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SUMMARY: The family of paired box (Pax) genes encodes the transcription factors that have been emphasized for the particular importance to embryonic development of the CNS, with the evidence obtained from various animal models. Human embryos have rarely been available for the detection of the expression of Pax family members. In this study 32 human embryos of Carnegie (CS) stages 10-20 were investigated to find the differences in the expression of Pax6 and Pax7 proteins in different regions of the neural tube and the caudal spinal cord. The expression of Pax6 and Pax7, as determined by immunohistochemistry, showed a tendency to increase in the later stages of the development both in the spinal cord and the brain. Significantly weaker expression of Pax6 and Pax7 was observed at CS 10 as compared to the later stages. At CS 10-12 weak expression of Pax6 was noticed in both dorsal and ventral parts of the developing spinal cord, while the expression of Pax7 was restricted to the cells in the roof plate and the dorsal part of the spinal cord. At CS 14-20 in the developing spinal cord Pax6 and Pax7 were detected mostly in the neuroepithelial cells of the ventricular layer, while only weak expression characterized the mantle and the marginal layers. At the same stages in the developing brain Pax6 and Pax7 were expressed in the different regions of the forebrain, the midbrain and the hindbrain suggesting for their involvement in the differentiation of neurons in specific parts of the developing brain. RESUMEN: La familia de genes Pax del inglés (Paired box) codifica los factores de transcripción debido a la particular importancia en el desarrollo embrionario del SNC, con la evidencia obtenida de varios modelos animales. Rara vez han estado disponibles embriones humanos para la detección de la expresión de genes de la familia Pax. En este estudio, se investigaron 32 embriones humanos de Carnegie (CS) etapas 10-20 para encontrar las diferencias en la expresión de las proteínas Pax6 y Pax7 en diferentes regiones del tubo neural y la médula espinal caudal. La expresión de Pax6 y Pax7, según la inmunohistoquímica, se observó una tendencia a aumentar en las etapas posteriores del desarrollo, tanto en la médula espinal como en el cerebro. Se observó una expresión significativamente más débil de Pax6 y Pax7 en CS 10 en comparación con las etapas posteriores. En CS 10-12 se notó una expresión débil de Pax6 en las partes dorsal y ventral de la médula espinal en desarrollo, mientras que la expresión de Pax7 se limitó a células en la placa del techo y dorsal de la médula espinal. En CS 14-20 en la médula espinal en desarrollo, Pax6 y Pax7 se observó principalmente en las células neuroepiteliales de la capa ventricular, mientras que expresión débil se caracterizó en las capas marginales. En las mismas etapas en el cerebro en desarrollo, Pax6 y Pax7 se expresaron en las diferentes áreas del prosencéfalo, el mesencéfalo y el mesencéfalo, lo que sugiere su participación en la diferenciación de las neuronas en partes específicas del cerebro en desarrollo.

SUMMARY: The family of paired box (Pax) genes encodes the transcription factors that have been emphasized for the particular importance to embryonic development of the CNS, with the evidence obtained from various animal models. Human embryos have rarely been available for the detection of the expression of Pax family members. In this study 32 human embryos of Carnegie (CS) stages 10-20 were investigated to find the differences in the expression of Pax6 and Pax7 proteins in different regions of the neural tube and the caudal spinal cord. The expression of Pax6 and Pax7, as determined by immunohistochemistry, showed a tendency to increase in the later stages of the development both in the spinal cord and the brain. Significantly weaker expression of Pax6 and Pax7 was observed at CS 10 as compared to the later stages. At CS 10-12 weak expression of Pax6 was noticed in both dorsal and ventral parts of the developing spinal cord, while the expression of Pax7 was restricted to the cells in the roof plate and the dorsal part of the spinal cord. At CS 14-20 in the developing spinal cord Pax6 and Pax7 were detected mostly in the neuroepithelial cells of the ventricular layer, while only weak expression characterized the mantle and the marginal layers. At the same stages in the developing brain Pax6 and Pax7 were expressed in the different regions of the forebrain, the midbrain and the hindbrain suggesting for their involvement in the differentiation of neurons in specific parts of the developing brain.

