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still life: Biological Overview | References

BIOLOGICAL OVERVIEW

TIAM Rac1-associated GEF 1 (TIAM1) regulates RAC1 signaling pathways that affect the control of neuronal morphogenesis and neurite outgrowth by modulating the actin cytoskeletal network. To date, TIAM1 has not been associated with a Mendelian disorder. This study describes five individuals with bi-allelic TIAM1 missense variants who have developmental delay, intellectual disability, speech delay, and seizures. Bioinformatic analyses demonstrate that these variants are rare and likely pathogenic. The Drosophila ortholog of TIAM1, still life (sif), is expressed in larval and adult central nervous system (CNS) and is mainly expressed in a subset of neurons, but not in glia. Loss of sif reduces the survival rate, and the surviving adults exhibit climbing defects, are prone to severe seizures, and have a short lifespan. The TIAM1 reference (Ref) cDNA partially rescues the sif loss-of-function (LoF) phenotypes. The function associated with three TIAM1 variants carried by two of the probands were assessed and they were compared to the TIAM1 Ref cDNA function in vivo. TIAM1 p.Arg23Cys has reduced rescue ability when compared to TIAM1 Ref, suggesting that it is a partial LoF variant. In ectopic expression studies, both wild-type sif and TIAM1 Ref are toxic, whereas the three variants (p.Leu862Phe, p.Arg23Cys, and p.Gly328Val) show reduced toxicity, suggesting that they are partial LoF variants. In summary, this study provides evidence that sif is important for appropriate neural function and that TIAM1 variants observed in the probands are disruptive, thus implicating loss of TIAM1 in neurological phenotypes in humans (Lu, 2022).

Many patients with rare diseases undergo a long and frustrating journey to obtain an accurate diagnosis, often referred to as the diagnostic odyssey. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) are effective approaches to identify diagnoses, as 80% or more of rare diseases are estimated to be caused by genomic abnormalities. However, these comprehensive sequencing methods also uncover many variants of uncertain significance (VUSs) with unknown clinical impact Drosophila melanogaster allows effective approaches to probe the functional impacts of these variants (Lu, 2022).

It is argued that bi-allelic loss-of-function (LoF) variants of TIAM Rac1-associated GEF 1 (TIAM1 [MIM: 600687]) cause a disease associated with developmental delay, intellectual disability, speech delay, and seizures. TIAM1 is a guanine nucleotide exchange factor (GEF). GEFs are positive regulators of small GTPases that promote their activation. Each individual GEF has a specificity profile, and TIAM1 is a Ras-related C3 botulinum toxin substrate 1 (RAC1)-specific GEF. RAC1 stimulates signaling pathways that regulate actin cytoskeleton organization, cell movement, differentiation, and proliferation (Lu, 2022).

TIAM1 is enriched in the brain. The rodent ortholog, Tiam1, is also expressed in the brain, is present in dendrites and spines, and is required to maintain proper outgrowth during development. When activated by neurotrophins such as brain-derived neurotrophic factor (BDNF), the TRKB receptor binds and activates TIAM1, which in turn activates RAC1, causing morphological changes by increasing neurite outgrowth. Similarly, when glutamate activates N-methyl-D-aspartate (NMDA) receptors, TIAM1 is also activated to control actin remodeling by inducing RAC1-dependent pathways. The localization of TIAM1 to spines is regulated by par-3 family cell polarity regulator and thereby controls proper spine formation. Mice lacking Tiam1 have simplified dendritic arbors, reduced dendritic spine density, and diminished excitatory synaptic transmission in the dentate gyrus. Taken together, TIAM1 controls neuronal morphogenesis and neurite outgrowth by RAC1-dependent actin cytoskeletal remodeling in rodents. However, alterations in TIAM1 have not yet been reported in the Online Mendelian Inheritance in Man (OMIM) database or the literature as causing human disease (Lu, 2022).

still life (sif) encodes the ortholog of TIAM1 in Drosophila. Previous studies showed that loss of sif leads to reduced locomotor activity. Sif was reported to be localized presynaptically and shown to genetically interact with Fasciclin II, a neural cell-adhesion molecule localized pre- and postsynaptially that controls synaptic growth, stabilization, and presynaptic structural plasticity. Its partial loss causes loss of some boutons at neuromuscular junctions. Also, Sif is highly enriched in lens-secreting cells in fly eyes and affects the distribution pattern of E-cadherin in pupal eyes (Lu, 2022).

