xylem and phloem. Xylem consists of vessel elements, tracheids, libriform fibres, fibre tracheids and axial parenchyma. In transverse view vessels appear oval to circular and solitary in G. lotoides and angular, narrow and mostly in radial multiples in G. oppositifolius. Xylem fibres retain their nucleus even after the formation of secondary walls in both the species. Storage of starch in the lumen is a characteristic feature of fibres. The significance of successive cambia, rayless xylem and nucleated xylem fibres is discussed in relation to the plant habit.

Asymmetric expression of a poplar ACC oxidase controls ethylene production during gravitational induction of tension wood.

Ethylene is produced in wood-forming tissues, and when applied exogenously, it has been shown to cause profound effects on the pattern and rate of wood development. However, the molecular regulation of ethylene biosynthesis during wood formation is poorly understood. We have characterised an abundant 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene (PttACO1) in the wood-forming tissues of Populus tremula (L.) x P. tremuloides (Michx). PttACO1 is primarily expressed in developing secondary xylem, and is specifically upregulated during secondary wall formation. Nevertheless, according to GC-MS analysis combined with tangential cryosectioning, the distribution of ACC was found to be fairly uniform across the cambial-region tissues. Gravitational stimulation, which causes tension wood to form on the upper side of the stem, resulted in a strong induction of PttACO1 expression and ACC oxidase activity in the tension wood-forming tissues. The ACC levels increased in parallel to the PttACO1 expression. However, the increase on the upper (tension wood) side was only minor, whereas large amounts of both ACC and its hydrolysable conjugates accumulated on the lower (opposite) side of the stem. This suggests that the relatively low level of ACC on the tension wood side is a result of its conversion to ethylene by the highly upregulated PttACO1, and the concurrent accumulation of ACC on the opposite side of the wood is because of the low PttACO1 levels. We conclude that PttACO1 and ACC oxidase activity, but not ACC availability, are important in the control of the asymmetric ethylene production within the poplar stem when tension wood is induced by gravitational stimulation.

Three-Dimensional Imaging and Analysis of Differentiating Secondary Xylem by Confocal Microscopy

We examined the three-dimensional (3-D) structure of differentiating xylem in a hardwood tree, Kalopanax pictus, by confocallaser scanning microscopy (CLSM) using relatively thick, hand-cut histological sections. 3-D studies of plant tissues by mechanical serial sectioning with a microtome are very time-con suming. By contrast, the preparation of samples for CLSM is easier and the 3-D structure of intact tissue is preserved during optical sectioning. We obtained extended-focus images of the surface of specimens and these images resembled the stereographic images obtained by scanning electron microscopy. In addition , we observed radial files of cambial derivative cells at various stages of differenti ation and the internal structure along the 'z' axis of specimens on serial optical sections. We analysed the developmental changes in the morphology of cambial derivat ive cells, for example, the 3-D shape and arrangement of cells, the readjustment of the position of cells, and the development of secondary walls, pits and perforation plates. Our results showed that the arrangement of the differentiating xylem cells mirror s that of the cambial cell s. Deviations from the longitudinal orientation of vessel elements were specified by similar patterns of orientation of fusiform and ray cambial cells. The development of vessel elements progressed more rapidly than that of other xylem elements. When secondary walls with bordered pits and perforation plates with membranes were present in vessel elements and their expansion ceased, no secondary wall formation was detected in adjacent ray cells. The delay in secondary wall formation by the ray parenchyma cells, as compared to that by vessel elements, might facilitate the readju stment of the position of cells in the developing xylem tissue that is a consequence of the considerable expan sion of the vessel elements.

