Developmental Neurobiology

The cortico-basal ganglia pathways that mediate vocal learning in zebra finches (Taeniopygia guttata) are localized in parallel circuits formed by CORE and SHELL subregions. These circuits traverse a specialized region of the basal ganglia essential for vocal learning (Area X), which includes intermixed striatal and pallidal neurons. The pallidal neurons within Area X exhibit analogs of mammalian direct and indirect pathways that may have opposing effects and thereby increase or inhibit thalamic activity respectively. Direct pallidal neurons of Area X send projections to the medial portion of the dorsolateral anterior thalamic nucleus (DLM), whereas indirect pallidal neurons form intrinsic connections onto DLM-projecting neurons. Expression of the transcription factor FoxP2 in the basal ganglia is necessary for normal vocal learning and production in both humans and songbirds. We used tract-tracing techniques to label direct pallidal Area X[->]DLM projection neurons and immunohistochemical techniques to label neurons expressing the transcription factor FoxP2 in adult and juvenile male zebra finches. Our results showed that DLM-projecting neurons did not express FoxP2 in either adults or juveniles. Measurements of nuclear sizes revealed a population of large neurons that expressed FoxP2 but were not retrogradely-labeled from DLM. A putative marker of striatal neurons (DARPP-32) did not co-localize with FoxP2 in many of these large neurons, suggesting that they form a class of indirect pallidal neurons. These findings offer FoxP2 as a possible marker for indirect pallidal neurons and support the existence of different subpopulations of neurons that correspond to direct and indirect pathways within Area X.

Striatal neurons play a central role in vertebrate action selection; however, their location in larval zebrafish is not well defined. We assayed for conserved striatal markers in the zebrafish subpallium using fluorescent in situ hybridization (FISH) and immunohistochemistry. Whole mount FISH revealed an inhibitory neuronal cluster rostral to the anterior commissure that expresses tac1, a gene encoding substance P. This molecular profile is shared by mammalian striatal direct pathway neurons. A second partially overlapping population of inhibitory neurons was identified that expresses penka, a gene encoding enkephalin. This molecular profile is shared by striatal indirect pathway neurons. Immunostaining for substance P and enkephalin confirmed the presence of these peptides in the subpallium. The tac1 and penka populations were both found to increase linearly across larval stages. Together, these findings support the existence of a striatal homologue in larval zebrafish that grows to match the development and increasing behavioural complexity of the organism.

Neural progenitor cells (NPCs) are the highly polarized dividing stem cells of the developing brain that give rise to all neurons and glia. Early on, NPCs divide symmetrically and expand the pool of progenitor cells, but as development continues the NPCs begin to asymmetrically divide to produce neurons. The mechanisms that govern this irreversible commitment to neurogenesis are not fully understood, but in other stem cell populations the regulation of mitochondria and cell metabolism is key to controlling stem cell fate. Here we use timelapse 3D confocal microscopy to observe NPCs, their cellular progeny, and their mitochondria in the developing Xenopus tectum. Our results track individual NPCs over days and show that they contain abundant mitochondria that form complicated networks distributed throughout the cells. We find that NPCs preparing to divide shift mitochondria toward the cell body where they become asymmetrically distributed, suggesting that the cells control which progeny inherit mitochondria. This uneven distribution of mitochondria in cell preparing to divide led us to test the role that mitochondria play in cell division. We overexpressed the mitochondrial biogenesis master regulator, PGC-1a, which induced the NPCs to asymmetrically divide and produce neurons, while PGC-1a knockdown limited neurogenesis. Together these data suggest that the regulation of mitochondria by NPCs prior to cell division and their unequal inheritance during cell division, contributes to the fate of the newborn cells in the developing brain.

Glia are irreplaceable components for the nervous system development and function. However, the cellular mechanisms each glial layer utilizes to communicate with each other and the extracellular environment is not well characterized. Here, we investigated the role of a heparan-sulfate proteoglycan, Syndecan (Sdc), in regulating glial cell function and development in the Drosophila nervous system. Sdc is expressed throughout multiple glial layers and loss of Sdc in all glia resulted in disruption of both central and peripheral glia. Within the CNS loss of Sdc in all glia lead to reduced brain lobes and disruption of neuroblast proliferation. In the PNS, loss of Sdc in different glial layers resulted in impaired ensheathment in wrapping glia and abnormal septate junction morphology in subperineurial glia. We focused on the outer layer of perineurial glia and found ensheathment defects and a reduction in glial numbers with Sdc loss. These phenotypes mirror those previously observed with the loss of integrins and a mutation in the integrin {beta}-subunit enhanced the phenotypes observed with loss of Sdc within the perineurial. Thus, our results indicate Sdc has multiple roles in Drosophila nervous system development including as an integral component in regulating glial cell morphology, maintaining neuroblast populations within the optic lobe and in mediating glial-ECM interactions.

