Nuclear LIM Research Articles

Lhx4 surpasses its paralog Lhx3 in promoting the differentiation of spinal V2a interneurons

Paralog factors are considered to ensure the robustness of biological processes by providing redundant activity in cells where they are co-expressed. However, the specific contribution of each factor is frequently underestimated. In the developing spinal cord, multiple families of transcription factors successively contribute to differentiate an initially homogenous population of neural progenitors into a myriad of neuronal subsets with distinct molecular, morphological, and functional characteristics. The LIM-homeodomain transcription factors Lhx3, Lhx4, Isl1 and Isl2 promote the segregation and differentiation of spinal motor neurons and V2 interneurons. Based on their high sequence identity and their similar distribution, the Lhx3 and Lhx4 paralogs are considered to contribute similarly to these processes. However, the specific contribution of Lhx4 has never been studied. Here, we provide evidence that Lhx3 and Lhx4 are present in the same cell populations during spinal cord development. Similarly to Lhx3, Lhx4 can form multiproteic complexes with Isl1 or Isl2 and the nuclear LIM interactor NLI. Lhx4 can stimulate a V2-specific enhancer more efficiently than Lhx3 and surpasses Lhx3 in promoting the differentiation of V2a interneurons in chicken embryo electroporation experiments. Finally, Lhx4 inactivation in mice results in alterations of differentiation of the V2a subpopulation, but not of motor neuron production, suggesting that Lhx4 plays unique roles in V2a differentiation that are not compensated by the presence of Lhx3. Thus, Lhx4 could be the major LIM-HD factor involved in V2a interneuron differentiation during spinal cord development and should be considered for in vitro differentiation of spinal neuronal populations.

17q12 Microdeletion Syndrome as a Rare Cause of Elevated Liver Enzymes: Case Report and Literature Review

17q12 deletion syndrome is a rare autosomal dominant inherited condition. It results from de novo mutation and can occur without a family history. Hepatocyte nuclear factor-1 beta (HNF1B) and LIM homeobox 1 (LXH1) genes are the most common genes to be deleted in this syndrome. It has unique clinical characteristics involving multiple systems in the body. The most common presentations are usually renal involvement and maturity-onset diabetes of the young type 5 (MODY5). Genetic study is the golden tool to diagnose patients with this syndrome. Our case presents the unique clinical features of 17q12 deletion syndrome along with a literature review.

Cytoplasmic LMO2-LDB1 Complex Activates STAT3 Signaling through Interaction with gp130-JAK in Glioma Stem Cells.

The oncogenic role of nuclear LIM domain only 2 (LMO2) as a transcriptional regulator is well established, but its function in the cytoplasm is largely unknown. Here, we identified LMO2 as a cytoplasmic activator for signal transducer and activator of transcription 3 (STAT3) signaling in glioma stem cells (GSCs) through biochemical and bioinformatics analyses. LMO2 increases STAT3 phosphorylation by interacting with glycoprotein 130 (gp130) and Janus kinases (JAKs). LMO2-driven activation of STAT3 signaling requires the LDB1 protein and leads to increased expression of an inhibitor of differentiation 1 (ID1), a master regulator of cancer stemness. Our findings indicate that the cytoplasmic LMO2-LDB1 complex plays a crucial role in the activation of the GSC signaling cascade via interaction with gp130 and JAK1/2. Thus, LMO2-LDB1 is a bona fide oncogenic protein complex that activates either the JAK-STAT signaling cascade in the cytoplasm or direct transcriptional regulation in the nucleus.

Cloning, characterization and subcellular localization of Nuclear LIM interactor interacting factor gene from Leishmania donovani

LIM domains are zinc-binding motifs that mediate protein–protein interactions and are found in a wide variety of cytoplasmic and nuclear proteins. The nuclear LIM domain family members have a number of different functions including transcription factors, gene regulation, cell fate determination, organization of the cytoskeleton and tumour formation exerting their function through various LIM domain interacting protein partners/cofactors. Nuclear LIM domain interacting proteins/factors have not been reported in any protozoan parasites including Leishmania. Here, we report for the first time cloning, characterization and subcellular localization of nuclear LIM interactor-interacting factor (NLI) like protein from Leishmania donovani, the causative agent of Indian Kala-azar. Primary sequence analysis of LdNLI revealed presence of characteristic features of nuclear LIM interactor-interacting factor. However, leishmanial NLI represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. The sub-cellular distribution of LdNLI revealed the discreet localization in nucleus and kinetoplast only, suggesting that the gene may have a role in parasite gene expression.

