Ezequiel Mendoza

Different types of germline de novo SETBP1 variants cause clinically distinct and heterogeneous neurodevelopmental disorders: Schinzel-Giedion syndrome (SGS, via missense variants at a critical degron region) and SETBP1-haploinsufficiency disorder. However, due to the lack of systematic investigation of genotype-phenotype associations of different types of SETBP1 variants, and limited understanding of its roles in neurodevelopment, the extent of clinical heterogeneity and how this relates to underlying pathophysiological mechanisms remains elusive. This imposes challenges for diagnosis. Here, we present a comprehensive investigation of the largest cohort to date of individuals carrying SETBP1 missense variants outside the degron region (n = 18). We performed thorough clinical and speech phenotyping with functional follow-up using cellular assays and transcriptomics. Our findings suggest that such variants cause a clinically and functionally variable developmental syndrome, showing only partial overlaps with classical SGS and SETBP1-haploinsufficiency disorder. We provide evidence of loss-of-function pathophysiological mechanisms impairing ubiquitination, DNA-binding, transcription, and neuronal differentiation capacity and morphologies. In contrast to SGS and SETBP1 haploinsufficiency, these effects are independent of protein abundance. Overall, our study provides important novel insights into diagnosis, patient care, and aetiology of SETBP1-related disorders.

The zebra finch is one of the most commonly used animal models for studying the genetic mechanisms underlying vocal learning. To investigate the genetic basis of vocal learning, various genes have been knocked down in Area X—a brain region involved in birdsong acquisition—during the critical learning period. All genes that affect speech when mutated in humans similarly impair song learning in zebra finches. To date, no study has demonstrated that not all genes downregulated in Area X result in decreased song learning. Therefore, we sought a candidate gene to knock down in Area X that could either have no effect or potentially improve song learning. STEP is a protein that dephosphorylates many targets in the brain, and its knockout in mice has resulted in enhanced learning. In this study, a lentiviral knockdown of STEP in Area X during song learning resulted in birds producing normal song in almost all parameters and levels of song analysis. This stands in contrast to the knockdown of other genes like all FoxP subfamily members, which resulted in diminished song learning in previous studies. The only parameter positively affected by STEP knockdown was song rhythmicity, as evidenced by a lower deviation of song element onsets from an isochronous pulse than even their tutors. These results demonstrate for the first time that not all knockdowns in Area X lead to deterioration of song learning and validate the specificity of the method and previous findings. Significance Statement This is the first coding gene knockdown in Area X without negatively affecting song learning.

Rare de novo heterozygous loss-of-function SETBP1 variants lead to a neurodevelopmental disorder characterized by speech deficits, indicating a potential involvement of SETBP1 in human speech. However, the expression pattern of SETBP1 in brain regions associated with vocal learning remains poorly understood, along with the underlying molecular mechanisms linking it to vocal production. In this study, we examined SETBP1 expression in the brain of male zebra finches, a well-established model for studying vocal production learning. We demonstrated that zebra finch SETBP1 exhibits a greater number of exons and isoforms compared to its human counterpart. We characterized a SETBP1 antibody and showed that SETBP1 colocalized with FoxP1, FoxP2, and Parvalbumin in key song nuclei. Moreover, SETBP1 expression in neurons in Area X is significantly higher in zebra finches singing alone, than those singing courtship song to a female, or non-singers. Importantly, we found a distinctive neuronal protein expression of SETBP1 and FoxP2 in Area X only in zebra finches singing alone, but not in the other conditions. We demonstrated SETBP1´s regulatory role on FoxP2 promoter activity in vitro. Taken together, these findings provide compelling evidence for SETBP1 expression in brain regions to be crucial for vocal learning and its modulation by singing behavior.

The efficiency of motor skill acquisition is age-dependent, making it increasingly challenging to learn complex manoeuvres later in life. Zebra finches, for instance, acquire a complex vocal motor programme during a developmental critical period after which the learned song is essentially impervious to modification. Although inhibitory interneurons are implicated in critical period closure, it is unclear whether manipulating them can reopen heightened motor plasticity windows. Using pharmacology and a cell-type specific optogenetic approach, we manipulated inhibitory neuron activity in a premotor area of adult zebra finches beyond their critical period. When exposed to auditory stimulation in the form of novel songs, manipulated birds added new vocal syllables to their stable song sequence. By lifting inhibition in a premotor area during sensory experience, we reintroduced vocal plasticity, promoting an expansion of the syllable repertoire without compromising pre-existing song production. Our findings provide insights into motor skill learning capacities, offer potential for motor recovery after injury, and suggest avenues for treating neurodevelopmental disorders involving inhibitory dysfunctions.