This study identified bi-allelic TIAM1 missense variants in five individuals from four families with developmental delay, intellectual disability, speech delay, and seizures. Functional studies in Drosophila revealed that the loss of sif reduces the survival rate of flies, and the surviving adult flies have a remarkably reduced lifespan and exhibit severe climbing defects. In addition, sif LoF mutants display severe seizure-like behaviors when stressed. Expression of the human TIAM1 reference (TIAM1 Ref) cDNA partially rescues the lethality of sif LoF mutants, whereas the variants observed in probands behave as partial LoF mutations in different phenotypic assays. The data support the hypothesis that the variants observed in the probands are the cause of the observed phenotypes (Lu, 2022).

Exome or genome sequencing of probands and parents with rare diseases combined with functional investigations in model organisms has led to the discovery of numerous novel Mendelian diseases. This study has identified TIAM1 as a disease-associated gene. Five individuals are presented with compound-heterozygous or homozygous coding variants in TIAM1. The main phenotypes observed in the probands are developmental delays, with severe deficits in speech and language, intellectual disability, and seizures. Using Drosophila, it was found that loss of sif, the ortholog of TIAM1, causes semi-lethality, and flies that eclose exhibit climbing defects and seizure-like behaviors and have a short lifespan. The sif^T2A-GAL4 allele allowed determination if the expression pattern of the gene and shows that sif is expressed in neurons but not in glia. It also allowed the 'humanization' of the fly by replacing fly sif with human TIAM1. TIAM1 Ref can partially rescue the survival rate and lifespan but not the climbing defects and seizure-like phenotypes of sif LoF mutants. The lack of rescue is likely due to the toxicity of the overexpression of TIAM1 Ref in flies. Alternatively, the limited rescue ability is due to functional diversity of the human homologs, as there are four other orthologs of sif in humans besides TIAM1 (Lu, 2022).

The variant pathogenicity prediction algorithm CADD indicates that these TIAM1 variants are rare and likely pathogenic. Since the TIAM1 variants carried by the probands reported in this study are bi-allelic, they are predicted to be LoF variants. To test this hypothesis, three of the six variants were assessed in vivo using two-pronged functional assays based on rescue and ectopic-expression experiments. TIAM1 p.Arg23Cys has reduced rescue ability, suggesting that it is a partial LoF variant. TIAM1 p.Leu862Phe and p.Gly328Val show rescue ability comparable to that of TIAM1 Ref and did not allow drawing a conclusion based on this assay, since TIAM1 cDNAs have different levels of toxicity that also affect viability. Therefore, ectopic-expression experiments were used in a WT background to further test the function of the variants, given that WT sif and TIAM1 Ref induce similar phenotypes, including wing defects, in ectopic-expression assays. Since TIAM1 Ref and the variants are inserted in the same genomic site, functional analyses of the variants can be perormed by comparing ectopic-expression-induced phenotypes. The three proband-associated variants exhibit reduced toxicity when compared to TIAM1 Ref. When the Ref causes a toxic phenotype and the variants are less toxic, the variants can typically be classified as LoF variants. Hence, the ectopic-expression data support the hypothesis that TIAM1 p.Arg23Cys, p.Leu862Phe, and p.Gly328Val are partial LoF variants. Taken together, the data suggest that the TIAM1 variants reported in this study result in a loss of function and are associated with neurodevelopmental phenotypes and seizures (Lu, 2022).

The bioinformatic data show that the Protein-Ligand Interaction (pLI) of TIAM1 is high (0.96), and the observed-to-expected ratio of LoF variants for TIAM1 is 0.20. However, there are 46 heterozygous individuals with TIAM1 LoF alleles observed in gnomAD, suggesting that loss of one copy of TIAM1 is tolerated. A possible reason why TIAM1 is associated with a high pLI score is that it is a very large gene (>400 kb). It has also been reported an autosomal-recessive disease associated with bi-allelic LoF variants in OXR1,65 a large gene (>400 kb) for which the observed-to-expected ratio of LoF variants is 0.19 and the pLI is 0.84 very similar to TIAM1 (Lu, 2022).

