Functional characterization and genetic mapping of alk8

The novel type I TGFβ family member receptor alk8 is expressed both maternally and zygotically. Functional characterization of alk8 was performed using microinjection studies of constitutively active (CA), kinase modified/dominant negative (DN), and truncated alk8 mRNAs. CA Alk8 expression produces ventralized embryos while DN Alk8 expression results in dorsalized phenotypes. Truncated alk8 expressing embryos display a subtle dorsalized phenotype closely resembling that of the identified zebrafish dorsalized mutant, lost-a-fin (laf). Single-strand conformation polymorphism (SSCP) analysis was used to map alk8 to zebrafish LG02 in a region demonstrating significant conserved synteny to Hsa2, and which contains the human alk2 gene, ACVRI. Altogether, these functional, gene mapping and phylogenetic analyses suggest that alk8 may be the zebrafish orthologue to human ACVRI (alk2), and therefore extend previous studies of Alk2 conducted in Xenopus.     

Lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation

TGFbeta signaling pathways of the bone morphogenetic protein (BMP) subclass are essential for dorsoventral pattern formation of both vertebrate and invertebrate embryos. Here we determine by chromosomal mapping, linkage analysis, cDNA sequencing and mRNA rescue that the dorsalized zebrafish mutant lost-a-fin (laf) is defective in the gene activin receptor-like kinase 8 (alk8), which encodes a novel type I TGFbeta receptor. The alk8 mRNA is expressed both maternally and zygotically. Embyros that lack zygotic, but retain maternal Laf/Alk8 activity, display a weak dorsalization restricted to the tail and die by 3 days postfertilization. We rescued the laf dorsalized mutant phenotype by alk8 mRNA injection and generated homozygous laf/alk8 mothers to investigate the maternal role of Laf/Alk8 activity. Adult fish lacking Laf/Alk8 activity are fertile, exhibit a growth defect and are significantly smaller than their siblings. Embryos derived from homozygous females, which lack both maternal and zygotic Laf/Alk8 activity, display a strongly dorsalized mutant phenotype, no longer limited to the tail. These mutant embryos lack almost all gastrula ventral cell fates, with a concomitant expansion of dorsal cell types. During later stages, most of the somitic mesoderm and neural tissue circumscribe the dorsoventral axis of the embryo. Zygotic laf/alk8 mutants can be rescued by overexpression of the BMP signal transducer Smad5, but not the Bmp2b or Bmp7 ligands, consistent with the Laf/Alk8 receptor acting within a BMP signaling pathway, downstream of a Bmp2b/Bmp7 signal. Antibodies specific for the phosphorylated, activated form of Smad1/5, show that BMP signaling is nearly absent in gastrula lacking both maternal and zygotic Laf/Alk8 activity, providing further evidence that Laf/Alk8 transduces a BMP signal. In total, our work strongly supports the role of Laf/Alk8 as a type I BMP receptor required for the specification of ventral cell fates.     

The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish

In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.     

Two Pax2/5/8-binding sites in Engrailed2 are required for proper initiation of endogenous mid-hindbrain expression

During early brain development mouse Engrailed2 (En2) is expressed in a broad band across most of the mid-hindbrain region. Evidence from gene expression data, promoter analysis in transgenic mice and mutant phenotype analysis in mice and zebrafish has suggested that Pax2, 5 and 8 play a critical role in regulating En2 mid-hindbrain expression. Previously, we identified two Pax2/5/8-binding sites in a 1.0 kb En2 enhancer fragment that is sufficient to directed reporter gene expression to the early mid-hindbrain region and showed that the two Pax2/5/8-binding sites are essential for the mid-hindbrain expression in transgenic mice. In the present study we have examined the functional requirements of these two Pax2/5/8-binding sites in the context of the endogenous En2 gene for directing mid-hindbrain expression. The two Pax2/5/8-binding sites were deleted from the En2 locus and replaced with the bacterial neo gene by homologous recombination in mouse embryonic stem cells. After transmitting the mutation into mice, the neo gene was removed by breeding with transgenic mice expressing cre from a CMV promoter. Embryos homozygous for this En2 Pax2/5/8-binding site deletion mutation had a mild reduction in En2 expression in the presumptive mid-hindbrain region at the 5-7 somite stage, when En2 expression is normally initiated. However, from embryonic day 9.0 onwards, the mutant embryos showed En2 expression indistinguishable from that seen in wild type embryos. Furthermore, the mutants did not show the cerebellar defect seen in mice with a null mutation in En2. This result demonstrates that the two Pax2/5/8-binding sites that were deleted, while being required for mid-hindbrain expression in the context of a 1.0 kb En2 enhancer, are only required for proper initiation of expression of the endogenous En2 gene. Interestingly, a comparison of the lacZ RNA and protein expression patterns directed by the 1.0 kb enhancer fragment revealed that lacZ protein was acting as a lineage marker in the mid-hindbrain region by persisting longer than the mRNA. The transgene expression directed by the 1.0 kb enhancer fragment therefore does not mimic the entire broad domain of En2 expression. Taken together, these two studies demonstrate that DNA binding sites in addition to the two Pax2/5/8-binding sites must be necessary for En2 mid-hindbrain expression.     

