The SCO is a brain gland that undergoes early development, yet the transcription factors responsible for its rapid differentiation and the nature of its secretory products have remained elusive. In this study, we conducted a transcriptomic analysis of the SCO at two key developmental stages (proliferation at HH23 and differentiation at HH30) and compared them with whole-brain transcriptomic data at the same stages. Our findings shed light on the molecular landscape of the SCO and its role as a secretory gland during embryonic development through the differential expression of numerous morphogens, axonal guidance molecules, proteases, and TFs depending on the developmental stage.

Early secretory activity of the SCO

The present transcriptomic characterization revealed that the SCO operates as a gland from early stages of development, expressing a myriad of morphogens and signaling molecules. GO analysis of DEGs in the SCO when compared with the whole brain indicated that the GO “extracellular region” (GO:0005576) was one of the most enriched. As we expected, SCO-spondin was a DEG on the SCO when comparing to the whole brain, in agreement with previous reports highlighting its unique production location (Fig. S1) [27], validating the transcriptomic analysis. In chickens, SCO-spondin is an important morphogenetic protein necessary for neurogenesis and the regulation of neuroepithelial cell proliferation and differentiation as well as for the proper formation of the PC [6, 28,29,30].

Morphogens and growth factors

In addition to SCO-spondin, our results showed that the SCO also expresses members of the FGF, BMP, and Wnt families. These molecules are associated with differentiation, migration, axonal guidance and proliferation [31,32,33,34].

Among the members of the BMP family, BMP5 and BMP7 were the most differentially expressed. BMP7 is a crucial morphogen secreted by the choroid plexus to the eCSF and is necessary for correct neurogenesis during brain development [35, 36]. However, in the chick embryo, the choroid plexus anlage is first detected in the lateral ventricles between HH29 and HH34 [37] several days after our BMP detection in the SCO. This finding positions the SCO as a possible initial source of morphogens to the eCSF, a fluid crucial for proper CNS development [38].

Several members of the Wnt family were highly expressed in SCO HH23 and to a lesser extent in HH30. This family is related to several differentiative and proliferative processes in different scenarios [31]. In fact, in zebrafish Wnt3 and Wnt3a are required for caudal forebrain development [39], the prospective SCO region. In chicks, hybridization in situ analysis at previous stages (HH13-HH20) revealed the expression of Wnt1, Wnt6, Wnt3, Wnt3A, Wnt2B, Wnt5B and Wnt9, but not Wnt7A and Wnt7B [20], in accordance with our results. In relation to Wnt5A, is the only Wnt family member with higher expression at HH30 when compared to HH23. Previously, it has been described its secretion to the eCSF by the choroid plexus, where it associates with lipoproteins such as ApoA and ApoE for transport and is required for morphogenesis of dorsal hindbrain [40]. Given that SCO expresses Wnt5A before the formation of the choroid plexus, and that SCO-spondin forms a complex with lipoproteins [28], it is possible that Wnt5A becomes part of this complex even before the formation of the choroid plexus.

In relation to FGF and Wnt members, it is interesting to note that the SCO also has receptors for these molecules, such as FGFR2 and Frizzled 10. This point opens the possibility that the SCO acts in an autocrine manner, as well as in response to morphogens from other sources. In this context, it has been reported that the FGF2 present in the eCSF may come from other areas of the embryonic brain wall, as well as from extra-neural origins [41].

Retinol Binding Protein 3 (RBP3) is also differentially expressed in the SCO especially at HH23. The presence of RBPs and all-trans retinol in eCSF has been previously described between HH20-HH29, indicating that RBPs reach their maximum concentration at HH20-HH24 and then gradually decline [42]. The authors suggested that the origin of this RBP is the yolk of the egg and do not discuss a possible local synthesis and secretion directly into the eCSF. RBPs bind specifically to all-trans retinol, which is then metabolized into retinoic acid, a well-established morphogen that acts as a crucial neurogenic agent in embryonic neural progenitors allowing a proper CNS development [42,43,44]. Our transcriptomics analysis reveals a high expression of RBP3 at HH23, suggesting that the SCO might be the first source of this transporter crucial for early CNS development.

Proteases

One of the most unexpected DEGs identified in the SCO are related with proteins with catalytic activity, such as ADAMTS-15, HTRA-1, and MMEL1. The presence of proteases in the eCSF of humans and rats was previously documented, where they constitute 7% and 6% of the total eCSF protein content respectively [45], although the origin and function of these enzymes are not clear. Interestingly, ADAM family members participate in the cleavage of the extracellular region of numerous tyrosine kinase receptors such as FGFR, Eph receptors and VEGFR, among others generating a negative regulation signals [46]. The occurrence of ADAM family members along with various tyrosine kinases receptors in the SCO suggests a potential regulatory mechanism in the signaling of these receptors.

