Non-thalamic origin of zebrafish sensory nuclei implies convergent evolution of visual pathways in amniotes and teleosts.

Ascending visual projections similar to the mammalian thalamocortical pathway are found in a wide range of vertebrate species, but their homology is debated. To get better insights into their evolutionary origin, we examined the developmental origin of a thalamic-like sensory structure of teleosts, the preglomerular complex (PG), focusing on the visual projection neurons. Similarly to the tectofugal thalamic nuclei in amniotes, the lateral nucleus of PG receives tectal information and projects to the pallium. However, our cell lineage study in zebrafish reveals that the majority of PG cells are derived from the midbrain, unlike the amniote thalamus. We also demonstrate that the PG projection neurons develop gradually until late juvenile stages. Our data suggest that teleost PG, as a whole, is not homologous to the amniote thalamus. Thus, the thalamocortical-like projections evolved from a non-forebrain cell population, which indicates a surprising degree of variation in the vertebrate sensory systems.

Amphibian thalamic nuclear organization during larval development and in the adult frog Xenopus laevis: Genoarchitecture and hodological analysis.

The early patterning of the thalamus during embryonic development defines rostral and caudal progenitor domains, which are conserved from fishes to mammals. However, the subsequent developmental mechanisms that lead to the adult thalamic configuration have only been investigated for mammals and other amniotes. In this study, we have analyzed in the anuran amphibian Xenopus laevis (an anamniote vertebrate), through larval and postmetamorphic development, the progressive regional expression of specific markers for the rostral (GABA, GAD67, Lhx1, and Nkx2.2) and caudal (Gbx2, VGlut2, Lhx2, Lhx9, and Sox2) domains. In addition, the regional distributions at different developmental stages of other markers such as calcium binding proteins and neuropeptides, helped the identification of thalamic nuclei. It was observed that the two embryonic domains were progressively specified and compartmentalized during premetamorphosis, and cell subpopulations characterized by particular gene expression combinations were located in periventricular, intermediate and superficial strata. During prometamorphosis, three dorsoventral tiers formed from the caudal domain and most pronuclei were defined, which were modified into the definitive nuclear configuration through the metamorphic climax. Mixed cell populations originated from the rostral and caudal domains constitute most of the final nuclei and allowed us to propose additional subdivisions in the adult thalamus, whose main afferent and efferent connections were assessed by tracing techniques under in vitro conditions. This study corroborates shared features of early gene expression patterns in the thalamus between Xenopus and mouse, however, the dynamic changes in gene expression observed at later stages in the amphibian support mechanisms different from those of mammals.

Specification of neurotransmitter identity by Tal1 in thalamic nuclei.

BACKGROUND: The neurons contributing to thalamic nuclei are derived from at least two distinct progenitor domains: the caudal (cTH) and rostral (rTH) populations of thalamic progenitors. These neural compartments exhibit unique neurogenic patterns, and the molecular mechanisms underlying the acquisition of neurotransmitter identity remain largely unclear. RESULTS: T-cell acute lymphocytic leukemia protein 1 (Tal1) was expressed in the early postmitotic cells in the rTH domain, and its expression was maintained in mature thalamic neurons in the ventrolateral geniculate nucleus (vLG) and the intergeniculate leaflet (IGL). To investigate a role of Tal1 in thalamic development, we used a newly generated mouse line driving Cre-mediated recombination in the rTH domain. Conditional deletion of Tal1 did not alter regional patterning in the developing diencephalon. However, in the absence of Tal1, rTH-derived thalamic neurons failed to maintain their postmitotic neuronal features, including neurotransmitter profile. Tal1-deficient thalamic neurons lost their GABAergic markers such as Gad1, Npy, and Penk in IGL/vLG. These defects may be associated at least in part with down-regulation of Nkx2.2, which is known as a critical regulator of rTH-derived GABAergic neurons. CONCLUSIONS: Our results demonstrate that Tal1 plays an essential role in regulating neurotransmitter phenotype in the developing thalamic nuclei. Developmental Dynamics 246:749-758, 2017. © 2017 Wiley Periodicals, Inc.