WALL STRUCTURE OF TERMINAL LATEWOOD TRACHEIDS OF HEALTHY AND DECLINING SILVER FIR TREES IN THE DINARIC REGION, SLOVENIA

Fir trees (Abies alba Mill.) in a permanently monitored forest in the Dinaric region in Slovenia respond to crown damages by distinctly reducing their ring widths. According to transmission electron microscopy (TEM) and UV-microspectrophotometry (UMSP) of cambium-adjacent latewood tracheids of affected trees, the secondary wall formation and lignification were completed by the middle of October. In samples taken at the same date from healthy looking silver firs, the S3 and the warty layer were not yet present in cambium-adjacent latewood tracheids. Additionally, their inner S2 showed lower lignin deposition, whereas the compound middle lamella, S1 and outer S2 were distinctly lignified as revealed by TEM and UMSP. It is assumed that these youngest tracheids of healthy trees will later undergo lignification or remain less lignified. From these observations we conclude that the cambial activity at breast height ends later in healthy silver fir trees as compared to declining trees.

Xyloglucan endotransglycosylases have a function during the formation of secondary cell walls of vascular tissues.

Xyloglucan transglycosylases (XETs) have been implicated in many aspects of cell wall biosynthesis, but their function in vascular tissues, in general, and in the formation of secondary walls, in particular, is less well understood. Using an in situ XET activity assay in poplar stems, we have demonstrated XET activity in xylem and phloem fibers at the stage of secondary wall formation. Immunolocalization of fucosylated xylogucan with CCRC-M1 antibodies showed that levels of this species increased at the border between the primary and secondary wall layers at the time of secondary wall deposition. Furthermore, one of the most abundant XET isoforms in secondary vascular tissues (PttXET16A) was cloned and immunolocalized to fibers at the stage of secondary wall formation. Together, these data strongly suggest that XET has a previously unreported role in restructuring primary walls at the time when secondary wall layers are deposited, probably creating and reinforcing the connections between the primary and secondary wall layers. We also observed that xylogucan is incorporated at a high level in the inner layer of nacreous walls of mature sieve tube elements.

Visualization by comprehensive microarray analysis of gene expression programs during transdifferentiation of mesophyll cells into xylem cells.

Plants have a unique transdifferentiation mechanism by which differentiated cells can initiate a new program of differentiation. We used a comprehensive analysis of gene expression in an in vitro zinnia (Zinnia elegans L.) culture model system to gather fundamental information about the gene regulation underlying the transdifferentiation of plant cells. In this model, photosynthetic mesophyll cells isolated from zinnia leaves transdifferentiate into xylem cells in a morphogenic process characterized by features such as secondary-wall formation and programmed cell death. More than 8,000 zinnia cDNA clones were isolated from an equalized cDNA library prepared from cultured cells transdifferentiating into xylem cells. Microarray analysis using these cDNAs revealed several types of unique gene regulation patterns, including: the transient expression of a set of genes during cell isolation, presumably induced by wounding; a rapid reduction in the expression of photosynthetic genes and the rapid induction of protein synthesis-associated genes during the first stage; the preferential induction of auxin-related genes during the subsequent stage; and the transient induction of genes closely associated with particular morphogenetic events, including cell-wall formation and degradation and programmed cell death during the final stage. This analysis also revealed a number of previously uncharacterized genes encoding proteins that function in signal transduction, such as protein kinases and transcription factors that are expressed in a stage-specific manner. These findings provide new clues to the molecular mechanisms of both plant transdifferentiation and wood formation.

Relationship of growth cessation with the formation of diferulate cross-links and p-coumaroylated lignins in tall fescue leaf blades.

We examined relationships among cell wall feruloylation, diferulate cross-linking, p-coumarate deposition, and apoplastic peroxidase (EC 1.11.1.7) activity with changes in the elongation rate of leaf blades of slow and rapid elongating genotypes of tall fescue ( Festuca arundinacea Schreb.). Growth was not directly influenced by ferulic acid deposition but leaf elongation decelerated as 8-5-, 8- O-4-, 8-8-, and 5-5-coupled diferulic acids accumulated in cell walls. Growth rapidly slowed and stopped with the deposition of p-coumarate, which is primarily associated with lignification in grass cell walls. Accretion of ferulate, diferulates and p-coumarate continued after growth ended, into the later stages of secondary wall formation. The concentration of 8-coupled diferulates dwarfed that of the more commonly measured 5-5-coupled isomer, suggesting that the latter dimer is a poor indicator of diferulate cross-linking in cell walls. Further work is required to clearly demonstrate the role of diferulate cross-linking and p-coumaroylated lignins in the cessation of leaf growth in grasses.