Abstract 14251: Nuclear Accumulation of Muscle LIM Protein is Regulated by Myocyte Contractility and Correlates With Cardiac Function in the Transition to Heart Failure

Prolonged increases in hemodynamic load lead to a loss of mechanosensing and heart failure because myocytes are unable to adapt to alterations in mechanical stress. We have previously shown that a decreased ratio of cytoplasmic to nuclear Muscle LIM protein (the MLP ratio) is associated with a loss of mechanosensitivity in human heart failure. However, the type and duration of mechanical stress that leads to impaired mechanosensitivity is unknown. In this study, we tested whether passive or active stress/strain was more important in modulating the MLP ratio (a measure of mechanosensitivity) and determined whether this correlated with heart function during the transition to failure. To test this, we exposed cultured neonatal rat myocytes to 48 h of 10% cyclic mechanical stretch at 1 Hz to mimic the passive forces associated with changes in blood pressure and volume. To mimic the forces generated by crossbridge activity during systole, we electrically paced myocytes at 6.8 V (1Hz) for 48 hours. The MLP ratio decreased 50% (p<0.05, n=4 cultures) only in response to electrical pacing, suggesting impaired mechanosensitivity. We then inhibited contractility by treating myocytes with 10μM blebbistatin which resulted in a 3 fold increase in the MLP ratio (n=8 cultures, p<0.05). This indicates that myocyte contractility regulates MLP nuclear shuttling and mechanosensitivity. To examine how mechanosensitivity changes during the transition to heart failure, we examined a guinea pig model of angiotensin II infusion (400ng/kg/min). The hearts were examined at 4, 8 and 12 weeks post-operatively. Using subcellular fractionation we showed that the MLP ratio increased 88% (n=4, P=0.009) during compensated phase at 8 weeks, but decreased significantly during heart failure at 12 weeks (p=0.0003, n=4). Furthermore, the MLP ratio correlated significantly with the E/A ratio (r=0.71, p<0.01 n=12), a clinical measure of diastolic function. This data indicate that impaired cardiac mechanosensitivity resulting in a reduced MLP ratio is regulated predominantly by myofilament crossbridge activity rather than passive strain. Increased mechanical stress within the contracting myofilaments contributes to the loss of mechanosensing during the transition to heart failure.

Critical Roles of the LIM Domains of Lhx3 in Recruiting Coactivators to the Motor Neuron-Specifying Isl1-Lhx3 Complex.

During spinal cord development, the LIM domains of the LIM homeodomain factor Lhx3 bind to either the LIM cofactor nuclear LIM interactor (NLI) or another LIM homeodomain factor, Isl1, assembling the tetrameric V2 interneuron-specifying Lhx3 complex (2NLI:2Lhx3) or the hexameric motor neuron-specifying Isl1-Lhx3 complex (2NLI:2Isl1:2Lhx3). However, the detailed molecular basis by which the Lhx3-LIM domains contribute to motor neuron specification still remains poorly understood. Here, we show that the Lhx3-LIM domains are essential for recruiting transcriptional coactivators to the Isl1-Lhx3 complex. Using a yeast genetic screening system, we identify Lhx3 point mutants that bind to NLI but not Isl1. Accordingly, these mutants fail to assemble the Isl1-Lhx3 complex. However, their interaction with coactivators is relatively intact, and they are fully functional in the Lhx3 complex and V2 interneuron specification. Interestingly, when these Lhx3 mutants are directly fused to Isl1, their transcriptional activity in the Isl1-Lhx3 complex is restored. We further show that this restoration reflects an unexpected role of the Lhx3-LIM domains, likely together with Isl1, to form an interaction interface for coactivators. Our results suggest that the Lhx3-LIM domains play critical roles in transactivation of the Isl1-Lhx3 complex by not only directing the assembly of the Isl1-Lhx3 complex but also recruiting coactivators to the complex.