Foxp2 Regulates Identities and Projection Patterns of Thalamic Nuclei During Development.

The molecular mechanisms underlying the formation of the thalamus during development have been investigated intensively. Although transcription factors distinguishing the thalamic primordium from adjacent brain structures have been uncovered, those involved in patterning inside the thalamus are largely unclear. Here, we show that Foxp2, a member of the forkhead transcription factor family, regulates thalamic patterning during development. We found a graded expression pattern of Foxp2 in the thalamic primordium of the mouse embryo. The expression levels of Foxp2 were high in the posterior region and low in the anterior region of the thalamic primordium. In Foxp2 (R552H) knockin mice, which have a missense loss-of-function mutation in the forkhead domain of Foxp2, thalamic nuclei of the posterior region of the thalamus were shrunken, while those of the intermediate region were expanded. Consistently, Foxp2 (R552H) knockin mice showed changes in thalamocortical projection patterns. Our results uncovered important roles of Foxp2 in thalamic patterning and thalamocortical projections during development.

Genetic Labeling of Nuclei-Specific Thalamocortical Neurons Reveals Putative Sensory-Modality Specific Genes.

The thalamus is a central brain structure with topographically ordered long-range axonal projections that convey sensory information to the cortex via distinct nuclei. Although there is an increasing knowledge about genes important for thalamocortical (TC) development, the identification of genetic landmarks of the distinct thalamic nuclei during the embryonic development has not been addressed systematically. Indeed, a more comprehensive understanding of how the axons from the individual nuclei find their way and connect to their corresponding cortical area is called for. Here, we used a genetic dual labeling strategy in mice to purify distinct principal sensory thalamic neurons. Subsequent genome-wide transcriptome profiling revealed genes specifically expressed in each nucleus during embryonic development. Analysis of regulatory regions of the identified genes revealed key transcription factors and networks that likely underlie the specification of individual sensory-modality TC connections. Finally, the importance of correct axon targeting for the specific sensory-modality population transcriptome was evidenced in a Sema6A mutant, in which visual TC axons are derailed at embryonic life. In sum, our data determined the developmental transcriptional profile of the TC neurons that will eventually support sensory processing.

Distinction in the immunoreactivities of two calcium-binding proteins and neuronal birthdates in the first and higher-order somatosensory thalamic nuclei of mice: Evolutionary implications.

Comparative embryonic studies are the most effective way to discern phylogenetic changes. To gain insight into the constitution and evolution of mammalian somatosensory thalamic nuclei, we first studied how calbindin (CB) and parvalbumin (PV) immunoreactivities appear during embryonic development in the first-order relaying somatosensory nuclei, i.e., the ventral posteromedial (VPM) and posterolateral (VPL) nuclei, and their neighboring higher-order modulatory regions, including the ventromedial or ventrolateral nucleus, posterior, and the reticular nucleus. The results indicated that cell bodies that were immunoreactive for CB were found earlier (embryonic day 12 [E12]) in the dorsal thalamus than were cells positive for PV (E14), and the adult somatosensory thalamus was characterized by complementary CB and PV distributions with PV dominance in the first-order relaying nuclei and CB dominance in the higher-order regions. We then labeled proliferating cells with [(3) H]-thymidine from E11 to 19 and found that the onset of neurogenesis began later (E12) in the first-order relaying nuclei than in the higher-order regions (E11). Using double-labeling with [(3) H]-thymidine autoradiography and CB or PV immunohistochemistry, we found that CB neurons were born earlier (E11-12) than PV neurons (E12-13) in the studied areas. Thus, similar to auditory nuclei, the first and the higher-order somatosensory nuclei exhibited significant distinctions in CB/PV immunohistochemistry and birthdates during embryonic development. These data, combined with the results of a cladistic analysis of the thalamic somatosensory nuclei, are discussed from an evolutionary perspective of sensory nuclei.