Besides TIAM1, sif is also the ortholog of other GEF-encoding genes, including TIAM2, DNMBP (MIM: 611282), ARHGEF37, and ARHGEF38.17 TIAM2 activates RAC1 and controls cell migration in neurons. DNMBP is a GEF for cell division control protein 42 that controls the shaping of cell junctions through binding to tight junction protein ZO-1, and knockdown of DNMBP results in a disorganized configuration of cell junctions. LoF variants of DNMBP cause infantile-onset cataracts in humans. Similarly, sif knockdown in the eye alters the distribution of septate junctions in adjacent cone cells and affects the function of the eye in young flies. Finally, ARHGEF37 and ARHGEF38 are Rho GEFs. ARHGEF37 assists dynamin 2 during clathrin-mediated endocytosis, while ARHGEF38 is an uncharacterized protein (Lu, 2022).

Tiam1 knockout mice have decreased spine density, simplified dendritic arbors, and decreased miniature excitatory postsynaptic currents in the hippocampus, but they exhibit only subtle behavior abnormalities, which may be due to redundancy of other GEFs. The related TIAM2 shares 37% overall identity and 71% Dbl homology (DH) domain identity with TIAM1. The expression levels of Tiam2 correlate with the stages of neuronal morphological development, and Tiam2 knockdown in neurons also causes reduced neurite outgrowth. TIAM1 promotes the formation and growth of spines and synapses by activating RAC1 signaling pathways that control the actin cytoskeleton. Dysfunction of the neuronal cytoskeleton has been implicated in a variety of diseases, including neurological developmental disorders as well as neurodegenerative diseases. Moreover, dysregulation of the neuronal cytoskeletal network also contributes to the pathogenesis of epilepsy (Lu, 2022).

Previous studies show that sif LoF mutants are viable and have reduced locomotor activity. This study generated a more severe LoF allele, sif^T2A-GAL4, which leads to semi-lethality, a highly reduced lifespan, and a severe sensitivity to seizure-like behaviors, in addition to the climbing defects. It is worth noting that fly mutants with such severe sensitivity to seizures are rarely observed. It was also shown that these phenotypes are mainly caused by neuronal sif loss based on RNAi knockdown assays. Interestingly, the sif RNAi- with ∼12% of the remaining sif transcripts based on real-time PCR show much stronger phenotypes, including semi-lethality, when compared to sif RNAi-2 with ∼24% remaining sif transcripts, indicating a threshold effect of sif expression between these two values, similar to what has been documented for other genes, like flower (Lu, 2022).

Finally, the limitations of this study are pointed out. The number of individuals described is small and some of the phenotypic features differ. Two of the individuals come from consanguineous families, and additional recessive conditions could be contributing to the phenotype. Additionally, the analysis was based on exome sequencing, and noncoding variants were not assessed and could contribute to variation in the phenotype. With additional identified individuals with bi-allelic TIAM1 variants, it should become more obvious which clinical features are core to the condition (Lu, 2022).

In summary, this study found that bi-allelic pathogenic TIAM1 variants are associated with a neurological disorder in humans. Functional analysis is provided in flies that supports an LoF model for TIAM1-associated variants. Further studies of the underlying mechanism will be necessary to provide a better understanding of the pathological mechanisms and may provide therapeutic strategies (Lu, 2022).

Synaptic development is controlled in the periactive zones of Drosophila synapses

A cell-adhesion molecule Fasciclin 2 (FAS2), which is required for synaptic growth and Still life (SIF), an activator of RAC, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. BetaPS integrin and discs large (DLG), both involved in synaptic development, also decorated the zone. However, Shibire (SHI), the Drosophila dynamin that regulates endocytosis, was found in the distinct region. Mutant analyses showed that sif genetically interacted with Fas2 in synaptic growth and that the proper localization of SIF required FAS2, suggesting that they are components in related signaling pathways that locally function in the periactive zones. It is proposed that neurotransmission and synaptic growth are primarily regulated in segregated subcellular spaces, active zones and periactive zones, respectively (Sone, 2000).