The nervous system is a complex network of cells whose interactions provide circuitry necessary for an organism to perceive and move through its environment. Revealing the molecular basis of how neurons and non-neuronal glia communicate is essential for understanding neural development, behavior, and abnormalities of the nervous system. BMP signaling in motor neurons, activated in part by retrograde signals from muscle expressed Gbb (BMP5/6/7) has been implicated in synaptic growth, function and plasticity in Drosophila melanogaster. Through loss-of-function studies, we establish Gbb as a critical mediator of glia to neuron signaling important for proper synaptic growth. Furthermore, the BMP2/4 ortholog, Dpp, expressed in a subset of motor neurons, acts by autocrine signaling to also facilitate neuromuscular junction (NMJ) growth at specific muscle innervation sites. In addition to signaling from glia to motor neurons, autocrine Gbb induces signaling in larval VNC glia which strongly express the BMP type II receptor, Wit. In addition to Dpps autocrine motor neuron signaling, Dpp also engages in paracrine signaling to adjacent glia but not to neighboring motor neurons. In one type of dorsal midline motor neuron, RP2, dpp transcription is under tight regulation, as its expression is under autoregulatory control in RP2 but not aCC neurons. Taken together our findings indicate that bi-directional BMP signaling, mediated by two different ligands, facilitates communication between glia and neurons. Gbb, prominently expressed in glia, and Dpp acting from a discrete set of neurons induce active Smad-dependent BMP signaling to influence bouton number during neuromuscular junction growth.

BackgroundCytoskeletal remodelling plays a pivotal role in the establishment of neuronal connectivity during development and in plasticity in adults. Mutations in the cytoskeleton regulatory protein Formin-2 (Fmn2) are associated with neurodevelopmental disorders like intellectual disability, though its function in neuronal morphogenesis has not been characterised in vivo. ResultsHere we develop a loss-of-function model for fmn2b, the zebrafish orthologue of Fmn2, using CRISPR/Cas9-mediated gene editing. fmn2b mutants display motor deficits starting from the earliest motor responses in the embryo. We find that fmn2b is expressed in spinal motor neurons and its loss reduces motor neuron innervation of the axial muscles without affecting myotome integrity. The translocation of caudal primary (CaP) motor neuron outgrowth is compromised in fmn2b mutants, while rostral primary (RoP) motor neurons have missing soma or stall at the horizontal myoseptum. Strikingly, axon collateral branching of the motor neurons is severely compromised and results in reduced synaptic coverage of the myotome. Rescue experiments identify the requirement for Fmn2-mediated actin nucleation for motor neuron outgrowth and arborisation. ConclusionsThe zebrafish loss-of-function model of Fmn2 reveals the specific requirement of F-actin polymerisation by Fmn2 in neuromuscular development. It also underscores the role of Fmn2 in motor neuropathies, especially as a proportion of individuals harbouring mutations in Fmn2 present with hypotonia.