Proteoglycans

Small Leucine-rich Proteoglycans (SLRPs) are a family of proteins that play important roles in regulating the extracellular matrix and tissue organization and have emerged as new neurogenic factor during brain development [47]. It has been demonstrated that decorin can interact with growth factors and extracellular matrix proteins, such as epidermal growth factor receptor [48] and Wnt7A [49], to modulate proliferation and differentiation of neuroepithelial cells. On the other hand, lumican has also been implicated in modulating the organization of the extracellular matrix in the developing brain, affecting neuronal migration and cortical morphogenesis [50].

In addition to the trophic influence exerted by growth factor and morphogens, the eCSF exerts an intraluminal osmotic pressure that stimulates the proliferation of neuroepithelial cells [51]. This osmotic pressure is attributed to the presence of proteoglycans, which, due to their negative charge, generate an increase in osmolarity facilitating the passage of water, increasing the eCSF volume and leading to the enlargement of the cerebral cavities [52]. Interestingly, the alteration of proteoglycans by the injection of B-D-Xyloside in the eCSF leads to an increase in intraluminal pressure, resulting in the enlargement of the brain, with the most affected area being the diencephalic/mesencephalic region, where the SCO is located [53]. At this respect, the overexpression of lumican, decorin, and keratocan in the SCO suggests their influence in the regulation of eCSF volume.

Axonal guidance molecules

Bilaterally symmetric organisms need to exchange information between the left and right sides of their bodies to integrate sensory input and to coordinate motor control. This exchange occurs through commissures formed by neurons that project axons across the midline [54]. In the chick brain, the first axons to traverse the brain midline are the PC axons, founding the pioneer axons at HH18 and fasciculate axons at HH23 (Fig S1) [55]. This early development is conserved in all vertebrates studied, including humans in which the PC is clearly distinguished in 12 mm embryos [54, 56]

SCO cells display long basal processes that cross the nerve bundles of the PC and attach to the pial membrane [1, 55, 57,58,59]. SCO cells grow concomitantly with the PC, and the roof of the fully differentiated caudal diencephalon consists almost entirely of the PC and the underlying SCO (Fig S1) [3]. The molecules involved in guiding the axons of the PC have not yet been fully described. Previously, our laboratory has described the complementary expression pattern of EphA7 and SCO-spondin in this region. Together, they participate in the guidance of axons from the ventral to the dorsal region of the caudal diencephalon by creating an axonal corridor bordered by repulsive boundaries [60]. In addition to SCO-spondin and EphA7, transcriptomic analysis reveals that 2.3% (in SCO HH23) and 3.2% (in SCO HH30) of the total counts were related to axonal guidance molecules, with members of the semaphorin, Eph, netrin, FGF, Wnt, and BMP families among others. For instance, the fibulin family (FBLN) comprises a secreted glycoproteins capable of binding calcium and interacting with numerous other proteins such as laminin and integrins [61]. Studies in chick embryos have demonstrated that FBLN2, in conjunction with semaphorin 3A, acts as an axonal growth repellent [62]. Additional studies will be required to clarify the localization of these molecules, and whether they are secreted toward the extracellular matrix in contact with the PC axons or to the apical region in contact with the CSF.

SCO express molecules related with dopaminergic neuron differentiation

A GO term enriched in SCO HH30 versus the whole brain was “Dopaminergic neuron differentiation”. The differentiation of dopaminergic neurons in the SCO region has been described in zebrafish early embryos, where pretectal dopaminergic neurons form a local arbor in the pretectum and projects into the ipsilateral tectum [63]. The differentiation of dopaminergic neurons has been studied principally in the ventral region of the diencephalic/mesencephalic boundary, which requires the expression of the transcription factors FOXA1/2, Lmx1A/B, Nr4a2 and Otx2 as well as Wnt and FGF families members, all of which are upregulated in the SCO at both stages studied. In fact, the forced expression of Lmx1A in embryonic stem cells is sufficient to promote dopaminergic differentiation [64]. Research involving conditional mutant mice of FOXA1/2, showed that these molecules exert a positive regulatory influence on the expression of Lmx1a and Lmx1b while concurrently inhibiting the expression of Nkx2.2 in mesodiencephalic dopaminergic progenitors in the ventral region [65]. Additionally, FOXA1/2 is required for the expression of Nurr1 (NR4A2) in immature mDA neurons during early differentiation [66] 67.

