Autism spectrum disorders (ASD) are more common among boys than girls. The mechanisms responsible for ASD symptoms and their sex differences remain mostly unclear. We previously identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sex-different expression during sexual differentiation of the hypothalamic sexually dimorphic nucleus. This study investigated the relationship between the sex-different development of autistic features and CRMP4 deficiency. Whole-exome sequencing detected a de novo variant (S541Y) of CRMP4 in a male ASD patient. The expression of mutated mouse CRMP4 S540Y, which is homologous to human CRMP4 S541Y, in cultured hippocampal neurons derived from Crmp4-knockout (KO) mice had increased dendritic branching, compared to those transfected with wild-type (WT) Crmp4, indicating that this mutation results in altered CRMP4 function in neurons. Crmp4-KO mice showed decreased social interaction and several alterations of sensory responses. Most of these changes were more severe in male Crmp4-KO mice than in females. The mRNA expression levels of some genes related to neurotransmission and cell adhesion were altered in the brain of Crmp4-KO mice, mostly in a gender-dependent manner. These results indicate a functional link between a case-specific, rare variant of one gene, Crmp4, and several characteristics of ASD, including sexual differences.

In tilapia, hormone treatment during the period of sexual differentiation can alter the phenotype of the gonads, indicating that endocrine factors can cause gonadal sex reversal. However, the endocrine mechanism underlying sex reversal of reproductive behaviors remains unsolved. In the present study, we detected sexual dimorphism of gonadotropin-releasing hormone type III (GnRH3) neurons in Mozambique tilapia Oreochromis mossambicus. Our immunohistochemical observations showed sex differences in the number of GnRH3 immunoreactive neurons in mature tilapia; males had a greater number of GnRH3 neurons in the terminal ganglion than females. Treatment with androgen (11-ketotestosterone (11-KT) or methyltestosterone), but not that with 17β-estradiol, increased the number of GnRH3 neurons in females to a level similar to that in males. Furthermore, male-specific nest-building behavior was induced in 70% of females treated with 11-KT within two weeks after the onset of the treatment. These results indicate androgen-dependent regulation of GnRH3 neurons and nest-building behavior, suggesting that GnRH3 is importantly involved in sex reversal of male-specific reproductive behavior.

Members of the collapsin response mediator protein (CRMP) family are reported to be involved in the pathogenesis of various neuronal disorders, including schizophrenia and autism. One of them, CRMP4, is reported to participate in aspects of neuronal development, such as axonal guidance and dendritic development. However, no physiological or behavioral phenotypes in Crmp4 knockout (Crmp4-KO) mice have been identified, making it difficult to elucidate the in vivo roles of CRMP4. Focusing on the olfaction process because of the previous study showing strong expression of Crmp4 mRNA in the olfactory bulb (OB) during the early postnatal period, it was aimed to test the hypothesis that Crmp4-KO pups would exhibit abnormal olfaction. Based on measurements of their ultrasonic vocalizations, impaired olfactory ability in Crmp4-KO pups was found. In addition, c-Fos expression, a marker of neuron activity, revealed hyperactivity in the OB of Crmp4-KO pups compared with wild-types following exposure to an odorant. Moreover, the mRNA and protein expression levels of glutamate receptor 1 (GluR1) and 2 (GluR2) were exaggerated in Crmp4-KO pups relative to other excitatory and inhibitory receptors and transporters, raising the possibility that enhanced expression of these excitatory receptors contributes to the hyperactivity phenotype and impairs olfactory ability. This study provides evidence for an animal model for elucidating the roles of CRMP4 in the development of higher brain functions as well as for elucidating the developmental regulatory mechanisms controlling the activity of the neural circuitry.

Collapsin response mediator protein 4 (CRMP4), a member of the CRMP family, is involved in the pathogenesis of neurodevelopmental disorders such as schizophrenia and autism. Here, we first compared layer thickness of the olfactory bulb between wild-type (WT) and CRMP4-knockout (KO) mice. The mitral cell layer (MCL) was significantly thinner, whereas the external plexiform layer (EPL) was significantly thicker in CRMP4-KO mice at postnatal day 0 (PD0) compared with WTs. However, differences in layer thickness disappeared by PD14. No apoptotic cells were found in the MCL, and the number of mitral cells (MCs) identified with a specific marker (i.e. Tbx21 antibody) did not change in CRMP4-KO neonates. However, DiI-tracing showed that the length of mitral cell apical dendrites was greater in CRMP4-KO neonates than in WTs. In addition, expression of CRMP4 mRNA in WT mice was most abundant in the MCL at PD0 and decreased afterward. These results suggest that CRMP4 contributes to dendritic elongation. Our in vitro studies showed that deletion or knockdown of CRMP4 resulted in enhanced growth of MAP2-positive neurites, whereas overexpression of CRMP4 reduced their growth, suggesting a new role for CRMP4 as a suppressor of dendritic elongation. Overall, our data suggest that disruption of CRMP4 produces a temporary alteration in EPL thickness, which is constituted mainly of mitral cell apical dendrites, through the enhanced growth of these dendrites.

The neuroplastic mechanisms in the fish brain that underlie sex reversal remain unknown. Gonadotropin-releasing hormone 3 (GnRH3) neurons control male reproductive behaviours in Mozambique tilapia and show sexual dimorphism, with males having a greater number of GnRH3 neurons. Treatment with androgens such as 11-ketotestosterone (KT), but not 17β-estradiol, increases the number of GnRH3 neurons in mature females to a level similar to that observed in mature males. Compared with oestrogen, the effect of androgen on neurogenesis remains less clear. The present study examined the effects of 11-KT, a non-aromatizable androgen, on cellular proliferation, neurogenesis, generation of GnRH3 neurons and expression of cell cycle-related genes in mature females. The number of proliferating cell nuclear antigen-positive cells was increased by 11-KT. Simultaneous injection of bromodeoxyuridine and 11-KT significantly increased the number of newly-generated (newly-proliferated) neurons, but did not affect radial glial cells, and also resulted in newly-generated GnRH3 neurons. Transcriptome analysis showed that 11-KT modulates the expression of genes related to the cell cycle process. These findings suggest that tilapia could serve as a good animal model to elucidate the effects of androgen on adult neurogenesis and the mechanisms for sex reversal in the fish brain.