This study aimed at identifying the role of angiopoietin 1 (angpt1) in brain development, the mode of action of angpt1, and the main targets in the zebrafish brain. We investigated embryonic brain angiogenesis and neural development in the angpt1sa14264, itgb1bmi371, tekhu1667 mutant fish, and the effects of transgenic overexpression of angpt1 in the larval brain. Lack of angpt1 was associated with downregulation of tek and upregulation of itgb1b. We found deficiencies in the patterning of proliferation, the vascular network and reticulospinal neurons in the hindbrain, and selective deficiencies in specific neurotransmitter systems. In the angpt1sa14264 and itgb1bmi371 larval brains, using microangiography, retrograde labeling, and immunostaining, we demonstrated that the targeted destruction of angpt1sa14264 and itgb1bmi371 mutant fish caused severe irregular cerebrovascular development, aberrant hindbrain patterning, downregulation of neural proliferation, expansion of the radial glial progenitors, deficiencies of dopaminergic, histaminergic, and GABAergic populations in the larval brain. In contrast, the tekhu1667 mutants regularly grew with no such apparent phenotypes. Neurally overexpressed angpt1 promoted opposite effects by increasing the vascular branching, increasing cell proliferation, and neuronal progenitors. Notably, zebrafish angpt1 showed neurogenic activity independent of its typical receptor tek, indicating the novel role of a dual regulation by angpt1 in embryonic neurogenesis and angiogenesis in zebrafish. The results show that angpt1 and its interaction with itgb1b are crucial in zebrafish brain neuronal and vascular development and suggest that angpt1 through itgb1b can act as a neurogenic factor in the neural proliferation fate in the developing brain.

The precise organization of neural circuits requires highly specific axon guidance, facilitated by cell-surface guidance receptors on axonal growth cones that help neurons reach their target destinations. Despite a limited repertoire of known guidance receptors and ligands, neural systems achieve complex axonal networks, suggesting that additional regulatory mechanisms exist. One proposed strategy is the co-expression of ligands and their receptors on the same axons, allowing modulation of receptor responsiveness to guidance cues. To investigate this mechanism, we studied the spinal lateral motor column (LMC) motor neurons, which make a binary axon pathfinding decision toward limb targets. We hypothesized that specificity protein 1 (Sp1), a transcription factor, regulates ephrin expression in LMC neurons, thereby modulating receptor functions via cis-attenuation to ensure accurate axonal pathfinding. Our results show that Sp1 is indeed expressed in LMC neurons during critical axonal extension periods. Manipulating Sp1 activity disrupted LMC axon trajectory selection, and RNA-Seq analysis indicated that Sp1 regulates genes associated with axon guidance, including ephrins. We found that Sp1 knockdown affected ephrin/Eph cis-binding and trans-signaling, highlighting Sp1s role in controlling axonal projections through ephrin gene regulation. Additionally, coactivators p300 and CBP are essential for Sp1s regulatory function. These findings identify Sp1 as a key transcription factor in LMC neurons, essential for ephrin expression and ephrin/Eph-mediated axon guidance, providing insights into the molecular mechanisms of neural circuit formation.

FMRFamide is a neuropeptide that is widely distributed in invertebrates and known to be involved in many physiological functions. Previously we noted marked differences in expression of the fmrf gene in the stellate ganglion of Doryteuthis pealei* compared to the central nervous system. In this study we aimed to examen the brain systems of Doryteuthis pealei* for the presence and distribution of fmrf-expressing cells and fiber networks. Late squid embryos and hatchlings were examined by in situ hybridization and immunohistochemistry in whole mounts and tissue sections. All central lobes contained limited numbers of scattered neurons expressing fmrf, but the FMRFamide-containing fiber systems were abundant and extensive, mostly present in the neuropil of lobes. Main clusters of neurons were located in the magnocellular and chromatophore lobes of the posterior subesophageal mass (PSM), and in dorsal aspects of the basal lobe (BL). Dense FMRFamide-immunoreactive fibers were particularly seen in the optic lobe (OL), medial and posterior supraesophageal masses (MSM and SPM) often with a commissural organization. The data show that the central lobes of Doryteuthis pealei hatchlings have a matured FMRFamide system organized in a limited number of centers, but with widely distributed efferents. This suggests that FMRFamide neurons are already functionally engaged in the late embryo. The localization indicates that control of chromatophores and fin movement are amongst these functions.

The vertebrate hypothalamus regulates physiological and behavioral responses to environmental stimuli through the function of evolutionarily-conserved neuronal subpopulations. Our previous work found that mutation of zebrafish lef1, which encodes a transcriptional mediator of the Wnt signaling pathway, leads to the loss of hypothalamic neurons and behavioral phenotypes that are both associated with stress-related human mood disorders However, the specific Lef1 target genes that link neurogenesis to behavior remain unknown. One candidate is otpb, which encodes a transcription factor with known roles in hypothalamic development. Here we show that otpb expression in the posterior hypothalamus is Lef1-dependent, and that like lef1, its function is required for the generation of crhbp+ neurons in this region. Transgenic reporter analysis of a crhbp conserved noncoding element suggests that otpb participates in a transcriptional regulatory network with other Lef1 targets. Finally, consistent with a role for crhbp in inhibiting the stress response, zebrafish otpb mutants exhibit decreased exploration in a novel tank diving assay. Together our findings suggest a potential evolutionarily-conserved mechanism for the regulation of innate stress response behaviors through Lef1-mediated hypothalamic neurogenesis.