Kisspeptins in reproductive biology: consensus knowledge and recent developments.

Kisspeptins, a family of neuropeptides encoded by the Kiss1 gene that are mainly expressed in discrete neuronal populations of the hypothalamus, have recently emerged as essential upstream regulatory elements of GnRH (gonadotropin-releasing hormone) neurons and, thereby, potent elicitors of gonadotropin secretion. Indeed, kisspeptins are now recognized as important regulators of key aspects of the maturation and function of the reproductive axis, including the sexual differentiation of the brain, the timing of puberty, the adult regulation of gonadotropin secretion by gonadal hormones, and the control of fertility by metabolic and environmental (e.g., photoperiod) cues. Appreciation of these fundamental biological features has led to the contention that kisspeptins are indispensable elements of the reproductive brain whose relevance goes beyond their crucial physiological roles and may pose potential pathophysiological and therapeutic interest. In spite of such a consensus, recent developments in the field have helped to expand, and somewhat challenged, our current understanding of the neuroendocrine and molecular mechanisms whereby some of the effects of kisspeptins are conducted. This review aims to provide a synoptic and balanced account of the consensus knowledge and recent findings in the field of kisspeptin physiology, which we predict will be crucial in shaping the progress of our understanding of the roles played by this family of neuropeptides in reproductive biology.

Thalamocortical pathfinding defects precede degeneration of the reticular thalamic nucleus in polysialic acid-deficient mice.

The modification of the neural cell adhesion molecule (NCAM) with polysialic acid (polySia) is tightly linked to neural development. Genetic ablation of the polySia-synthesizing enzymes ST8SiaII and ST8SiaIV generates polySia-negative but NCAM-positive (II(-/-)IV(-/-)) mice characterized by severe defects of major brain axon tracts, including internal capsule hypoplasia. Here, we demonstrate that misguidance of thalamocortical fibers and deficiencies of corticothalamic connections contribute to internal capsule defects in II(-/-)IV(-/-) mice. Thalamocortical fibers cross the primordium of the reticular thalamic nucleus (Rt) at embryonic day 14.5, before they fail to turn into the ventral telencephalon, thus deviating from their normal trajectory without passing through the internal capsule. At postnatal day 1, a reduction and massive disorganization of fibers traversing the Rt was observed, whereas terminal deoxynucleotidyl transferase dUTP nick end labeling and cleaved caspase-3 staining indicated abundant apoptotic cell death of Rt neurons at postnatal day 5. Furthermore, during postnatal development, the number of Rt neurons was drastically reduced in 4-week-old II(-/-)IV(-/-) mice, but not in the NCAM-deficient N(-/-) or II(-/-)IV(-/-)N(-/-) triple knock-out animals displaying no internal capsule defects. Thus, degeneration of the Rt in II(-/-)IV(-/-) mice may be a consequence of malformation of thalamocortical and corticothalamic fibers providing major excitatory input into the Rt. Indeed, apoptotic death of Rt neurons could be induced by lesioning corticothalamic fibers on whole-brain slice cultures. We therefore propose that anterograde transneuronal degeneration of the Rt in polysialylation-deficient, NCAM-positive mice is caused by defective afferent innervation attributable to thalamocortical pathfinding defects.

Specificity and plasticity of thalamocortical connections in Sema6A mutant mice.

