Zhong-Min Dai

Stage-specific regulation of oligodendrocyte development by Wnt/β-catenin signaling.

Oligodendrocytes are myelin-forming glia that ensheath the axons of neurons in the CNS. Recent studies have revealed that Wnt/β-catenin signaling plays important roles in oligodendrocyte development and myelin formation. However, there are conflicting reports on the specific function of Wnt signaling components in oligodendrocyte specification and differentiation. In the present study, we demonstrate that activation of β-catenin in neural progenitor cells before gliogenesis inhibits the generation of oligodendrocyte progenitors (OLPs) in mice. Once OLPs are formed, β-catenin becomes necessary for oligodendrocyte differentiation. Disruption of β-catenin signaling instead leads to a significant delay of oligodendrocyte maturation. These findings suggest that Wnt/β-catenin pathway regulates oligodendrocyte development in a stage-dependent manner.

BMP-FGF Signaling Axis Mediates Wnt-Induced Epidermal Stratification in Developing Mammalian Skin

Epidermal stratification of the mammalian skin requires proliferative basal progenitors to generate intermediate cells that separate from the basal layer and are replaced by post-mitotic cells. Although Wnt signaling has been implicated in this developmental process, the mechanism underlying Wnt-mediated regulation of basal progenitors remains elusive. Here we show that Wnt secreted from proliferative basal cells is not required for their differentiation. However, epidermal production of Wnts is essential for the formation of the spinous layer through modulation of a BMP-FGF signaling cascade in the dermis. The spinous layer defects caused by disruption of Wnt secretion can be restored by transgenically expressed Bmp4. Non-cell autonomous BMP4 promotes activation of FGF7 and FGF10 signaling, leading to an increase in proliferative basal cell population. Our findings identify an essential BMP-FGF signaling axis in the dermis that responds to the epidermal Wnts and feedbacks to regulate basal progenitors during epidermal stratification.

Gpr177-mediated Wnt Signaling is Required for Fungiform Placode Initiation

Fungiform papillae are formed as patterned rows on the surface of the anterior tongue at early organogenesis and contain one taste bud in each papilla to form one of the important sensory organs. Despite the essential role of Wnt/β-catenin signaling in controlling the development of fungiform taste papillae, the universal function of Wnt ligands in the initiation of the fungiform placode has not been completely elucidated. Here, by ShhCre -mediated oral epithelial deletion of Wntless (Gpr177), a regulator essential for intracellular Wnt trafficking, we demonstrate that an overall function of Wnts is required for initiation of the fungiform placode. Multiple Wnts are expressed in the tongue epithelium at E11.5 before initiation of the fungiform placodes. Epithelial Gpr177 loss-of-function, associated with reduction of canonical Wnt signaling in lingual epithelium as exhibited by a loss of TopGal activity and Axin2 expression, results in the failure of fungiform placode initiation, as assessed by diminished expression of several taste placode molecular markers. Moreover, LiCl treatment of Gpr177 epithelial-deficient tongue explants at E11.5, but not at E12.5, restores tongue placode formation, demonstrating that Wnt ligands in the tongue surface prior to but not after fungiform placode initiation are responsible for fungiform papilla initiation. Epithelium-specific expression of an active β-catenin in the Gpr177-deficient tongue leads to fungiform papillae generation, suggesting that an intra-epithelial response to Wnts is required for placode initiation. Together, these results suggest that Gpr177 controls epithelial initiation of the fungiform placode through signaling via epithelial Wnt ligands.

Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis

Traditional cut-paste DNA cloning is often limited by the availability of restriction enzyme sites. Here, we described the complementary annealing mediated by exonuclease (CAME), in which the insert or vector fragment is amplified to carry sequences complementary to the other, and both fragments are modified by exonuleases to create directional single-stranded overhangs. The two recessed DNA fragments are joined through complementary strand annealing. The CAME is highly efficient for cloning the DNA of at least 12 kb and single DNA fragment out of a complex DNA sample. Moreover, the application of CAME greatly improved the efficiency of site-directed mutagenesis.

Guanine-rich sequences inhibit proofreading DNA polymerases

DNA polymerases with proofreading activity are important for accurate amplification of target DNA. Despite numerous efforts have been made to improve the proofreading DNA polymerases, they are more susceptible to be failed in PCR than non-proofreading DNA polymerases. Here we showed that proofreading DNA polymerases can be inhibited by certain primers. Further analysis showed that G-rich sequences such as GGGGG and GGGGHGG can cause PCR failure using proofreading DNA polymerases but not Taq DNA polymerase. The inhibitory effect of these G-rich sequences is caused by G-quadruplex and is dose dependent. G-rich inhibitory sequence-containing primers can be used in PCR at a lower concentration to amplify its target DNA fragment.

T4 DNA polymerase improves the efficiency of multiple site-directed mutagenesis

ABSTRACT Site-directed mutagenesis (SDM) is a useful tool to study the functions of regulatory sequences of DNA and RNA, and the structures and functions of proteins. Numerous methods have been developed for either single or multiple SDM (MSDM). However, MSDM is sometimes difficult. Here we demonstrated that T4 DNA polymerase greatly enhanced the efficiency of MSDM. Moreover, we have also showed that it is efficient to clone multiple specific mutation-containing sequences simultaneously.