Glial cells perform many functions in the nervous system, including maintaining the blood-brain/nerve barriers and structurally supporting axons. While their functions are well-characterized, the complex molecular mechanisms important for their development are less known. Here, we investigated whether microRNA-mediated post-transcriptional regulation is involved during glial development, ensheathment and blood-nerve-barrier formation in Drosophila. In this study, we systematically knocked down 120 different microRNAs by competitive inhibition using microRNA-sponges and analyzed peripheral glial morphology. Knockdown of miRNA-125 in the blood-nerve barrier-forming glia (subperineurial glia) resulted in the most penetrant morphological defects. Since microRNA-125 is co-transcribed with miRNAs-let7 and -100 in a genetic cluster, our further verification for subperineurial glia function included miRNA-125 plus all other members of this cluster. However, the loss of each individual gene and the entire cluster did not lead to any morphological defects in the subperineurial glia. To test the efficiency of the microRNA sponge approach in subperineurial glia, we expressed a sponge targeting a microRNA established to be vital for blood-brain barrier formation (microRNA-285) and found no defects in brain lobes and peripheral nerves. Given that a scrambled-sponge control also generated morphological defects, this suggests that using miRNA sponge lines may not be an effective approach to study miRNA function in Drosophila peripheral glia.

Construction and maturation of the postsynaptic apparatus are crucial for synapse and dendrite development. The fundamental mechanisms underlying these processes are most often studied in glutamatergic central synapses in vertebrates. Whether the same principles apply to excitatory cholinergic synapses in the insect central nervous system (CNS) is not known. To address this question, we investigated Drosophila ventral lateral neurons (LNvs) and identified nAchR1 (D1) and nAchR6 (D6) as the main functional nicotinic acetylcholine receptor (nAchR) subunits in these cells. With morphological and calcium imaging studies, we demonstrated their distinct roles in supporting dendrite morphogenesis and synaptic transmission. Furthermore, our analyses revealed a transcriptional upregulation of D1 and downregulation of D6 during larval development, indicating a close association between the temporal regulation of nAchR subunits and synapse maturation. Together, our findings show transcriptional regulation of nAchR composition is a core element of developmental and activity-dependent regulation of central cholinergic synapses.

Toxcast/Tox21 is a massive federally run research effort dedicated to better understanding the potential toxicity of thousands of compounds in a high throughput manner. Among this list of compounds is equilin, an estrogen-like compound that was flagged as a potential thyroid hormone agonist. Here we examine if equilin acts like a thyroid hormone agonist on cellular and molecular mechanisms of brain development in Xenopus laevis tadpoles. To examine the effect of equilin, tadpoles were divided into eight groups and received 4 days of exposure. The experimental groups were as follows: 1 L, 10 L, and 100 L of equilin, 1 L, 10M, and 100 M of 17-{beta} estradiol as an estrogen control, 15 g/mL thyroxine (T4) as a thyroid hormone control, and a no-exposure control. After 4 days of treatment, animals were treated with CldU to label dividing cells for 2hr and then euthanized in MS-222. After fixation, body length was measured and the brains dissected out. IHC was performed on brains for CldU to label proliferating neural progenitor cells. Brains were then whole-mounted and analyzed using confocal microscopy. We found that equilin did not increase the number of dividing progenitor cells in a T4-like manner. Instead, equilin decreased proliferation in a dose-dependent manner, as did estradiol. The same paradigm was performed separately staining for caspase-3 and h2ax, finding that equilin increased cell death in contrast to CNTL and T4. In another experiment, RNA was extracted from tadpole brains in each group and qPCR was performed to assess change in expression of thyroid hormone-sensitive genes, Equilin did not affect gene expression in a thyroid hormone-like manner. Our data indicate that equilin does not act as a thyroid hormone agonist in the Xenopus laevis nervous system but instead acts similarly to estradiol. Our data strongly suggest that equilin is not a TH disruptor, contrary to the findings of the ToxCast/Tox21 dataset.