The establishment of connectivity between specific thalamic nuclei and cortical areas involves a dynamic interplay between the guidance of thalamocortical axons and the elaboration of cortical areas in response to appropriate innervation. We show here that Sema6A mutants provide a unique model to test current ideas on the interactions between subcortical and cortical guidance mechanisms and cortical regionalization. In these mutants, axons from the dorsal lateral geniculate nucleus (dLGN) are misrouted in the ventral telencephalon. This leads to invasion of presumptive visual cortex by somatosensory thalamic axons at embryonic stages. Remarkably, the misrouted dLGN axons are able to find their way to the visual cortex via alternate routes at postnatal stages and reestablish a normal pattern of thalamocortical connectivity. These findings emphasize the importance and specificity of cortical cues in establishing thalamocortical connectivity and the spectacular capacity of the early postnatal cortex for remapping initial sensory representations.

Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice.

The mammalian thalamus is located in the diencephalon and is composed of dozens of morphologically and functionally distinct nuclei. The majority of these nuclei project axons to the neocortex in unique patterns and play critical roles in sensory, motor, and cognitive functions. It has been assumed that the adult thalamus is derived from neural progenitor cells located within the alar plate of the caudal diencephalon. Nevertheless, how a distinct array of postmitotic thalamic nuclei emerge from this single developmental unit has remained largely unknown. Our recent studies found that these thalamic nuclei are in fact derived from molecularly heterogeneous populations of progenitor cells distributed within at least two distinct progenitor domains in the caudal diencephalon. In this study, we investigated how such molecular heterogeneity is established and maintained during early development of the thalamus and how early signaling mechanisms influence the formation of postmitotic thalamic nuclei. By using mouse genetics and in utero electroporation, we provide evidence that Sonic hedgehog (Shh), which is normally expressed in ventral and rostral borders of the embryonic thalamus, plays a crucial role in patterning progenitor domains throughout the thalamus. We also show that increasing or decreasing Shh activity causes dramatic reorganization of postmitotic thalamic nuclei through altering the positional identity of progenitor cells.

Brn3a-expressing retinal ganglion cells project specifically to thalamocortical and collicular visual pathways.

Retinal ganglion cells (RGCs) innervate several specific CNS targets serving cortical and subcortical visual pathways and the entrainment of circadian rhythms. Recent studies have shown that retinal ganglion cells express specific combinations of POU- and LIM-domain transcription factors, but how these factors relate to the subsequent development of the retinofugal pathways and the functional identity of RGCs is mostly unknown. Here, we use targeted expression of an genetic axonal tracer, tau/beta-galactosidase, to examine target innervation by retinal ganglion cells expressing the POU-domain factor Brn3a. Brn3a is expressed in RGCs innervating the principal retinothalamic/retinocollicular pathway mediating cortical vision but is not expressed in RGCs of the accessory optic, pretectal, and hypothalamic pathways serving subcortical visuomotor and circadian functions. In the thalamus, Brn3a ganglion cell fibers are primarily restricted to the outer shell of the dorsal lateral geniculate, providing new evidence for the regionalization of this nucleus in rodents. Brn3a RGC axons have a relative preference for the contralateral hemisphere, but known mediators of the laterality of RGC axons are not repatterned in the absence of Brn3a. Brn3a is coexpressed extensively with the closely related factor Brn3b in the embryonic retina, and the effects of the loss of Brn3a in retinal development are not severe, suggesting partial redundancy of function in this gene class.

Severe defects in dorsal thalamic development in low-density lipoprotein receptor-related protein-6 mutants.

Mice with mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein-6 (LRP6) have a smaller and severely disorganized dorsal thalamus and lack thalamocortical projections. Using molecular markers, we showed that most dorsal thalamic and epithalamic neurons were missing, and most of the major dorsal thalamic nuclei were not identifiable. However, the ventral thalamus was essentially unaffected, although the dorsal thalamic defect leads to rostral displacement of portions of the ventral thalamus. Analysis of younger embryos showed that epithalamic and dorsal thalamic neurons were not produced at early stages of development, whereas ventral thalamic neurons were still produced. These defects were accompanied by improper formation of the boundary between dorsal and ventral thalamus, the zona limitans interthalamica (ZLI). Furthermore, the expression of an early marker of posterior forebrain development that marks the compartment from the midbrain-hindbrain junction to the ZLI (including the future dorsal thalamus, pretectum, and midbrain) was disrupted, supporting the idea that diencephalic development is abnormal from very early in embryogenesis. This study provides compelling in vivo evidence that thalamic development requires normal activity of the LRP6-mediated canonical Wnt signaling pathway.