Identification and Characterization of a Stress-Inducible Gene OsNLI-IF Enhancing Drought Tolerance in Transgenic Tobacco

Plants respond to the adverse environment by activat-ing a series of stress-inducible genes, including genes encoding transcription factors. The expression of these genes is regulated by a core DNA sequence con-tained in their promoter region called cis -acting element. The promoter region of several stress-responsive genes contains several stress-regulated cis -acting elements such as dehydration-responsive element, C-repeat, low-temperature-responsive element, NAC recognition sequence and ZFHD recognition sequence. In this study we isolated a cDNA for a transcription factor named nuclear LIM interactor-interacting fac-tor from rice cDNA library by yeast one-hybrid screening using two target sequences of 50 nucleotides derived from two stress-inducible promoters, JRC0528 and JRC0332, of cold-inducible genes OsZF1 and OsNAC6 respectively, as baits. The NLI-IF protein showed both DNA-binding and transcriptional activities in yeast experiments. Expression of OsNLI-IF was found to be induced by cold, heat, salt and drought stresses. The OsNLI-IF gene overexpressing transgenic tobacco plants showed improvement in drought tolerance. The present study emphasizes that OsNLI-IF could be useful for development of drought-tolerant transgenic crop plants.

Identification of loci and genes for growth related traits from a genome-wide association study in a slow- × fast-growing broiler chicken cross

Identification of functional genes for multifactorial traits is an ongoing priority. A genome-wide association study and gene expression study for weight and carcass composition traits is presented here using a F2 resource population derived from a cross between slow- and fast-growing chicken breeds. For weight traits, eight SNPs on GGA4 plus one SNP on GGA27 were significantly associated with two or more target traits, including body weight (BW) at day 56, BW93, carcass weight, eviscerated weight, breast weight (BrW), thigh muscle weight (ThW) and wing weight. Of 11candidate genes identified near significant SNPs, three were shown by Q-PCR to be differentially expressed in breast muscle between chickens selected from phenotypic extremes of carcass weight and BrW, including ligand dependent nuclear receptor corepressor-like protein and LIM domain-binding factor 2. For major carcass composition traits, nine significant SNPs on GGA1, GGAZ and GGA6 were identified as being associated with BrW and/or percentage. The wings apart-like homolog (Drosophila, WAPAL) on GGA6 had the most significant association with percentage of ThW.

STAT3 promotes motor neuron differentiation by collaborating with motor neuron-specific LIM complex

The motor neuron (MN)-hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN-hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN-hexamer-bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN-hexamer, enhancing the transcriptional activity of the MN-hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN-hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord.

Expression and functional analysis of novel stress-involved transcription factor NLI-IF in rice

We reported previously that one transcription factor NLI-IF (Nuclear LIM interactor-interacting factor)-encoded cDNA clone was identified by yeast one hybrid assay, using 2 target sequences JRC0528 and JRC0332. In this paper we reported some results of in vivo analysis of NLI-IF. The core sequence-specific DNA binding ability of NLI-IF was identified, using yeast one hybrid assay. To identify of protein-protein interaction, the rice stress-treated cDNA library was screened using yeast two hybrid assay and 8 cDNA clones were isolated. These results support to our expectation that transcription factor NLI-IF is involved in stress responses in plant.

Expression and purification of recombinant protein NLI-IF from Escherichia coli

In order to investigate the expression level of NLI-IF (Nuclear LIM Interactor-Interacting Factor) transcription factor - encoding gene which involved in drought tolerance in rice and identiflied in vitro DNA - binding ability of NLI-IF, we expressed and purified recombinant protein NLI-IF from E. coli Rossetta. ­1,3 kb NLI-IF - encoding sequence was cut from pGEMT/NLI-IF and inserted into EcoRI/XhoI site in MCS of expression vector pET28a. pET28a/NLI-IF was transformed into E. coli Rossetta. 52 kDa recombinant protein NLI-IF was expressed optimally in E. coli using 0.1 mM IPTG as an inducer, at 20oC, for 5 h. We were successful in purification of recombinant protein NLI-IF using Ni-NTA affinity chromatography systerm. Purified protein has an ability of binding, specifically, to anti-His-tag antibody in Westerrn blot assay.