TT(N)mGCCTC inhibits archaeal family B DNA polymerases

The proofreading activity of the archaeal family B DNA polymerases enables PCR with high fidelity. However, thermostable proofreading DNA polymerases occasionally failed to amplify target fragment that could be amplified by Taq DNA polymerase. We have previously showed that G-rich sequences, which form G-quadruplex, can bind to and inhibit proofreading DNA polymerases. Here we showed that single-stranded oligonucleotides containing sequences of TT(N)mGCCTC can bind and inhibit archaeal family B DNA polymerases but not Taq DNA polymerase. It is very likely that TT(N)mGCCTC inhibits thermostable DNA polymerases during PCR in a single-stranded form. To the best of our knowledge, this is the first example of DNA sequence that could inhibit DNA polymerase in its single-stranded form.

Wntless, a conserved Wnt-transport protein, is involved in the innate immune response of Macrobrachium rosenbergii

&NA; Wnt signaling plays important roles in a variety of developmental and pathological processes. Here we show that Wntless, the main regulator for Wnt secretion, is involved in the innate immune response of the giant freshwater prawn, Macrobrachium rosenbergii. The full‐length cDNA of the prawn Wntless (named MrWntless) is 2173 bp in length and contains a 1602‐bp open reading frame (ORF), which is conceptually translated into a 533‐amino acids sequence. MrWntless protein contains a highly conserved Wnt‐binding domain which is required for secretion of Wnt ligands, and exhibits 57–67% identity with known Wntless proteins of other animals. MrWntless was found to be expressed in a variety of prawn tissues including heart, gill, muscle, gut, hepatopancreas and ovary. Moreover, MrWntless expression was significantly increased in the hepatopancreas and gill of the prawns challenged by the bacterial pathogen Aeromonas hydrophila and Vibrio parahaemolyticus. Knockdown of MrWntless by RNA interference in prawns led to dramatically decreased MrWntless expression of approximately 70%. Furthermore, the cumulative mortality rate of the prawn injected with MrWntless dsRNA was greatly increased in response to A. hydrophila challenge compared with the control prawns. Taken together, we provide evidence that prawn Wntless is important for their innate immune response against bacterial pathogens. HighlightsA Wntless homologue is isolated from Macrobrachium rosenbergii (termed MrWntless).Expression of MrWntless is found in a variety of prawn tissues.MrWntless expression is upregulated in prawn tissues upon bacterial infection.MrWntless plays a protective role in prawns against Aeromonas hydrophila infection.

Molecular cloning and expression analysis of a prawn ( Macrobrachium rosenbergii ) juvenile hormone esterase-like carboxylesterase following immune challenge

&NA; Methyl farnesoate (MF), the crustacean juvenile hormone (JH), plays critical roles in various physiological processes in crustaceans. The titer of MF is precisely regulated by specific carboxylesterase. Here, we report for the first time that the cloning and expression analysis of a JH esterase‐like carboxylesterase from the prawn Macrobrachium rosenbergii (named as MrCXE). MrCXE contained a 1935‐bp open reading frame (ORF) conceptually translated into a 644‐amino acids protein. MrCXE protein shared the highest identity (36%) with JH esterase‐like carboxylesterase from the swimming crab, Portunus trituberculatus and exhibited the typical motifs of JH esterase‐like carboxylesterases. MrCXE was most abundantly expressed in hepatopancreas, the major tissue for MF metabolism. MrCXE was expressed at a low level in gut and was not detected in other tissues. Additionally, MrCXE expression was upregulated in hepatopancreas by eyestalk ablation to increase MF level. Furthermore, the mRNA level of MrCXE was significantly increased in the hepatopancreas when challenged by the bacterial pathogens Aeromonas hydrophila and Vibrio parahaemolyticus. To our knowledge, this is the first report that the JH esterase‐like carboxylesterase is involved in the innate immune response of the crustaceans. HighlightsFull‐length cDNA of a juvenile hormone esterase‐like carboxylesterase (MrCXE) is isolated from Macrobrachium rosenbergii.MrCXE is most abundantly expressed in hepatopancreas, the major tissue for MF metabolism.MrCXE expression is upregulated in hepatopancreas upon Aeromonas hydrophila and Vibrio parahaemolyticus infection.

Disruption of Wnt production in Shh lineage causes bone malformation in mice, mimicking human Malik–Percin‐type syndactyly

Here, we show that Shh‐Cre‐mediated deletion of Wntless, the Wnt cargo protein, in mouse posterior limb mesenchyme causes bone syndactyly of the 3rd and 4th digits, resembling the human Malik–Percin type. The Shh descendants gradiently distributed from digit 5 to posterior half of digit 3 in wild‐type limbs, however, they abnormally increased in posterior digit 3 in WntlessShh‐Cre. WntlessShh‐Cre limbs displayed altered expression of hedgehog pathway genes and impaired noncanonical Wnt signaling activity. We further showed that the anterior limb mesenchymal cells in the WlsShh‐Cre served as a source of Wnt5a to reorientate the adjacent Wls‐lacking Shh lineage cells to move anteriorly and subsequently led to syndactyly, suggesting that aberrant mesenchymal cell movement/condensation may underlie the pathogenesis of syndactyly.