A Thesis for applying for the degree of Doctor of Philosophy in Veterinary Science The roles of BMP and Pax in the embryonic development have been extensively studied in recent years and the formation of the neural tube is usually described as a self-evident process, but formation of nervous system in human embryos has actually not been examined in detail. In the present study 40 human embryos at Carnegie stages (CS) 10-20 were obtained, and the expression of BMP-2, BMP-4, Pax2, Pax6 and Pax7 proteins were examined in the developing brain. 22 rat embryos of CS 14, 18 and 20 were employed to compare the BMP-2, BMP-4 and Pax2 expression in the developing spinal cord of human and rat embryos throughout early stages of the nervous system development. To detect expression of proteins the method of immunohistochemistry was used. BMP-2 and BMP-4 are essential signalling molecules for the formation of the neural tube in human embryos as their expression was seen throughout all studied developmental stages 10-20. The expression of both proteins, BMP-2 and BMP-4, had a tendency to decline in the later stages of the development. In the developing CNS of rat embryos BMP-2 and BMP-4 have a similar spatial and temporal expression pattern compared to the corresponding stages of the human embryos. However, slight differences were noted as in the spinal cord of rat embryos BMP-2 and BMP-4 expression was weaker compared to human embryos. In the human embryos Pax2, Pax6 and Pax7 were identified as signalling molecules that are involved in the formation of the early spinal cord and brain. The expression of Pax2, Pax6 and Pax7 had a tendency to increase in the later stages of the development both in the spinal cord and the brain. Significantly weaker expression of the Pax proteins was observed at CS 10. The similar temporal expression pattern of Pax2 to human embryos was seen in the forming spinal cord of rat embryos, as the Pax2 expression was found to increase at the later stages of the development. Differently from human embryos, no clear regional differences in Pax2 expression in the developing spinal cord of the rat embryos were not seen. It can be said that in the human and rat embryos BMP-2, BMP-4, Pax2, Pax6 and Pax7 proteins are associated with the establishment of neuroepithelial cells within the developing neural tube and with the migration and the differentiation of specific neural cell populations. In particular, the studied signalling molecules play an essentially important role in the determining of the dorsal-ventral and rostral-caudal axis of the developing brain. BMP ja Pax valgud on ühed olulisemad signaalmolekulid imetajate neuraaltoru moodustumisel ja edasises kesknärvisüsteemi arengus. BMP ja Pax valkude osalusest inimembrüo närvisüsteemi varajases arengus on siiani ilmunud üksikud teadusartiklid. Käesoleva töö eesmärgiks oli uurida BMP-2, BMP-4, Pax2, Pax6 ja Pax7 ekspressiooni inimembrüo arenevas pea- ja seljaajus. Lisaks inimembrüotele analüüsiti BMP-2, BMP-4 ja Pax2 avaldumist rottide embrüote seljaajus. Uuringuteks kogutud embrüod jäid Carnegie arenguetappidesse 10–20, roti embrüod vastavalt 14, 18 ja 20 arenguetappi. BMP ja Pax valkude ekspressiooni hindamiseks kasutati immunohistokeemilist meetodit. BMP ja Pax signaalmolekulide ekspressioon avaldus kõigis uuritud etappides 10–20. Nii BMP-2 kui ka BMP-4 avaldumisel ilmnes hilisemates arenguetappides ekspressiooni nõrgenemise tendents. BMP-2 ja BMP-4 on olulised kasvufaktorid inimembrüo seljaaju dorsoventraalse telje väljakujunemisel, eriti etappides 16–18, kus antud töö tulemused näitasid BMP-de tugevamat ekspressiooni areneva seljaaju dorsaalosas võrreldes ventraalosaga. Roti arenevas seljaajus ilmnes võrreldes inimembrüote vastavate arenguetappidega ajalise ja ruumilise BMP-2 ja BMP-4 ekspressiooni sarnasus. Nii Pax2, Pax6 kui Pax7 avaldumise hindamisel selgus ekspressiooni tugevnemine hilisematel arenguetappidel. Pax valkude ekspressioon etappidel 10–14 näitab antud signaalmolekulide vajalikkust seljaaju dorsoventraalse telje ja ajuvatsakeste moodustumisel. Hilisemates etappides 16–20 on Pax2, Pax6 ja Pax7 kaasatud ees-, kesk- ja tagaaju mitmete erinevate piirkondade arengusse. Roti ja inimembrüo arenevas seljaajus avaldus sarnane Pax2 ajaline ekspressioon – Pax2 signaal tugevnes hilisematel arenguetappidel. Erinevalt inimembrüotest, ei ilmnenud rottide embrüotel arenevas seljaajus Pax2 avaldumises selgeid regionaalseid erinevusi dorsaalse ja ventraalse poole vahel. Kokkuvõtlikult võib öelda, et BMP-2, BMP-4, Pax2, Pax6 ja Pax7 on olulisel määral seotud neuroepiteeli rakkude formeerumise, migreerumise ja diferentseerumisega erinevateks närvirakkude populatsioonideks. Uuritud singnaalmolekulid on inimese ja roti embrüote varajases kesknärvisüsteemi arengus määrava tähtsusega dorsoventraalse ja kraniokaudaalse telje väljakujunemisel. Publication of this dissertation is granted by the Estonian University of Life Sciences