Dendritic spines are the principal site of excitatory synapse formation in the human brain. Impaired formation of spines during development has been observed in several autism spectrum disorders (ASDs), including Fragile X syndrome. Fragile X is caused by transcriptional silencing of the Fmr1 gene encoding the RNA-binding protein FMRP (Fragile X mental retardation protein). While spine development has been well characterized in the mammalian CNS, spines are not unique to mammals. Pyramidal neurons (PyrNs) of the zebrafish optic tectum form an apical dendrite containing a dense array of dendritic spines. We employed a genetic labeling system to monitor PyrN dendritic spine development in larval zebrafish. Our findings identify a developmental window when PyrN dendrite growth is concurrent with spine formation. Throughout this period, motile, transient filopodia gradually transform into stable spines containing postsynaptic specializations. fmr1 mutant zebrafish larvae exhibit pronounced defects in both PyrN dendrite growth and the formation of morphologically mature spines. Live imaging of PyrN dendrites suggests these defects are caused by an inability to stabilize nascent contacts. These findings indicate spine stabilization is essential for PyrN dendritic arborization and establish zebrafish larvae as a model system to study spine development in vivo.

Glioblastoma (GB) is the most aggressive, lethal and frequent primary brain tumor. It originates from glial cells and is characterized by rapid expansion through infiltration. GB cells interact with the microenvironment and healthy surrounding tissues, mostly neurons and vessels. GB cells project tumor microtubes (TMs) that contact with neurons and exchange signaling molecules related to Wingless/WNT, JNK, Insulin or Neuroligin-3 pathways. This cell to cell communication promotes GB expansion and neurodegeneration. Moreover, healthy neurons form glutamatergic functional synapses with GB cells which facilitate GB expansion and premature death in mouse GB xerograph models. Targeting signaling and synaptic components of GB progression may become a suitable strategy against glioblastoma. In a Drosophila GB model, we have determined the post-synaptic nature of GB cells with respect to neurons, and the contribution of post-synaptic genes expressed in GB cells to tumor progression. In addition, we document the presence of intratumoral synapses between GB cells, and the functional contribution of pre-synaptic genes to GB calcium dependent activity and expansion. Finally, we explore the relevance of synaptic genes in GB cells to the lifespan reduction caused by GB advance. Our results indicate that both presynaptic and postsynaptic proteins play a role in GB progression and lethality.

AbstractNeural stem cell (NSC) differentiation is controlled by cell-intrinsic and external signals from the stem cell niche including niche surface glia (SG). However, the mechanisms by which transcription factors drive NSC differentiation within the niche remain largely unknown. Here, we show that the Drosophila melanogaster transcription factor, Chromatin-linked adaptor for MSL proteins (CLAMP) is required for regulation of stemness and proliferation of NSCs, especially of the optic lobe (OL). CLAMP promotes transcription of genes involved in stemness, proliferation, and glial development and represses transcription of genes involved in neurogenesis and niche survival. Consistent with transcriptional changes, CLAMP promotes NSC proliferation and niche SG production, while lack of CLAMP severely and specifically impacts OL development. To identify potential mechanisms by which CLAMP may regulate brain development, we examined CLAMP motifs and available CLAMP ChIP-seq data to determine which genes may be direct versus indirect targets. CLAMP motifs are present at many target genes including the glial-determining gene, glial cells missing, while Tailless, the master regulator of OL-development is directly bound by CLAMP. In accordance to these results, in larval OL NSCs lacking CLAMP, Tailless levels are decreased dramatically, suggesting that CLAMP controls OL neurogenesis via Tailless. Overall, our results suggest that CLAMP regulates a transcriptional program which drives NSC proliferation and differentiation via cell-intrinsic and niche-dependent mechanisms that involve transcriptional regulation of Tailless and niche glia.