Immuno Electron Microscopic Localization of Peroxidases in the Differentiating Xylem of Populus spp.

Summary Peroxidases were localized in differentiating xylem cells of Populus spp. by means of immunogold labelling in combination with transmission electron microscopy (TEM). Polyclonal antibodies raised in rabbits against horseradish peroxidase were used for these experiments. Within the cytoplasm, TEM revealed a distinct labelling of the dictyosomes, indicating that these organelles are involved in the transport of peroxidases to the plasma membrane. During the formation of primary and secondary walls, cell corner regions became distinctly labelled, the developing secondary wall layers to a much lesser degree. Significant differences in the immunogold labelling between the intercorner middle lamella and the secondary cell wall layers were not observed. According to the immunogold labelling, peroxidases are incorporated into the secondary wall more or less simultaneously with deposition of polysaccharides. Correlations between peroxidase incorporation and lignification are discussed.

A small GTP-binding host protein is required for entry of powdery mildew fungus into epidermal cells of barley.

Small GTP-binding proteins such as those from the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture, secondary wall formation, meristem signaling, and defense against pathogens. We isolated a RacB homolog from barley (Hordeum vulgare) to study its role in resistance to the barley powdery mildew fungus (Blumeria graminis f.sp. hordei). RacB was constitutively expressed in the barley epidermis and its expression level was not strongly influenced by inoculation with B. graminis. However, after biolistic bombardment of barley leaf segments with RacB-double-stranded RNA, sequence-specific RNA interference with RacB function inhibited fungal haustorium establishment in a cell-autonomous and genotype-specific manner. Mutants compromised in function of the Mlo wild-type gene and the Ror1 gene (genotype mlo5 ror1) that are moderately susceptible to B. graminis showed no alteration in powdery mildew resistance upon RacB-specific RNA interference. Thus, the phenotype, induced by RacB-specific RNA interference, was apparently dependent on the same processes as mlo5-mediated broad resistance, which is suppressed by ror1. We conclude that an RAC small GTP-binding protein is required for successful fungal haustorium establishment and that this function may be linked to MLO-associated functions.