This study characterized the periactive zone in three respects. Initially, the periactive zone was defined on the basis of the distribution patterns of SIF and FAS2, both clearly surrounding the electron-dense region marked with anti-DPAK antibody. The concentric staining patterns that represent pairs of the periactive zone and electron-dense region were often separated from the adjacent pairs and therefore suggested that these two regions constitute a structural unit in the NMJ. Secondly, the biochemical properties of the molecules found in the periactive zone characterize this zone. Cell adhesion molecules, FAS2, and integrin, were found on the plasma membrane, and DLG and SIF were found inside the membrane of the zone, although βPS integrin and DLG were more widely distributed, at least on the postsynaptic side. These findings suggest that the periactive zone may link the information from cell adhesion molecules to the intracellular signaling pathways. Finally, the role for the periactive zone was suggested by the genetic evidence for the molecules localized in the zone. The mutant analyses in this and the previous studies have shown that all these molecules are involved in synaptic development. In Fas2 null mutants, neuromuscular synapses fail to grow and eventually retract. In the mutants affecting βPS integrin, the growth of larval NMJ is increased or decreased dependent on the alleles. The mutation in dlg caused the reduction in size of the subsynaptic reticulum in postsynapses and the increase in the number of active zones in presynapses. Also, the sif mutation exhibited a modulatory effect on synaptic growth when combined with the Fas2 mutation as shown in this report. Taken together, all gene products localized in the peri-active zone participate in the synaptic development. These findings provide genetic evidence suggesting that the periactive zone serves as a membrane domain for the structural control of synaptic terminals (Sone, 2000).

In addition, the data indicate that the periactive zone is distinct from the zone for endocytosis recently reported. Staining for SHI, which is in a donut-like pattern, does not overlap with most of the FAS2-positive region, and is nearly within the hole of the FAS2 rings. This observation suggests that endocytosis actively occurs near or within the electron-dense region, which is consistent with the previous finding that one type of endocytosis occurs near the active zone in the Drosophila optic lobe synapses. Although it is possible that endocytosis occurs in the periactive zone at some frequency, these data suggest that the major role for the zone is directed to events other than shi-dependent endocytosis (Sone, 2000).

Genetic data suggested that FAS2 and SIF are components in the related signaling pathways that locally function in the periactive zone. The altered localization of SIF in the Fas2 mutants suggests that a certain level of FAS2 expression is required to maintain the configuration of the intracellular molecules including SIF in the periactive zone. Further evidence that shows the functional interaction between SIF and FAS2 was also obtained by mutant analyses. While each of the sif and Fas2 mutations causes the reduction in bouton number, the sif and Fas2 double mutations exhibited a suppressive genetic interaction in synaptic growth. This suppression suggests that the two pathways for FAS2 and SIF signals are both involved in synaptic development and the balance between the pathways is important for the regulation of synaptic growth. These notions were supported by the exacerbated reduction of the bouton number when SIF was overexpressed in the Fas2^e76 background (Sone, 2000).

It has been shown that FAS2 mediates synaptic stabilization, the varying extent of which seems to cause the increase or decrease in synaptic growth. The current data may be therefore consistent with the idea that SIF acts to regulate synaptic stabilization that is mediated by FAS2 or other adhesion molecules. Moreover, SIF may modulate synaptic growth in an inhibitory manner when the FAS2-mediated synapse stabilization is reduced. It may be further noteworthy that the Fas2^e76 sif^ES11 double mutants exhibit low viability and are difficult to maintain as a stock even as heterozygotes. This observation may be also interpreted as indicating that eliminating the doses of both sif and Fas2 impairs a regulatory cascade that is established by the balance of the two signaling pathways (Sone, 2000).

In summary, the cell adhesion molecule FAS2 and intracellular signaling molecule SIF interact with each other, both controlling synaptic development in the periactive zone. This finding enforces the idea that the periactive zone is the functional membrane domain where various types of proteins constitute signaling networks or protein complexes that control synapse formation. This notion is further supported by the fact that DLG regulates the localization of FAS2 in the periactive zone. In addition, FAS2, in turn, may function to organize the arrangement of the molecules including SIF in the periactive zones (Sone, 2000).