The proper functioning of the nervous system is dependent on the establishment and maintenance of intricate networks of neurons that form functional neural circuits. Once neural circuits are assembled during development, a distinct set of molecular programs is likely required to maintain their connectivity throughout the lifetime of the organism. Here, we demonstrate that Fasciclin 3 (Fas3), an axon guidance cell adhesion protein, is necessary for the maintenance of the olfactory circuit in adult Drosophila. We utilized the TARGET system to spatiotemporally knockdown Fas3 in selected populations of adult neurons. Our findings show that Fas3 knockdown results in the death of olfactory circuit neurons and reduced survival of adults. We also demonstrated that Fas3 knockdown activates caspase-3 mediated cell death in olfactory local interneurons, which can be rescued by overexpressing p35, an anti-apoptotic protein. This work adds to the growing set of evidence indicating a critical role for axon guidance proteins in the maintenance of neuronal circuits in adults. SUMMARY STATEMENTLittle is known about the maintenance of adult neural circuits. We show that the continuous expression of Fasciclin 3, a cell adhesion protein involved in axon guidance, is required for neuronal survival in the adult olfactory circuit.

PFTK1/Eip63E is a member of the Cyclin-dependent kinases (CDKs) family and plays an important role in normal cell cycle progression. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. We sought to understand the function of Eip63E in the CNS by studying the fly ventral nerve cord during development. Our results demonstrate that Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. Functional interaction studies performed using the same system identify an interaction between Eip63E and the small GTPase Rho1. Furthermore, deficiency of Eip63E homolog in mice, PFTK1, in a newly generated PFTK1 knockout mice results in increased axonal outgrowth confirming that the developmental defects observed in the fly model are due to defects in axogenesis. Importantly, RhoA phosphorylation and activity is affected by PFTK1 in primary neuronal cultures. We here report that GDP bound inactive RhoA is a substrate of PFTK1 and PFTK1 phosphorylation is required for RhoA activity. In conclusion, our work establishes an unreported neuronal role of PFTK1 in axon development mediated by phosphorylation and activation of GDP-bound RhoA. The results presented add to our understanding of the role of Cdks in the maintenance of RhoA mediated axon growth and its impact on CNS development and axonal regeneration.

In the cerebellum of juvenile Oncorhynchus masou, proliferating BrdU+ and HuCD+ cells and constitutive neurogenic niches were detected in different zones; the largest number of labeled cells were found in the dorsal part of the molecular layer and the dorsal matrix zone (DMZ). Cells labeled with glutamine synthetase (GS) and radial glia were also present in the intact O. masou cerebellum. The most intensive proliferation was detected in the rostral part of cerebellum. This part is assumed to contain active zones of constitutive neurogenesis. After an injury inflicted to the cerebellum, the number of BrdU+ and HuCD+ cells increased significantly. The number of BrdU+ cells after this type of injury was much greater than after a telencephalon trauma. A quantitative analysis revealed that after the cerebellum injury the proliferative activity in the caudal part of CCb is increased compared to that in the control. A reactivation of neurogenic and neuroepithelial niches and their transformation into reactive neurogenic domains, with an increased distribution density of intensely labeled HuCD+ cells of different types, were observed. The increase in the number of HuCD+ differentiated cells in the basal area suggests that the processes of neuronal differentiation are intensified in the cerebellum of juvenile O. masou after injury. The number of GS positive cells (GS+) and fibers increased in all the zones of cerebellum. The most intensive astrocytic response was noted in the dorsal part of cerebellum. The data of the enzyme immunoassay confirm the multiple variations in the level of GS after a traumatic injury to cerebellum in O. masou.

Alternative promoter usage generates multiple transcription factor isoforms during brain development, yet their functional significance remains poorly defined. One such example is TCF7L2, a transcription factor critical for the development of the thalamus and recurrently affected by de novo mutations in autism spectrum disorder. TCF7L2 exists in two isoforms driven from different promoters: the long isoform (L-TCF7L2) with the {beta}-catenin-binding domain, and the shorter isoform (S-TCF7L2), lacking this domain and classically considered a dominant-negative regulator of the L isoform. We investigated the role of TCF7L2 isoforms in thalamic development using total and isoform-specific knockout strategies. Integrated phenotypic and transcriptomic analyses revealed functional redundancy of TCF7L2 isoforms during embryogenesis. {beta}-catenin subcellular localization and chromatin occupancy uncovered a developmental switch in TCF7L2 activity, from a {beta}-catenin-independent and isoform-redundant mode in the embryonic thalamus to a {beta}-catenin-dependent program postnatally. More broadly, these findings point to distinct embryonic and postnatal regulatory strategies, with alternative promoter usage potentially supporting robust availability of regulatory proteins during brain development.