Construction of rice drought cDNA library and identification of NLI-IF1 using yeast one hybrid screening

Rice database search on promoter sequences revealed a target sequence (CCTCCTCC) that commonly appears on stress inducible promoters of functional genes involved in abiotic stress, such as, drought, high salt and cold. In addition, promoter analysis of two stress inducible promoters, it shows that JRC0332 express specifically in cold stress and JRC0528 in ABA, high-salinity, drought and cold stresses respectively and both promoters contain the target sequence. In this study, we construct a drought cDNA library from total RNA of Niponpare variety for gene screening. Yeast one-hybrid screening technique using two target sequences of 50 nucleoties on JRC0528 and JRC0332 promoters contain the target sequence for drought cDNA library screening. We identify a cDNA encoding a Nuclear LIM interactor-interacting factor (NLI-IF1). In-vivo specific binding in Yeast suggested that NLI-IF1 bind specifically to the target sequence.

LMO4 functions as a co-activator of neurogenin 2 in the developing cortex

The proneural protein neurogenin 2 (NGN2) is a key transcription factor in regulating both neurogenesis and neuronal radial migration in the embryonic cerebral cortex. However, the co-factors that support the action of NGN2 in the cortex remain unclear. Here, we show that the LIM-only protein LMO4 functions as a novel co-factor of NGN2 in the developing cortex. LMO4 and its binding partner nuclear LIM interactor (NLI/LDB1/CLIM2) interact with NGN2 simultaneously, forming a multi-protein transcription complex. This complex is recruited to the E-box containing enhancers of NGN2-target genes, which regulate various aspects of cortical development, and activates NGN2-mediated transcription. Correspondingly, analysis of Lmo4-null embryos shows that the loss of LMO4 leads to impairments of neuronal differentiation in the cortex. In addition, expression of LMO4 facilitates NGN2-mediated radial migration of cortical neurons in the embryonic cortex. Our results indicate that LMO4 promotes the acquisition of cortical neuronal identities by forming a complex with NGN2 and subsequently activating NGN2-dependent gene expression.

Islet-to-LMO stoichiometries control the function of transcription complexes that specify motor neuron and V2a interneuron identity

LIM transcription factors bind to nuclear LIM interactor (Ldb/NLI/Clim) in specific ratios to form higher-order complexes that regulate gene expression. Here we examined how the dosage of LIM homeodomain proteins Isl1 and Isl2 and LIM-only protein Lmo4 influences the assembly and function of complexes involved in the generation of spinal motor neurons (MNs) and V2a interneurons (INs). Reducing the levels of Islet proteins using a graded series of mutations favored V2a IN differentiation at the expense of MN formation. Although LIM-only proteins (LMOs) are predicted to antagonize the function of Islet proteins, we found that the presence or absence of Lmo4 had little influence on MN or V2a IN specification. We did find, however, that the loss of MNs resulting from reduced Islet levels was rescued by eliminating Lmo4, unmasking a functional interaction between these proteins. Our findings demonstrate that MN and V2a IN fates are specified by distinct complexes that are sensitive to the relative stoichiometries of the constituent factors and we present a model to explain how LIM domain proteins modulate these complexes and, thereby, this binary-cell-fate decision.

LMO4 Controls the Balance between Excitatory and Inhibitory Spinal V2 Interneurons

Multiple excitatory and inhibitory interneurons form the motor circuit with motor neurons in the ventral spinal cord. Notch signaling initiates the diversification of immature V2-interneurons into excitatory V2a-interneurons and inhibitory V2b-interneurons. Here, we provide a transcriptional regulatory mechanism underlying their balanced production. LIM-only protein LMO4 controls this binary cell fate choice by regulating the activity of V2a- and V2b-specific LIM complexes inversely. In the spinal cord, LMO4 induces GABAergic V2b-interneurons in collaboration with SCL and inhibits Lhx3 from generating glutamatergic V2a-interneuons. In LMO4;SCL compound mutant embryos, V2a-interneurons increase markedly at the expense of V2b-interneurons. We further demonstrate that LMO4 nucleates the assembly of a novel LIM-complex containing SCL, Gata2, and NLI. This complex activates specific enhancers in V2b-genes consisting of binding sites for SCL and Gata2, thereby promoting V2b-interneuron fate. Thus, LMO4 plays essential roles in directing a balanced generation of inhibitory and excitatory neurons in the ventral spinal cord.