The role of bone morphogenetic proteins (BMP-s) in the development of the nervous system has been widely studied on avian and rodent embryos. Human embryos have rarely been available for detection of BMP expression. In this study 39 human embryos of Carnegie stages (CS) 10-20 were investigated. The embryos were fixed in paraformaldehyde, embedded in paraffin and sectioned serially in transverse direction. BMP-2 and BMP-4 protein expression in the developing neural tube and the caudal spinal cord was determined by immunohistochemistry. Our data show that BMP-s tend to be more expressed in the neural tube in earlier stages; in particular, BMP-4 staining was found to be higher at CS10 compared to CS20. More detailed analysis was performed on embryos of CS14-18. Stronger BMP-2 and BMP-4 expression was found in the dorsal part than in the ventral part of the spinal cord. No differences were seen in the staining intensity of BMP-s in the spinal ganglia. Interestingly, in neural crest cells BMP-2 staining was stronger at CS16 and CS18 as compared to CS14, while no differences were found in BMP-4 staining. On the other hand, in the non-neural ectoderm BMP-4 staining was found to be stronger at CS16 than at CS14, while no differences were seen for BMP-2. In conclusion, expression of BMP-s in the developing neural tube and spinal cord of human embryos is generally in accordance with the findings made in rodents and birds.

The role of bone morphogenetic proteins (BMP-s) in the development of the nervous system has been widely studied on avian and rodent embryos. Human embryos have rarely been available for detection of BMP expression. In this study 39 human embryos of Carnegie stages (CS) 10-20 were investigated. The embryos were fixed in paraformaldehyde, embedded in paraffin and sectioned serially in transverse direction. BMP-2 and BMP-4 protein expression in the developing neural tube and the caudal spinal cord was determined by immunohistochemistry. Our data show that BMP-s tend to be more expressed in the neural tube in earlier stages; in particular, BMP-4 staining was found to be higher at CS10 compared to CS20. More detailed analysis was performed on embryos of CS14-18. Stronger BMP-2 and BMP-4 expression was found in the dorsal part than in the ventral part of the spinal cord. No differences were seen in the staining intensity of BMP-s in the spinal ganglia. Interestingly, in neural crest cells BMP-2 staining was stronger at CS16 and CS18 as compared to CS14, while no differences were found in BMP-4 staining. On the other hand, in the non-neural ectoderm BMP-4 staining was found to be stronger at CS16 than at CS14, while no differences were seen for BMP-2. In conclusion, expression of BMP-s in the developing neural tube and spinal cord of human embryos is generally in accordance with the findings made in rodents and birds. El papel de las proteínas morfogenéticas óseas (BMP-s) ha sido ampliamente estudiado en el desarrollo del sistema nervioso en embriones de aves y roedores. Los embriones humanos rara vez han estado disponibles para la detección de la expresión de BMP. En este estudio se investigaron 39 embriones humanos de los estadios Carnegie (CS) 10-20. Los embriones fueron fijados en paraformaldehído, embebidos en parafina y seccionados en serie en dirección transversal. Se determinó por inmunohistoquímica BMP-2 la expresión de la proteína BMP-4 en el tubo neural y en la médula espinal caudal en desarrollo. Nuestros resultados mostraron que la BMP-s tienden a ser más expresadas en el tubo neural en etapas tempranas, en particular, se encontró tinción BMP-4 más alta en comparación con CS10 CS20. Un análisis más detallado se realizó en embriones de CS14-18. En la parte dorsal se observó mayor expresión de BMP-2 y de BMP-4 que en la parte ventral de la médula espinal. No se observaron diferencias en la intensidad de la tinción de BMP-s en los ganglios espinales. Curiosamente, en las células de la cresta neural BMP-2 la tinción fue más fuerte en CS16 y CS18 en comparación con CS14, mientras que no se encontraron diferencias en la tinción de BMP-4. Por otro lado, en el ectodermo no neural se encontró tinción BMP-4 más fuerte en CS16 que en CS14, mientras que no se observaron diferencias para BMP-2. En conclusión, la expresión de BMP-s en el tubo neural en desarrollo y la médula espinal de embriones humanos está generalmente de acuerdo con los hallazgos realizados en roedores y aves.