Expression of regulatory genes during differentiation of thalamic nuclei in mouse and monkey.

Expression patterns of genes implicated in development of the thalamus were examined in mice and monkeys, using in situ hybridization with RNA probes complementary to Cad6, Dlx1, Dlx2, Dlx5, Gbx2, Id2, and Lef1 cDNAs. Expression patterns were related to the evolving cytoarchitecture in mice at birth (P0) and in adulthood, and in fetal monkeys early and late in the period of gestation when thalamic nuclei are becoming histologically differentiated out of a series of pronuclear masses. At the earlier developmental stage, each gene was expressed in a pattern that appeared to be pronucleus-specific and maintained a nucleus-specific pattern into adulthood, with the possible exception of Gbx2. Each gene displayed a unique expression pattern in the dorsal thalamus, ventral thalamus, and epithalamus, and no gene was expressed throughout all three divisions or in every nucleus of a division. With the exception of Dlx2, whose expression disappeared at the later time point, all continued to be expressed into adulthood at higher levels and with identical patterns. Despite late appearance of gamma-aminobutyric acid (GABA)ergic cells in the dorsal lateral geniculate nucleus of mice, no Dlx genes, which promote formation of a GABAergic phenotype elsewhere, were detected in dorsal thalamus. Each thalamic nucleus was distinguished by expression of a combination of genes, and homologous nuclei in mouse and monkey exhibited the same combination. The presence of a centre médian nucleus and four pulvinar nuclei in monkeys was marked by patterns of expression not found in mice. The centre médian nucleus was marked by high expression of Id2, which was expressed only weakly in very few nuclei of mice.

Development of the human motor-related thalamic nuclei during the first half of gestation, with special emphasis on GABAergic circuits.

This study analyzed the expression of differentiation markers (Calbindin D28K: CaBP; parvalbumin: PARV; calretinin: CalR), gamma-aminobutyric acid (GABA) markers (GABA, glutamic acid decarboxylases: GAD65, GAD67; and GABA transporters: GAT1, GAT3), and other markers (neurotensin: NT, and neurofilament-specific protein: SMI32) in the human thalamus at 8-23 gestation weeks (g.w.), focusing on the motor-related nuclei. From 8-13 g.w. mainly CaBP was expressed in the cells while fiber bundles traversing the thalamus in addition to CaBP expressed all GABA markers except GAD67. CaBP and PARV expression patterns in different nuclei changed over the time course studied, whereas NT was expressed consistently along the anterior-lateral curvature of the thalamus. CalR and SMI were detectable at 23 g.w. in the ventral parts of the dorsal thalamus. Most remarkably, punctate GAD65 immunoreactivity in the neuropil was confined to the nigro- and pallidothalamic afferent receiving nuclei from 16 to about 21 g.w., overlapping with that of CaBP in some of these nuclei (subdivisions of the ventral anterior and mediodorsal nuclei) and with PARV in others (centromedian nucleus). During this period, GAD65 immunoreactivity can be considered a marker of the basal ganglia afferent receiving territory in the motor thalamus. GAD67-positive local circuit neurons were first detected at 12-13 g.w. in the thalamic nuclei outside the basal ganglia afferent receiving territory. In the ventral anterior and centromedian nuclei, GAD-containing local circuit neurons were not conspicuous even at 22-23 g.w. The cells of the reticular nucleus expressed GAD67 and PARV from 12 g.w. on starting in the lateral-posterior regions. By 23 g.w., both markers were expressed in about two-thirds of the nucleus except for its most medial-anterior part. The results imply spatially and temporally differential expression of GABA and differentiation markers in the developing human thalamus.