Genetic evidence for involvement of maternally derived Wnt canonical signaling in dorsal determination in zebrafish

In zebrafish, the program for dorsal specification begins soon after fertilization. Dorsal determinants are localized initially to the vegetal pole, then transported to the blastoderm, where they are thought to activate the canonical Wnt pathway, which induces the expression of dorsal-specific genes. We identified a novel maternal-effect recessive mutation, tokkaebi (tkk), that affects formation of the dorsal axis. Severely ventralized phenotypes, including a lack of dorso-anterior structures, were seen in 5-100% of the embryos obtained from tkk homozygous transmitting females. tkk embryos displayed defects in the nuclear accumulation of beta-catenin on the dorsal side, and reduced or absent expression of dorsal-specific genes. Mesoderm and endoderm formation outside the dorsal axis was not significantly affected. Injection of RNAs for activated beta-catenin, dominant-negative forms of Axin1 and GSK3beta, and wild-type Dvl3, into the tkk embryos suppressed the ventralized phenotypes and/or dorsalized the embryos, and restored or induced an ectopic and expanded expression of bozozok/dharma and goosecoid. However, dorsalization by wnt RNAs was affected in the tkk embryos. Inhibition of cytoplasmic calcium release elicited an ectopic and expanded expression of chordin in the wild-type, but did not restore chordin expression efficiently in the tkk embryos. These data indicate that the tkk gene product functions upstream of or parallel to the beta-catenin-degradation machinery to control the stability of beta-catenin. The tkk locus was mapped to chromosome 16. These data provide genetic evidence that the maternally derived canonical Wnt pathway upstream of beta-catenin is involved in dorsal axis formation in zebrafish.     

Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation

A bone morphogenetic protein (BMP) signaling pathway acts in the establishment of the dorsoventral axis of the vertebrate embryo. Here we demonstrate the genetic requirement for two different Bmp ligand subclass genes for dorsoventral pattern formation of the zebrafish embryo. From the relative efficiencies observed in Bmp ligand rescue experiments, conserved chromosomal synteny, and isolation of the zebrafish bmp7 gene, we determined that the strongly dorsalized snailhouse mutant phenotype is caused by a mutation in the bmp7 gene. We show that the original snailhouse allele is a hypomorphic mutation and we identify a snailhouse/bmp7 null mutant. We demonstrate that the snailhouse/bmp7 null mutant phenotype is identical to the presumptive null mutant phenotype of the strongest dorsalized zebrafish mutant swirl/bmp2b, revealing equivalent genetic roles for these two Bmp ligands. Double mutant snailhouse/bmp7; swirl/bmp2b embryos do not exhibit additional or stronger dorsalized phenotypes, indicating that these Bmp ligands do not function redundantly in early embryonic development. Furthermore, overexpression experiments reveal that Bmp2b and Bmp7 synergize in the ventralization of wild-type embryos through a cell-autonomous mechanism, suggesting that Bmp2b/Bmp7 heterodimers may act in vivo to specify ventral cell fates in the zebrafish embryo.     