An in vitro assay shows that SIF catalyzes the guanine- nucleotide exchange reaction for mammalian RAC1. This is consistent with the previous observation that SIF induces ruffling membranes in human KB cells, as does the constitutive active form of RAC1. In addition, Drosophila RHO family G proteins are more than 85% identical in amino acid sequences to the corresponding mammalian G proteins. These lines of evidence suggest that SIF activates Drosophila RAC in the periactive zones. As SIF contains multiple domains that potentially mediate interaction with other molecules, RAC and several other molecules may be recruited to make a protein complex in the zone. In mice, TIAM1 and STEF have been identified as GEFs that specifically activate RAC1, and both are highly related to SIF in domain organization and amino acid sequence in several domains, indicating that these two proteins are likely to be the mouse orthologs of SIF. Interestingly, both Tiam1 and Stef are expressed in the brain, and Tiam1 expression is observed in the adult hippocampus. It would be important therefore to examine whether TIAM1 and STEF are localized in the synaptic terminals (Sone, 2000).

RAC is well known as a regulator of the actin-based cytoskeleton and cell adhesion in various cells. RAC is also implicated in the structural changes of nerve terminals including growth cones and dendrites. Therefore, in the periactive zones, activated RAC may locally regulate the processes of the structural change in the synaptic terminals, which include reorganization of the actin-based cytoskeleton and cell adhesion (Sone, 2000).

Previous studies have shown that RAC acts in the neurite outgrowth of neuroblastoma cells that depends on the signal from integrin on the cell surface. The mammalian SIF homolog TIAM1, which functions as a RAC GEF, recruits integrin to specific adhesive contacts at the cell periphery. Moreover, expression of TIAM1 increases cadherin-mediated cell adhesion in epithelial MDCK cells. Therefore, there appear to be signaling links between the RAC and cell-adhesion molecules. This study shows that SIF activates RAC, sif genetically interacts with Fas2 in synaptic growth and the SIF localization was perturbed in the Fas2 mutants. Taken together, these data suggest that the SIF-RAC pathway is linked to the cell-adhesion molecule FAS2 in close vicinity in the periactive zone (Sone, 2000).

This study has indicated the periactive zone as a region for the control of synaptic development. The periactive zone surrounds the active zone, which is the site for vesicle exocytosis or neurotransmission. This concentric organization suggests that the two zones specialized for the different cellular functions constitute an elemental unit for the presynaptic structure. Investigation of how these zones are incorporated into the synaptic bouton during development will be interesting but remains to be carried out (Sone, 2000).

The segregated distribution of the two zones suggests that the mechanisms controlling synaptic development and neurotransmission may be separable. This view was supported by the mutant analyses for FAS2 and SIF; both mutations affect structural properties of synapses without changing basic electrophysiological functions. In the NMJs of Fas2 mutants, the bouton number is decreased or increased depending on the alleles but the total synaptic strength is maintained at the normal level (Sone, 2000).

Functional strength of the synapse is regulated only through the activity of a transcription factor, cAMP-response-element- binding protein (CREB), which functions independently of FAS2. Also in sif mutants, basic electrophysiological properties of NMJs are normal. These observations clearly contrast with the mutant phenotypes for the proteins controlling vesicle exocytosis: synaptotagmin, cysteine string protein, n-synaptobrevin and syntaxin 1A. All these mutants show the impaired EJPs. Taken together, these results indicate that synaptic development and neurotransmission are genetically separable phenomena and are regulated by independent pathways. It is proposed that these genetically separable phenomena are spatially segregated into the two zones on the presynaptic plasma membrane, although the possibility that the two zones interact with each other cannot be excluded (Sone, 2000).

Thia study identified new members to be added to the periactive zone proteins. In these studies, Drosophila highwire protein and its C. elegans homolog, RPM-1, were demonstrated to function in the growth or structural development of synapses, and highwire was found to localize in the periactive zone. Discovery of these proteins further enforces the current view and highlights the importance of periactive zones for synaptic growth and stability (Sone, 2000).

In the nervous system, various cell-adhesion molecules and signaling molecules may provide characteristic attributes for each synapse. In addition to the molecules described in this article, cadherins and catenins are likely to play such a role in synapses. Notably, N-cadherin and SIF are localized laterally to the active zones in the neuron-neuronal synapses of the mammalian and Drosophila brain, respectively. This suggests that the presence of the specialized area surrounding the active zone is a general feature among synapses. Further examination of signaling pathways that function locally in the periactive zone would provide insights as to how synapses grow or retract during development and plasticity (Sone, 2000).

