Kosei Sato

Specification of cell fate along the proximal-distal axis in the developing chick limb bud.

Pattern formation along the proximal-distal (PD) axis in the developing limb bud serves as a good model for learning how cell fate and regionalization of domains, which are essential processes in morphogenesis during development, are specified by positional information. In the present study, detailed fate maps for the limb bud of the chick embryo were constructed in order to gain insights into how cell fate for future structures along the PD axis is specified and subdivided. Our fate map revealed that there is a large overlap between the prospective autopod and zeugopod in the distal limb bud at an early stage (stage 19), whereas a limb bud at this stage has already regionalized the proximal compartments for the prospective stylopod and zeugopod. A clearer boundary of cell fate specifying the prospective autopod and zeugopod could be seen at stage 23, but cell mixing was still detectable inside the prospective autopod region at this stage. Detailed analysis of HOXA11 AND HOXA13 expression at single cell resolution suggested that the cell mixing is not due to separation of some different cell populations existing in a mosaic. Our findings suggest that a mixable unregionalized cell population is maintained in the distal area of the limb bud, while the proximal region starts to be regionalized at the early stage of limb development.

Fruitless Recruits Two Antagonistic Chromatin Factors to Establish Single-Neuron Sexual Dimorphism

The Drosophila fruitless (fru) gene encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. However, the mechanism whereby fru establishes the sexual fate of neurons remains enigmatic. Here, we show that Fru forms a complex with the transcriptional cofactor Bonus (Bon), which, in turn, recruits either of two chromatin regulators, Histone deacetylase 1 (HDAC1), which masculinizes individual sexually dimorphic neurons, or Heterochromatin protein 1a (HP1a), which demasculinizes them. Manipulations of HDAC1 or HP1a expression change the proportion of male-typical neurons and female-typical neurons rather than producing neurons with intersexual characteristics, indicating that on a single neuron level, this sexual switch operates in an all-or-none manner.

Neuroethology of male courtship in Drosophila: from the gene to behavior

Neurogenetic analyses in the fruit fly Drosophila melanogaster revealed that gendered behaviors, including courtship, are underpinned by sexually dimorphic neural circuitries, whose development is directed in a sex-specific manner by transcription factor genes, fruitless (fru) and doublesex (dsx), two core members composing the sex-determination cascade. Via chromatin modification the Fru proteins translated specifically in the male nervous system lead the fru-expressing neurons to take on the male fate, as manifested by their male-specific survival or male-specific neurite formations. One such male-specific neuron group, P1, was shown to be activated when the male taps the female abdomen. Moreover, when artificially activated, P1 neurons are sufficient to induce the entire repertoire of the male courtship ritual. These studies provide a conceptual framework for understanding how the genetic code for innate behavior can be embodied in the neuronal substrate.

Sex-switching of the Drosophila brain by two antagonistic chromatin factors.

In Drosophila melanogaster, the fruitless (fru) gene encoding BTB-Zn-finger transcription factors organizes male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. However, the molecular mechanism by which fru controls the sexual fate of neurons has been unknown. Our recent study represents a first step toward clarification of this mechanism. We have shown that: (1) Fru forms a complex with the transcriptional cofactor Bonus (Bon), which recruits either of two chromatin regulators, Histone deacetylase 1 (HDAC1) or Heterochromatin protein 1a (HP1a), to Fru-target sites; (2) the Fru-Bon complex has a masculinizing effect on single sexually-dimorphic neurons when it recruits HDAC1, whereas it has a demasculinizing effect when it recruits HP1a; (3) HDAC1 or HP1a thus recruited to Fru-target sites determines the sexual fate of single neurons in an all-or-none manner, as manipulations of HDAC1 or HP1a expression levels affect the proportion of male-typical neurons and female-typical neurons without producing neurons of intersexual characteristics. Here, we hypothesize that chromatin landscape changes induced by ecdysone surges direct the HDAC1- or HP1a-containing Fru complex to distinct targets, thereby allowing them to switch the neuronal sexual fate in the brain.

Morphogenetic change of the limb bud in the hand plate formation.

The vertebrate hand plate is flattened and paddle shaped; that is, it is wide along the anteroposterior (AP) axis (thumb to little finger) and thin along the dorsoventral axis (back of hand to palm). To learn how the hand plate develops its three-dimensional architecture, we observed morphological changes in the distal limb bud of the chick embryo at stages 23-27 and the gecko embryo 11-13 days after oviposition. Cell population of the posterior distal limb bud expanded more than that of the anterior one in the chick embryo. Taken together with the observation that these two cell populations did not show significant differences in their expansion along the proximodistal axis, we propose that the cell population in the posterior limb bud contributes more to the morphogenetic increase along the AP axis, which widens the limb bud for the formation of the hand plate. Our observation that more mitoses were oriented anteroposteriorly than dorsoventrally in the chick embryo at around stage 25 suggests that the oriented cell division contributes to the morphogenetic increase along the AP axis.

An epigenetic switch of the brain sex as a basis of gendered behavior in Drosophila.