Quantitative features of the nucleus rotundus in the brain of pre- and post-hatch chicks.

The nucleus rotundus (ROT) is a major relay station in the tectofugal pathway of the avian visual system. In this study, some quantitative features of ROT in developing chicks were analysed using new stereological methods. Total neuron number (N) and mean volume (V) of ROT were estimated by the optical fractionator method and by the Cavalieri principle, respectively. Neuronal density of neurons in ROT was calculated from these data. The eyes of the chick embryo are not normally stimulated by light until days E19/20. Therefore in this study, chicks at three developmental stages were investigated: on the 17th embryonic day (E17), that is before light stimulation of the visual system, at the time of hatch (0-day, stimulated by light) and 10 days after hatch (10-day). The results showed that N was reduced by 27% between E17 and 0-day, and 7.8% between 0- and 10-day while neuronal density was reduced by 15% and 32% over the same periods. It is concluded that the reduction of neuronal density during the pre-hatch period may be due to neuron loss, whereas the post-hatch decrease of neuronal density may be the result of an increase in ROT total volume. Cell loss was more prominent in the pre-hatch than in the post-hatch period. Estimates of neuronal density in the developing ROT are not useful indicators of developmental status, since they do not relate to total neuron number.

Organisation and maturation of the human thalamus as revealed by CD15.

The distribution of the CD15 antigen (CD15, 3-fucosyl-N-acetyl-lactosamine, Lewis x) has been studied immunohistochemically in the fetal human thalamus. Its changing patterns could be related to three successive, but overlapping, periods primarily due to its association with radial glial cells, neuropil, and neural cell bodies, respectively. From 9 weeks of gestation (wg), a subset of CD15-positive radial glial cells distinguished the neuroepithelium of the ventral thalamus, a characteristic also seen in the developing mouse. Distal processes of the radial glial cells converged at the root of the forebrain choroid tenia, which was also CD15 positive. From 13 wg until approximately 20 wg, CD15-positive neuropil labeling marked the differentiation areas of prospective nuclei within the dorsal thalamus and progressively outlined their territories in a time sequence, which appeared specific for each nucleus. CD15 labeling of differentiating nuclei of the ventral, medial, anterior, and intralaminar thalamic divisions showed a transient topographic relationship with restricted areas of the ventricular wall. After 26 wg, CD15 immunoreactivity was observed in subpopulations of glial cells and neurons. Transient CD15 immunoreactivity was also found in delimited compartments within the subventricular region. The time of CD15 expression, its location, and cellular association suggest that CD15 is involved in segmentation of diencephalon, in the specification of differentiating nuclear areas and initial processes regarding the formation of intercellular contacts and cellular maturation.

Target-derived neurotrophic factors regulate the death of developing forebrain neurons after a change in their trophic requirements.

Many neurons die as the normal brain develops. How this is regulated and whether the mechanism involves neurotrophic molecules from target cells are unknown. We found that cultured neurons from a key forebrain structure, the dorsal thalamus, develop a need for survival factors including brain-derived neurotrophic factor (BDNF) from their major target, the cerebral cortex, at the age at which they innervate it. Experiments in vivo have shown that rates of dorsal thalamic cell death are reduced by increasing cortical levels of BDNF and are increased in mutant mice lacking functional BDNF receptors or thalamocortical projections; these experiments have also shown that an increase in the rates of dorsal thalamic cell death can be achieved by blocking BDNF in the cortex. We suggest that the onset of a requirement for cortex-derived neurotrophic factors initiates a competitive mechanism regulating programmed cell death among dorsal thalamic neurons.

Combinatorial expression patterns of LIM-homeodomain and other regulatory genes parcellate developing thalamus.