Still life, a protein in synaptic terminals of Drosophila homologous to GDP-GTP exchangers

The morphology of axon terminals changes with differentiation into mature synapses. A molecule that might regulate this process was identified by a screen of Drosophila mutants for abnormal motor activities. The still life (sif) gene encodes a protein homologous to guanine nucleotide exchange factors, which convert Rho-like guanosine triphosphatases (GTPases) from a guanosine diphosphate-bound inactive state to a guanosine triphosphate-bound active state. The SIF proteins are found adjacent to the plasma membrane of synaptic terminals. Expression of a truncated SIF protein resulted in defects in neuronal morphology and induced membrane ruffling with altered actin localization in human KB cells. Thus, SIF proteins may regulate synaptic differentiation through the organization of the actin cytoskeleton by activating Rho-like GTPases (Sone, 1997).

Search PubMed for articles about Drosophila Still Life

Cheng, J., Scala, F., Blanco, F. A., Niu, S., Firozi, K., Keehan, L., Mulherkar, S., Froudarakis, E., Li, L., Duman, J. G., Jiang, X. and Tolias, K. F. (2021). The Rac-GEF Tiam1 Promotes Dendrite and Synapse Stabilization of Dentate Granule Cells and Restricts Hippocampal-Dependent Memory Functions. J Neurosci 41(6): 1191-1206. PubMed ID: 33328293

Lambert, J. M., Lambert, Q. T., Reuther, G. W., Malliri, A., Siderovski, D. P., Sondek, J., Collard, J. G. and Der, C. J. (2002). Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism. Nat Cell Biol 4(8): 621-625. PubMed ID: 12134164

Leeuwen, F. N., Kain, H. E., Kammen, R. A., Michiels, F., Kranenburg, O. W. and Collard, J. G. (1997). The guanine nucleotide exchange factor Tiam1 affects neuronal morphology; opposing roles for the small GTPases Rac and Rho. J Cell Biol 139(3): 797-807. PubMed ID: 9348295

Lu, S., Hernan, R., Marcogliese, P. C., Huang, Y., Gertler, T. S., Akcaboy, M., Liu, S., Chung, H. L., Pan, X., Sun, X., Oguz, M. M., Oztoprak, U., de Baaij, J. H. F., Ivanisevic, J., McGinnis, E., Guillen Sacoto, M. J., Chung, W. K. and Bellen, H. J. (2022). Loss-of-function variants in TIAM1 are associated with developmental delay, intellectual disability, and seizures. Am J Hum Genet 109(4): 571-586. PubMed ID: 35240055

Miyamoto, Y., Yamauchi, J., Tanoue, A., Wu, C. and Mobley, W. C. (2006). TrkB binds and tyrosine-phosphorylates Tiam1, leading to activation of Rac1 and induction of changes in cellular morphology. Proc Natl Acad Sci U S A 103(27): 10444-10449. PubMed ID: 16801538

Sone, M., Hoshino, M., Suzuki, E., Kuroda, S., Kaibuchi, K., Nakagoshi, H., Saigo, K., Nabeshima, Y. and Hama, C. (1997). Still life, a protein in synaptic terminals of Drosophila homologous to GDP-GTP exchangers. Science 275(5299): 543-547. PubMed ID: 8999801

Sone, M., Suzuki, E., Hoshino, M., Hou, D., Kuromi, H., Fukata, M., Kuroda, S., Kaibuchi, K., Nabeshima, Y. and Hama, C. (2000). Synaptic development is controlled in the periactive zones of Drosophila synapses. Development 127(19): 4157-4168. PubMed ID: 10976048

Tolias, K. F., Bikoff, J. B., Burette, A., Paradis, S., Harrar, D., Tavazoie, S., Weinberg, R. J. and Greenberg, M. E. (2005). The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines. Neuron 45(4): 525-538. PubMed ID: 15721239

Tolias, K. F., Bikoff, J. B., Kane, C. G., Tolias, C. S., Hu, L. and Greenberg, M. E. (2007). The Rac1 guanine nucleotide exchange factor Tiam1 mediates EphB receptor-dependent dendritic spine development. Proc Natl Acad Sci U S A 104(17): 7265-7270. PubMed ID: 17440041

date revised: 12 November 2022

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