Rare de novo heterozygous loss-of-function SETBP1 variants lead to a neurodevelopmental disorder characterized by speech deficits, indicating a potential involvement of SETBP1 in human speech. However, the expression pattern of SETBP1 in brain regions associated with language remains poorly understood, along with the underlying molecular mechanisms linking it to speech. In this study, we examined SETBP1 expression in the brain of male zebra finches, a well-established model for studying vocal production learning. We demonstrated that zebra finch SETBP1 exhibits a greater number of exons and isoforms compared to its human counterpart. We characterized a SETBP1 antibody and showed that SETBP1 colocalized with FoxP1, FoxP2, and Parvalbumin in key song nuclei. Moreover, SETBP1 expression in neurons in Area X is significantly higher in zebra finches singing alone, than those singing courtship song to a female, or non-singers. Importantly, we found a distinctive neuronal protein expression of SETBP1 and FoxP2 in Area X only in zebra finches singing alone, but not in the other conditions. We demonstrated SETBP1’s regulatory role on FoxP2 promoter activity in vitro . Taken together, these findings provide compelling evidence for SETBP1 expression in brain regions to be crucial for vocal learning and its modulation by singing behavior.

The efficiency of motor skill acquisition is age-dependent, making it increasingly challenging to learn complex maneuvers later in life ^1–6 . Zebra finches, for instance, acquire a complex vocal motor program during a developmental critical period ^7,8 after which the learned song is essentially impervious to modification ⁹ . Although inhibitory interneurons are implicated in critical period closure ^10–13 , it is unclear whether manipulating them can reopen heightened motor plasticity windows. Using pharmacology and a novel cell-type specific optogenetic approach, we manipulated inhibitory neuron activity in a premotor area of adult zebra finches beyond their critical period. When exposed to auditory stimulation in the form of novel song, manipulated birds added new vocal syllables to their stable song sequence. By lifting inhibition in a premotor area during sensory experience, we reintroduced vocal plasticity, promoting an expansion of the syllable repertoire without compromising pre-existing song production. Our findings provide insights into motor skill learning capacities, offer potential for motor recovery after injury, and suggest avenues for treating neurodevelopmental disorders involving inhibitory dysfunctions.

Singing in birds is accompanied by beak, head and throat movements. The role of these visual cues has long been hypothesised to be an important facilitator in vocal communication, including social interactions and song acquisition, but has seen little experimental study. To address whether audio‐visual cues are relevant for birdsong we used high‐speed video recording, 3D scanning, 3D printing technology and colour‐realistic painting to create RoboFinch, an open source adult‐mimicking robot which matches temporal and chromatic properties of songbird vision. We exposed several groups of juvenile zebra finches during their song developmental phase to one of six singing robots that moved their beaks synchronised to their song and compared them with birds in a non‐synchronised and two control treatments. Juveniles in the synchronised treatment approached the robot setup from the start of the experiment and progressively increased the time they spent singing, contra to the other treatment groups. Interestingly, birds in the synchronised group seemed to actively listen during tutor song playback, as they sung less during the actual song playback compared to the birds in the asynchronous and audio‐only control treatments. Our open source RoboFinch setup thus provides an unprecedented tool for systematic study of the functionality and integration of audio‐visual cues associated with song behaviour. Realistic head and beak movements aligned to specific song elements may allow future studies to assess the importance of multisensory cues during song development, sexual signalling and social behaviour. All software and assembly instructions are open source, and the robot can be easily adapted to other species. Experimental manipulations of stimulus combinations and synchronisation can further elucidate how audio‐visual cues are integrated by receivers and how they may enhance signal detection, recognition, learning and memory.

Visual (and probably also magnetic) signal processing starts at the first synapse, at which photoreceptors contact different types of bipolar cells, thereby feeding information into different processing channels. In the chicken retina, 15 and 22 different bipolar cell types have been identified based on serial electron microscopy and single‐cell transcriptomics, respectively. However, immunohistochemical markers for avian bipolar cells were only anecdotally described so far. Here, we systematically tested 12 antibodies for their ability to label individual bipolar cells in the bird retina and compared the eight most suitable antibodies across distantly related species, namely domestic chicken, domestic pigeon, common buzzard, and European robin, and across retinal regions. While two markers (GNB3 and EGFR) labeled specifically ON bipolar cells, most markers labeled in addition to bipolar cells also other cell types in the avian retina. Staining pattern of four markers (CD15, PKCα, PKCβ, secretagogin) was species‐specific. Two markers (calbindin and secretagogin) showed a different expression pattern in central and peripheral retina. For the chicken and European robin, we found slightly more ON bipolar cell somata in the inner nuclear layer than OFF bipolar cell somata. In contrast, OFF bipolar cells made more ribbon synapses than ON bipolar cells in the inner plexiform layer of these species. Finally, we also analyzed the photoreceptor connectivity of selected bipolar cell types in the European robin retina. In summary, we provide a catalog of bipolar cell markers for different bird species, which will greatly facilitate analyzing the retinal circuitry of birds on a larger scale.