The anatomical and functional organization of dorsal thalamus (dTh) and ventral thalamus (vTh), two major regions of the diencephalon, is characterized by their parcellation into distinct cell groups, or nuclei, that can be histologically defined in postnatal animals. However, because of the complexity of dTh and vTh and difficulties in histologically defining nuclei at early developmental stages, our understanding of the mechanisms that control the parcellation of dTh and vTh and the differentiation of nuclei is limited. We have defined a set of regulatory genes, which include five LIM-homeodomain transcription factors (Isl1, Lhx1, Lhx2, Lhx5, and Lhx9) and three other genes (Gbx2, Ngn2, and Pax6), that are differentially expressed in dTh and vTh of early postnatal mice in distinct but overlapping patterns that mark nuclei or subsets of nuclei. These genes exhibit differential expression patterns in dTh and vTh as early as embryonic day 10.5, when neurogenesis begins; the expression of most of them is detected as progenitor cells exit the cell cycle. Soon thereafter, their expression patterns are very similar to those that we observe postnatally, indicating that unique combinations of these genes mark specific cell groups from the time they are generated to their later differentiation into nuclei. Our findings suggest that these genes act in a combinatorial manner to control the specification of nuclei-specific properties of thalamic cells and the differentiation of nuclei within dTh and vTh. These genes may also influence the pathfinding and targeting of thalamocortical axons through both cell-autonomous and non-autonomous mechanisms.

Ontogeny of the tectorotundal pathway in chicks (Gallus gallus): birthdating and pathway tracing study.

The avian tectorotundal system has been suggested as a homologue of the mammalian colliculopulvinar system. In the tectorotundal system, neurons of the stratum griseum centrale (SGC) of the optic tectum send their axons bilaterally to the nucleus rotundus (Rt). In transit to the Rt, the axons of the SGC neurons collateralize in the nuclei posteroventralis thalami (PV), subpretectalis (SP), and interstitiopretectosubpretectalis (IPS) of the tectothalamic tract (TT). The current study used birthdating and pathway-tracing methods to investigate the neurogenesis and time course of neuronal connections of the tectorotundal pathway in chicks during embryogenesis. By using tritiated thymidine autoradiography, we observed that the SGC neurons of the tectum were generated by embryonic days 3.0-5.5 (E3.0-E5.5), the Rt by E3.5-E5.0, and the nuclei of TT by E3.5-4.5. To trace the tectorotundal pathway, we injected cholera toxin B subunit (CTb) into the tectum, and the CTb-like immunoreactivity was examined. By E4.5-E5.5, some CTb-like immunoreactive (CTb-LI) axons terminated in the ipsilateral SP/IPS. By E6.0-E6.5, CTb-LI axon bundles were seen ipsilaterally in the TT. Increased numbers of labeled axons were seen terminating in the SP/IPS. By E7.0-E7.5, heavily labeled axons in the TT were observed with diffuse terminals in areas ventral to the presumptive Rt and PV. By E7.5-E8.0, the tectal axons innervated the ipsilateral Rt, in which some of the collaterals crossed the midline to the contralateral diencephalon. The crossed tectorotundal projection was seen first by E8.0-E8.5. Also, during this stage, a few CTb-LI collaterals terminated in the contralateral SP/IPS. Between E10 and E13, the pattern of bilateral tectorotundal projections became more regionalized, whereas labeling continued to increase in the SP/IPS. At E16, the labeling pattern of all tectorecipient structures resembled that of the hatchling. The current study revealed the temporal order of development of the tectorotundal pathway during embryogenesis. The SGC cells first innervate ipsilaterally the SP/IPS and then the Rt/PV. The schedule of the crossed tectorotundal connections coincides with the schedule of tectal projections onto the contralateral intrinsic nuclei of the TT. We conclude that E8.0 (+/- E0.5) is a critical stage for the development of the tectofugal pathway. Moreover, the current study provides important insights into the relative ontogeny of the mammalian tectofugal pathway.