积雪草对早期糖尿病肾病大鼠TGF-β1表达及相关下游信号的影响

目的：通过研究积雪草（CA）对早期糖尿病肾病大鼠转化生长因子β₁（TGF-β₁）表达及相关下游信号的影响,阐明积雪草防治早期糖尿病肾病（DN）的分子机制。方法：60只雄性SD大鼠,按体重随机分为假手术组（n=10）和造模组（n=50）。造模组大鼠进行右肾切除术,1周后给以腹腔注射链脲佐菌素（STZ）30 mg/kg,连续给3 d;72 h后测血糖,以 ≥ 16.7 mmol/L,尿糖+++以上及尿量大于对照组的50%为DN模型成模标准。假手术组进行右肾被膜损伤,并注射相应量生理盐水。造模组通过灌胃给药,分为：DN模型组（模型组）、DN+福辛普利组（蒙组1.6 mg/kg·d）、DN+积雪草高剂量组（高剂量组16.8 mg/kg·d）、DN+积雪草中剂量组（中剂量组11.2 mg/kg·d）和DN+积雪草低剂量组（低剂量组5.6 mg/kg·d）（n=10）,连续给药16周,每日上午1次灌胃。利用实时荧光定量PCR和Western blot分别检测肾组织中TGF-β₁、TβR1、TβR2、Smad2/3、p-Smad2/3及Smad7 mRNA和蛋白的表达。结果：与假手术组相比,DN组TGF-β₁、TβR1、TβR2、Smad2/3 mRNA和蛋白表达及Smad2/3蛋白的磷酸化水平显著增加（P<0.05）、Smad7 mRNA和蛋白表达明显减少（P<0.05）,而福辛普利和高剂量积雪草能倒转DN引起的TGF-β₁、TβR1、TβR2、Smad2/3 mRNA和蛋白表达增加（P<0.05）及Smad7 mRNA和蛋白表达降低（P<0.05）。结论：积雪草可能通过调控TGF-β₁/Smad信号通路起到防治DN的作用。     

Inhibitor-resistant type I receptors reveal specific requirements for TGF-beta signaling in vivo

Activin/nodal-like TGF-beta superfamily ligands signal through the type I receptors Alk4, Alk5, and Alk7, and are responsible for mediating a number of essential processes in development. SB-431542, a chemical inhibitor of activin/nodal signaling, acts by specifically interfering with type I receptors. Here, we use inhibitor-resistant mutant receptors to examine the efficacy and specificity of SB-431542 in Xenopus and zebrafish embryos. Treatment with SB-431542 eliminates Smad2 phosphorylation in vivo and generates a phenotype very similar to those observed in genetic mutants in the nodal signaling pathway. Inhibitor-resistant Alk4 efficiently rescues Smad2 signaling, developmental phenotype, and marker gene expression after inhibitor treatment. This system was used to examine type I receptor specificity for several activin/nodal ligands. We find that Alk4 can efficiently rescue signaling by a wide range of ligands, while Alk7 can only weakly rescue signaling by the same ligands. In whole embryos, nodal signaling during gastrulation can be rescued with Alk4, but not Alk7, while Alk5 can only mediate signaling by ligands expressed later in development. The combination of the ALK inhibitor SB-431542 with inhibitor-resistant ALKs provides a powerful set of tools for examining nodal/activin signaling during embryogenesis.     

The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos

The pattern of RNA expression of the murine Dlx-2 (Tes-1) homeobox gene is described in embryos ranging in age from E8.5 through E11.5. Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. Dlx-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the Dlx genes.     

Expression of Zkrml2, a homologue of the Krml1/val segmentation gene, during embryonic patterning of the zebrafish (Danio rerio)

We have identified Zkrml2, a novel homologue of the segmentation gene Krml/val in zebrafish (Danio rerio). Zkrml2 shows 72% and 92% identity in its basic leucine zipper domain with mouse Krml1 and zebrafish val, respectively. Zkrml2 is expressed coincident with MyoD throughout the somites starting at the three somite stage, becomes restricted to the dermomyotome, and subsequently disappears. Transient expression is also detected in the reticulospinal and oculomotor neurons. Zkrml2 maps to the Oregon linkage group 11 (Boston Linkage group 14) with no mapped zebrafish mutations nearby.     

Mouse arylamine N-acetyltransferase 2 (Nat2) expression during embryogenesis: a potential marker for the developing neuroendocrine system

Arylamine N-acetyltransferase (NAT) genes in humans and in rodents encode polymorphic drug metabolizing enzymes. Human NAT1 (and the murine equivalent mouse Nat2) is found early in embryonic development and is likely to have an endogenous role. We report the detailed expression of the murine gene (Nat2) and encoded protein in mouse embryos, using a transgenic mouse model bearing a lacZ transgene inserted into the coding region of mouse Nat2. In mouse embryos, the transgene was expressed in sensory epithelia, epithelial placodes giving rise to visceral sensory neurons, the developing pituitary gland, sympathetic chain and urogenital ridge. In Nat2 ^+/+ mice, the presence and activity of Nat2 protein was detected in these tissues and their adult counterparts. Altered expression of the human orthologue in breast tumours, in which there is endocrine signalling, suggests that human NAT1 should be considered as a potential biomarker for neuroendocrine tissues and tumours.