Two transcription factor genes, fruitless (fru) and doublesex (dsx), are the primary factors that direct the development of brain sex differences in Drosophila. In the nervous system, the dsx gene produces different proteins, DsxM and DsxF, respectively, in males and females, whereas the fru gene produces proteins (FruM) only in males. Thus, the dsx-dependent sex differences in the nervous system likely reflect the distinct target specificity of DsxM and DsxF, whereas the fru-dependent sex differences rely on the presence and absence of FruM. Some neurons express both fru and dsx and others express either fru or dsx, while the majority of neurons express neither. By studying the sexual dimorphism of single neurons, insights into the molecular mechanism whereby FruM specifies the neuronal sex have been obtained. The sexually dimorphic morphologies of a fru-expressing neural cluster in males are completely feminized when FruM is lost, whereas the same cluster has an intersexual appearance under moderate reductions in FruM in fru hypomorphic mutants. Single-cell labeling demonstrates that even in such fru hypomorphic mutants, each neuron in the cluster has a complete male-type structure or a complete female-type structure, indicating that the intersexual appearance of the cluster results from the mixed presence of the male-type and female-type neurons. This all-or-none mode of sex-type determination by FruM is mediated by a competitive recruitment to the FruM-target genomic sites of two antagonistic chromatin regulators, histone deacetylase 1 and heterochromatin protein 1a, each of which masculinizes or demasculinizes single neurons in concert with FruM. These findings open up a new avenue for the study of epigenetic bases for sexual differentiation.

Serotonergic neuronal death and concomitant serotonin deficiency curb copulation ability of Drosophila platonic mutants

Drosophila platonic (plt) males court females, but fail to copulate. Here we show that plt is an allele of scribbler (sbb), a BMP signalling component. sbb knockdown in larvae leads to the loss of approximately eight serotonergic neurons, which express the sex-determinant protein Doublesex (Dsx). Genetic deprivation of serotonin (5-HT) from dsx-expressing neurons results in copulation defects. Thus, sbb+ is developmentally required for the survival of a specific subset of dsx-expressing neurons, which support the normal execution of copulation in adults by providing 5-HT. Our study highlights the conserved involvement of serotonergic neurons in the control of copulatory mechanisms and the key role of BMP signalling in the formation of a sex-specific circuitry.

The Drosophila lingerer protein cooperates with Orb2 in long-term memory formation.

Abstract Recently mated Drosophila females were shown to be reluctant to copulate and to exhibit rejecting behavior when courted by a male. Males that experience mate refusal by a mated female subsequently attenuate their courtship effort toward not only mated females but also virgin females. This courtship suppression persists for more than a day, and thus represents long-term memory. The courtship long-term memory has been shown to be impaired in heterozygotes as well as homozygotes of mutants in orb2, a locus encoding a set of CPEB RNA-binding proteins. We show that the impaired courtship long-term memory in orb2-mutant heterozygotes is restored by reducing the activity of lig, another putative RNA-binding protein gene, yet on its own the loss-of-function lig mutation is without effect. We further show that Lig forms a complex with Orb2. We infer that a reduction in the Lig levels compensates the Orb2 deficiency by mitigating the negative feedback for Orb2 expression and thereby alleviating defects in long-term memory.

Optogenetic activation of the fruitless-labeled circuitry in Drosophila subobscura males induces mating motor acts

It remains an enigma how the nervous system of different animal species produces different behaviors. We studied the neural circuitry for mating behavior in Drosophila subobscura, a species that displays unique courtship actions not shared by other members of the genera including the genetic model D. melanogaster, in which the core courtship circuitry has been identified. We disrupted the D. subobscura fruitless (fru) gene, a master regulator for the courtship circuitry formation in D. melanogaster, resulting in complete loss of mating behavior. We also generated frusoChrimV, which expresses the optogenetic activator Chrimson fused with a fluorescent marker under the native fru promoter. The fru-labeled circuitry in D. subobscura visualized by frusoChrimV revealed differences between females and males, optogenetic activation of which in males induced mating behavior including attempted copulation. These findings provide a substrate for neurogenetic dissection and manipulation of behavior in non-model animals, and will help to elucidate the neural basis for behavioral diversification. SIGNIFICANCE STATEMENT How did behavioral specificity arise during evolution? Here we attempted to address this question by comparing the parallel genetically definable neural circuits controlling the courtship behavior of Drosophila melanogaster, a genetic model, and its relative, D. subobscura, which exhibits a courtship behavioral pattern unique to it, including nuptial gift transfer. We found that the subobscura fruitless circuit, which is required for male courtship behavior, was slightly but clearly different from its melanogaster counterpart, and that optogenetic activation of this circuit induced subobscura-specific behavior, i.e., regurgitating crop contents, a key element of transfer of nuptial gift. Our study will pave the way for determining how and which distinctive cellular elements within the fruitless circuit determine the species-specific differences in courtship behavior.

The core-promoter factor TRF2 mediates a Fruitless action to masculinize neurobehavioral traits in Drosophila

In fruit flies, the male-specific fruitless (fru) gene product FruBM plays a central role in establishing the neural circuitry for male courtship behavior by orchestrating the transcription of genes required for the male-type specification of individual neurons. We herein identify the core promoter recognition factor gene Trf2 as a dominant modifier of fru actions. Trf2 knockdown in the sexually dimorphic mAL neurons leads to the loss of a male-specific neurite and a reduction in male courtship vigor. TRF2 forms a repressor complex with FruBM, strongly enhancing the repressor activity of FruBM at the promoter region of the robo1 gene, whose function is required for inhibiting the male-specific neurite formation. In females that lack FruBM, TRF2 stimulates robo1 transcription. Our results suggest that TRF2 switches its own role from an activator to a repressor of transcription upon binding to FruBM, thereby enabling the ipsilateral neurite formation only in males.fruitless (fru) is an important sex-determinant gene that controls the expression of neuroanatomical sex differences in Drosophila. Here the authors report that a core-promoter factor, TRF2, suppresses a male-type neurite specification through direct interaction with FruBM isoform at the robo1 target gene promoter.