Transgene-Induced Silencing of the Zoosporogenesis-Specific NIFC Gene Cluster of Phytophthora infestans Involves Chromatin Alterations

Clustered within the genome of the oomycete phytopathogen Phytophthora infestans are four genes encoding spore-specific nuclear LIM interactor-interacting factors (NIF proteins, a type of transcriptional regulator) that are moderately conserved in DNA sequence. NIFC1, NIFC2, and NIFC3 are zoosporogenesis-induced and grouped within 4 kb, and 20 kb away resides a sporulation-induced form, NIFS. To test the function of the NIFC family, plasmids expressing full-length hairpin constructs of NIFC1 or NIFC2 were stably transformed into P. infestans. This triggered silencing of the cognate gene in about one-third of transformants, and all three NIFC genes were usually cosilenced. However, NIFS escaped silencing despite its high sequence similarity to the NIFC genes. Silencing of the three NIFC genes impaired zoospore cyst germination by 60% but did not affect other aspects of the life cycle. Silencing was transcriptional based on nuclear run-on assays and associated with tighter chromatin packing based on nuclease accessibility experiments. The chromatin alterations extended a few hundred nucleotides beyond the boundaries of the transcribed region of the NIFC cluster and were not associated with increased DNA methylation. A plasmid expressing a short hairpin RNA having sequence similarity only to NIFC1 silenced both that gene and an adjacent member of the gene cluster, likely due to the expansion of a heterochromatic domain from the targeted locus. These data help illuminate the mechanism of silencing in Phytophthora and suggest that caution should be used when interpreting silencing experiments involving closely spaced genes.

Cofactor CLIM2 promotes the repressive action of LIM homeodomain transcription factor Lhx2 in the expression of porcine pituitary glycoprotein hormone α subunit gene

We have cloned a porcine orthologue of cofactor CLIM2 (Ldb1/NLI) from the porcine pituitary cDNA library by protein-protein interaction with the Yeast Two-Hybrid System using porcine Lhx2 as a bait protein. Porcine CLIM2 shows a high identity (99%) in the dimerization domain, nuclear localization signal and LIM binding domain with those of man and mouse. The expression of CLIM2 gene in the anterior pituitary lobe was detected during the porcine fetal and postnatal period by RT-PCR analysis, suggesting that this protein is constitutively expressing and plays a basic role in the anterior pituitary. Transfection assay to the pituitary tumor derived LbetaT2 cells, and the Chinese hamster ovary cells demonstrated that CLIM2 acts as a corepressor of the porcine Lhx2 function. Interestingly, CLIM2 alone apparently repressed the high level of alphaGSU gene expression in LbetaT2 cells. These data suggest that CLIM2 is a basic factor in the pituitary development and function, and plays the role of repressor to modify the function of Lhx2 on the alphaGSU gene expression.

Structure and sequential gene expression of in vitro-induced pronephros (kidney) in the amphibian development