In addition to the TFs described, some members of the Wnt family are also involved in the early dopaminergic differentiation. In this way, Wnt1(−/−) mice results in a loss of LMX1A expression, with the subsequent loss of mDA neurons, an effect enhanced in Wnt1(−/−) Wnt5a (−/−) double mutants [68].

The differential expression in the SCO of Wnt1 and Wnt5a secretory molecules as well as dopaminergic TFs (FOXA1/2, Lmx1A/B, NR4A2 and OTX2) suggest that the development of dopaminergic neurons in this region may be orchestrated by the same factors than in the ventral region of the diencephalic/mesencephalic boundary.

Transcription factors differentially expressed in the SCO

Several studies have reported the expression and relevance of different TFs during the formation of the SCO and adjacent regions, such as Pax6 and Msx in mice [16, 18]; Zic1, Pax7 and Pax3 in chick and Xenopus SCO medial region; and Pax6, Meis1 and Dmbx1 in chick and Xenopus SCO lateral region [31, 69, 70]. The present transcriptomic analysis validated the expression of these genes and identified several other enriched TFs. Further elucidating the specific genes and pathways regulated by these TFs could enhance our understanding of the molecular mechanisms underlying SCO development.

For instance, the involvement of Sox14, a member of the Sox family, in regulating neural development [71] and its exceptionally high expression levels at stage HH30 raise intriguing questions about its specific functions during embryogenesis. In this study, we successfully identified more than 150 genes harboring a putative binding site for Sox14. This gene dataset offers valuable insights into the potential biological processes associated with Sox14, particularly the expression of secretion molecules and membrane components. Additionally, the upregulation of Sox14, along with its associated lncRNA, suggests a coordinated regulatory mechanism that may influence the differentiation and maturation of neural cells within the SCO region.

LncRNAs related to gene regulation in the SCO

Over the last decade, extensive documentation emphasizing the crucial regulatory role of lncRNAs in various biological processes has been reported [23]. Intergenic lncRNAs are known to exhibit more tissue-specific expression than to protein-coding genes [72]. This transcriptomic analysis revealed sophisticated coordination in the regulation of gene expression throughout the developmental stages of SCO, which might be orchestrated by distinct mechanisms involving both, differentially expressed TFs and lncRNAs.

Our analysis identified several genes, including axonal guidance molecules and receptors, such as FGFR2, Lhx5, OLIG2, Sox11, among others, which exhibit differential expression and are potentially regulated by lncRNAs (Table S4). For instance, FGFR2, a receptor highly expressed in early stages of brain development, contributes to processes such as proliferation and differentiation of neural cells [73] and is regulated by lncRNAs during rabbit development [74] possibly via the modulation of chromatin signatures [75].

The potential regulatory role of lncRNAs in gene expression suggests a coordinated action with TFs, thereby enhancing the complexity of the regulatory network underlying SCO development.

SCO at stage HH23 showed a high level of proliferative activity

As stated before, the transcriptomic data revealed that at HH23 the SCO functions as a gland. In addition to its secretory activity, the GO analysis shown a strong proliferative potential within the SCO at stage HH23, which is consistent with the rapid growth and development typically observed during embryonic stages. Interestingly, a recent study revealed a heterochronic pattern of proliferation in the caudal diencephalic region, which gives rise to prosomere 1. The proliferation analysis shown that at HH11 stage, the alar plate significantly enlarged compared to the ventral plate [76]. This observation suggests the presence of heterochronic characteristics specifically in this region of prosomere 1, which may persist into stage HH23.

The identified biological processes provide insights into the molecular mechanisms underlying these developmental events, highlighting the importance of DNA synthesis and cellular restructuring in facilitating the expansion of the SCO region during early embryonic stages.

One of the limitations of this study is that it revealed the expression of several transcripts but not their location in the SCO. Previous reports have shown that the SCO is not a homogeneous structure. In fact, it has been divided into a medial region (which expresses EphA7 and transitin but not SCO-spondin) and lateral region (which expresses SCO-spondin but not EphA7) [60]. Additionally, SCO cells contact different compartments such as the ventricular CSF, meningeal CSF, blood vessels and extracellular matrix. Electronic microscopy revealed that most of the secretory granules are located towards the apical region, in contact with the ventricular CSF, although it is possible to find granules in the basal prolongation [3]. In this way, it will be interesting to analyze the destiny of the different secreted molecules.