Transcript profiling takes a stroll through the wood

Xylogenesis, the process of wood formation, has fascinated biologists for many years. Not only is it an astonishing feat of natural structural engineering, providing the basis for water transport and support, essential prerequisites for the evolution of modern woody species, but wood is also an important industrial raw material. Its formation is a highly ordered, complex, developmental sequence starting with the division and expansion of cambial derivatives, followed by primary and secondary wall formation, lignification and ultimately cell death. Together these processes involve hundreds of genes, of which only a proportion have been identified and ascribed functions. In an elegant new study, Magnus Hertzberg and colleagues [1xA transcriptional roadmap to wood formation. Hertzberg, M. et al. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14732–14737Crossref | PubMed | Scopus (300)See all References[1] comprehensively profiled the transcript population across this developmental sequence, providing new insight into the genes governing the structure and synthesis of wood.The authors took 30-μm-thick tangential sections through the cambial region of poplar stems (Populus spp.), spanning five distinct developmental zones (A–E) from the meristem through to mature cells with secondary thickening. The transcript population in each sample was amplified and hybridized to a hybrid aspen (Populus tremula × P. tremuloides) cDNA microarray of expressed sequence tags. Over the sampled region, 1246 genes exhibited a fourfold differential in expression, whereas hierarchical cluster analysis revealed gene families subject to stage-specific developmental regulation with up to eightfold differentials in expression. The poplar genes ranged from those involved in cell-cycle control, including cyclins showing maximal expression in the cambial meristem and zone of early cell expansion (zones A–B), through enzymes influencing primary cell wall characteristics, such as pectin esterases and pectate lyases (zones A–C), to tubulins involved in the arrangement of the secondary cell wall (zone D) and phenylalanine ammonia-lyase, a key enzyme in lignin biosynthesis (zones D and E). Although many of the expressed sequences were homologous to previously identified genes from other species, their expression profiles reveal valuable information about their relative importance at different stages in xylogenesis and indicate possible unknown additional functions. For example, one of the cyclin homologues, cyclin A, was expressed not only in zones A and B, but throughout the entire developmental sequence, which suggests that it has additional roles in differentiation. Seventy-eight sequences that show no similarity to any database entries and 211 sequences that share significant homology with Arabidopsis genes of unknown function were also differentially expressed during xylem development. This finding illustrates the value of expression profiles that span complete developmental gradients because the kinetics of any given gene's profile should provide important clues as to its function. Furthermore, combining analysis of novel cell-specific transcript profiles with phenotypic analysis of the corresponding Arabidopsis mutants should facilitate functional analysis of these genes and identify new gene targets for the manipulation of wood properties.As the first comprehensive analysis of gene expression spanning an entire developmental process within a multicellular organism, this study represents a model system not only for further investigation of cell wall biosynthesis but also for several other developmental phenomena. The characterization of sequences expressed in the cambial meristem will, for example, facilitate the identification of cellular components that are involved in cell specification, whereas analysis of those expressed beyond the maturation of xylem vessels might help to reveal the molecular and biochemical basis of programmed cell death in plants.

Comparative subcellular immunolocation of polypeptides associated with xylan and callose synthases in French bean (Phaseolus vulgaris) during secondary wall formation

The Golgi apparatus of plant cells is thought to be the main site of synthesis of cell wall matrix polysaccharides and the terminal glycosylation of glycoproteins. Much of this evidence still depends on earlier biochemical studies employing subcellular fractionation. However acquiring pure Golgi membranes is still difficult and the question of spatial organisation of glycosyl transferases can be addressed by immunolocation of the enzymes. An antibody to a xylan synthase-associated polypeptide from French bean, the enzyme which synthesises the core polysaccharide for secondary wall xylan, has been raised and shown to inhibit its activity. Xylan is deposited in secondary thickenings and the xylan synthase was only detected in appreciable amounts in developing xylem cells. The location within the Golgi stack was observed throughout the dictyosomes. Some enzyme subunits were also detected in post-Golgi vesicles. A second antibody to a non-catalytic Mr 65 000 subunit of β 1,3- glucan (callose) synthase was used for a comparative study. Although the bulk of this enzyme has been detected in previous studies at plasmamembrane-wall interfaces in sieve plates and stressed tissue, a Golgi-location can be observed in root tip meristematic cells during cell plate formation. The enzyme was present throughout the stacks. Callose was also immunolocated in a similar manner to xylan in secondary walls and thickenings and in pits in developing xylem. In these cells, the callose synthase was detected at the surface of the growing thickenings and the plasmamembrane within the pits.

Interrelation between Lignin Deposition and Polysaccharide Matrices during the Assembly of Plant Cell Walls

Abstract: The modifications caused by genetic down‐regulation of the enzyme cinnamoyl CoA reductase (CCR) from monolignol biosynthetic pathways on tobacco and Arabidopsis thaliana were investigated at the ultrastructural level. A typical result was that the same transformation led to similar abnormality in secondary wall formation of fibres in both plants. The cell wall alterations mainly consisted in an important disorganization and loosening of cellulose microfibrils in the inner part of the S2 layer. This inability of the transformants to form a coherent cell wall coincided with a lack of synthesis of non‐condensed forms of lignin in this disorganized region of the wall, as demonstrated by immunolabelling of lignin subunits. A similar disorganization was observed during fibre wall formation in the differentiating tissues of young Populus and A. thaliana plants. The transitory lack of organization of cellulose microfibrils, also coincided with a depletion in non‐condensed forms of lignins. These results suggest that such lignin substructures may be involved in the cohesion of secondary walls during cell wall biogenesis. The mutual influence of the cellulose‐hemicellulose environment and monolignol local polymerization is discussed.