We have successfully induced the formation of 20 different organs in vitro from animal caps (undifferentiated or multipotent cells) of Xenopus blastulae and six different tissues from mice embryonic stem cells. For the Xenopus embryo, we subsequently have attempted to transplant these in vitro-generated organs into embryos to determine their functional capacity. In the experiment we induced the pronephros (nephron) consisting of renal tubule, glomerulus, and renal duct from animal caps treated with activin and retinoic acid. Then we transplanted these cell mass treated with activin and retinoic acid into an embryo in which the renal rudiment had been removed. We found that the transplant developed into pronephros normally and function in vivo. Using this system, we have cloned and analyzed the pronephros-specific genes in Xenopus development. Sequential gene expressions of pronephros in vivo and in vitro resemble the mesonephros formation in mammals. In the screening for genes expressed in pronephroi, we isolated a novel gene, named dullard. Dullard protein contains the C-terminal conserved domain of nuclear LIM interactor-interacting factor (NLI-IF), a protein whose function is unknown. The spatial expression was in neural regions at neurula stages, and localized to neural tissues, branchial arches, and pronephroi. The translational knockdown of dullard resulted in failure of neural tube development and structural deformation of the pronephric tubules, and the embryos consequently showed a reduction of head development. In the same screening method for the pronephros-specific genes, we isolated the XTRAP-gamma gene that codes translucent-associated protein, one of the subunit of the translocon-associated protein complex. XTRAP is one of the proteins proposed to aid in the translocation of nascent polypeptides into the lumen of the endothelium reticulum. XTRAP-gamma is expressed in pronephros tubules during pronephros development. The translational knockdown of XTRAP-gamma caused the suppression of tubulogenesis, the increase of Xlim-1 expression, and the decrease of Xpax-2 and Xwnt-4 expression. This translational knockdown of XTRAP-gamma also inhibited differentiation of the pronephros in animal caps by the treatment with activin and retinoic acid. These results showed that XTRAP-gamma plays an important role in the pronephric development. As another approach to clarify the molecular mechanisms of renal development, we investigated the role of Xenopus frizzled-8 ( Xfz8 ) in pronephros development to study the relation between Wnt signaling and nephric development. Translational inhibition of Xfz8 caused reduction in the staining of a duct-specific antibody and defects in pronephric tubule branching, while there was no effect on the expression of early pronephric maker genes in the duct region. Histologic analysis showed that the Xfz8-inhibited cells failed to form a normal epithelial structure. These results suggest that Xfz8 is involved in the formation of epithelium in the developing pronephric duct and tubules.

The LIM domain-only protein LMO4 is required for neural tube closure

Nuclear LIM domain-only proteins (LMOs), which consist of two closely spaced 50 amino acid Zn 2+–finger protein interaction modules mediate interactions between several classes of transcription factors important for development. LMO2 is necessary for development of the entire hematopoietic system and overexpression of LMO1 or LMO2 results in human acute T cell leukemia. LMO4 is the most widely expressed LMO but its normal function is unknown. During development, LMO4 is expressed in dividing neuroepithelial cells within the ventricular zone along the entire rostrocaudal axis of the nervous system. In telencephalic and spinal cord regions of the CNS, LMO4 is highly expressed in ventral but is low in dorsal proliferating neuroepithelial cells. To understand the role of LMO4 during mouse development, we generated a homozygous null mutation in the gene. We found that LMO4 is required for proper closure of the anterior neural tube. In the absence of LMO4, elevation, bending, and proliferation of the ventral neural epithelium and consequent fusion of the prospective dorsal ends of the neural tube do not occur. LMO4 mutant mice die embryonically and exhibit exencephaly, which is associated with abnormal patterns of cell proliferation and with high levels of apoptotic cell death within the neuroepithelium. LMO4 is thus essential for normal patterns of proliferation and for survival of neural epithelial cells in the rostral neural tube. LMO4 is also expressed in Schwann cell progenitors after these contact neurites, a process mediated in part by neuregulin (Nrg).

A cluster of NIF transcriptional regulators with divergent patterns of spore-specific expression in Phytophthora infestans

Seven genes encoding putative nuclear LIM interactor-interacting factors (NIFs) from Phytophthora infestans were characterized. Four exhibited spore-specific expression and clustered within 20 kb, and three hyphal forms were also identified. Pi-nifS mRNA accumulated during sporulation, and Pi-nifC1, Pi-nifC2, and Pi-nifC3 mRNA during zoosporogenesis. By expressing fusions with the β-glucuronidase reporter gene in transformants, promoter fragments determining patterns of transcription specific to sporangia formation or zoosporogenesis were identified. Such studies also indicated that Pi-nifS was expressed at a late stage of sporulation. Pi-nifC1, Pi-nifC2, and Pi-nifC3 induction involved inositol-trisphosphate signaling based on studies with 2-aminoethoxydiphenylborate and U-73122, was independent of verapamil-sensitive signals, and required the same cool temperatures that trigger zoosporogenesis. In contrast, warmer temperatures favoring direct sporangial germination caused Pi-nifS mRNA to rapidly decay, while verapamil increased mRNA levels. The divergent expression and clustering of the spore-specific genes may help reveal how genomes and developmental pathways evolved within the genus.