Differentiation of xylem cells in rolC transgenic aspen trees - a study of secondary cell wall development

Xylem cell differentiation of 35S-rolC transgenic aspen was analyzed and compared with wild type trees. The transgenic trees, main- ly characterized by dwarfism and physiological alterations, did not show distinctive differences in the structure and chemical composition of xy- lem cell walls as compared with the wild type trees. However, there was a difference in the dynamics of differentiation. In the transgenics the formation of xylem cells was delayed and the differentiation zone reduced to a few rows. PATAg and KMnO4 staining in electron microscopy as well as UV-microspectrophotometry revealed that in the transgenics during secondary wall formation polysaccharides and lignin were simulta- neously deposited close to the cambium. Immunocytochemical techniques have visualized the first deposition of condensed lignin in cell corners and of non-condensed lignin in secondary walls near cell corners. Because of altered formation and differentiation of xylem cells, 35S-rolC transgenic aspen may be useful to study factors controlling the differentiation continuum. Populus / rolC / wood formation / lignification / electron microscopy / immunogoldlabelling / UV-microspectrophotometry

Immunocytochemical Localization of Enzymes Involved in Lignification of the Cell Wall

-methyltransferase, and cinnnamyl alcohol dehydrogenase were localized to differentiating xylem. These enzymes are particularly abundant during secondary wall formation. Immunolabeling was observed on polysomes and in the cytosol of the cells during secondary wall formation, indicating that these enzymes are synthesized in the polysomes and released in the cytosol. The synthesis of monolignols might occur in the cytosol. Immunolabeling of anionic peroxidase was also localized to the differentiating xylem, particularly during secondary wall formation. The labeling, however, was observed in the rough endoplasmic reticulum (r-ER), the Golgi apparatus, and the plasma membrane, indicating that peroxidase is synthesized in the r-ER, transported to the Golgi apparatus, and localized on the plasma membrane by fusion of the Golgi vesicles to the membrane.

Hoxb2 and Hoxb4 Act Together to Specify Ventral Body Wall Formation

Three different alleles of the Hoxb4 locus were generated by gene targeting in mice. Two alleles contain insertions of a selectable marker in the first exon in either orientation, and, in the third, the selectable marker was removed, resulting in premature termination of the protein. Presence and orientation of the selectable marker correlated with the severity of the phenotype, indicating that the selectable marker induces cis effects on neighboring genes that influence the phenotype. Homozygous mutants of all alleles had cervical skeletal defects similar to those previously reported for Hoxb4 mutant mice. In the most severe allele, Hoxb4PolII, homozygous mutants died eitherin utero at approximately E15.5 or immediately after birth, with a severe defect in ventral body wall formation. Analysis of embryos showed thinning of the primary ventral body wall in mutants relative to control animals at E11.5, before secondary body wall formation. Prior to this defect, both Alx3 and Alx4 were specifically down regulated in the most ventral part of the primary body wall in Hoxb4PolII mutants. Hoxb4loxp mutants in which theneo gene has been removed did not have body wall or sternum defects. In contrast, both the Hoxb4PolII and the previously described Hoxb2PolII alleles that have body wall defects have been shown to disrupt the expression of bothHoxb2 and Hoxb4 in cell types that contribute to body wall formation. Our results are consistent with a model in which defects in ventral body wall formation require the simultaneous loss of at least Hoxb2 and Hoxb4, and may involve Alx3 and Alx4.